Pichia

Ann N Y Acad Sci, 1998 Dec 13, 864, 14 - 22
A toolbox of recombinant lipases for industrial applications; Schmidt-Dannert C et al.; We created a toolbox of recombinant, microbial lipases, which allows us in combination with a lipase database to choose among the overexpressed lipases the most appropriate for a specific application and to improve it further via mutagenesis . By systematic comparison of geometry and properties of the scissile fatty acid binding site of five representative lipases of each family of structurally homologous lipases, three subgroups can be defined . Hence, efficient expression systems for the functional production of large amounts of microbial lipases, representing different lipase subgroups, were developed . In particular, recombinant lipases from Bacillus thermocatenulatus and Pseudomonas cepacia were functionally overexpressed in E . coli . The lipase genes from Geotrichum candidum CMICC 335426 and Rhizopus oryzae were overexpressed in Pichia pastoris . Due to an unusual codon usage that prevents heterologous expression, the LIP1 gene (1647 nt) of Candida rugosa was completely synthesized and overexpressed in Pichia pastoris.

FEBS Lett, 1999 Jan 8, 442(1), 95 - 8
Recombinant human amyloid precursor-like protein 2 (APLP2) expressed in the yeast Pichia pastoris can stimulate neurite outgrowth; Cappai R et al.; The human amyloid precursor-like protein 2 (APLP2) is a member of the Alzheimer's disease amyloid precursor protein (APP) gene family . The human APLP2 ectodomain (sAPLP2) was expressed in the yeast Pichia pastoris and the recombinant sAPLP2 was purified from the culture medium in a single step by metal-chelating Sepharose chromatography . The neuritotrophic activity of APLP2 was compared to the APP isoforms sAPP695 and sAPP751 on chick sympathetic neurones . APLP2 had neurite outgrowth-promoting activity similar to that of the APP isoforms . This suggests that APP and APLP2 have a similar or related role and supports the idea of a redundancy in function between the APP-gene family proteins.

Biochemistry, 1998 Dec 22, 37(51), 17745 - 53
The ice-binding site of sea raven antifreeze protein is distinct from the carbohydrate-binding site of the homologous C-type lectin; Loewen MC et al.; Antifreeze proteins lower the freezing point of their solution by binding to ice and inhibiting its growth . One of several structurally different antifreeze proteins in fishes (type II) is homologous to the carbohydrate-recognition domain of Ca2+-dependent lectins and adopts the same three-dimensional fold . Type II antifreeze proteins from herring and smelt require Ca2+ for binding to ice, whereas this same antifreeze protein in sea raven binds to ice in the absence of Ca2+ and has only two of the five Ca2+-liganding amino acids that are present in the lectin . To locate the ice-binding site, site-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven antifreeze protein were produced by secretion from Pichia pastoris . Pairs of amino acid replacements, insertions, and a peptide loop swap were made in the region equivalent to the sugar-binding site of the lectin that encompasses loops 3 and 4 and beta-sheets 7 and 8 . Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca2+-binding site are only peripherally involved in ice binding . When adjacent surface residues were mutated, the replacement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation . This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corresponding to the proposed CaCO3-binding surface of a third homologue, the pancreatic stone protein . Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein gene than to the lectin gene . These results support the notion that this family of proteins has evolved more than one binding surface from the same protein scaffold.

Biochemistry (Mosc), 1998 Dec, 63(12), 1407 - 13
Formation and distribution of modified FAD between isozymes of alcohol oxidase in the methylotrophic yeast pichia methanolica; Ashin VV et al.; The content of modified FAD (mFAD) in isoforms of alcohol oxidase (AO) in the methylotrophic yeast Pichia methanolica MH4 is increased in the stationary growth phase of the culture or under lower dilution rates . The isoform with slower electrophoretic mobility has the higher mFAD content . The results of in vitro experiments and the occurrence of mFAD in AO-lacking mutants of Hansenula polymorpha DL-1 imply a biosynthetic origin of this cofactor . HPLC analysis of tryptic hydrolysates of the first and the ninth isoforms of AO from P . methanolica MH4 revealed two different types of subunits . Possible mechanism of mFAD formation and distribution between the AO isozymes is discussed.

Biochemistry, 1999 Jan 19, 38(3), 1111 - 8
Calcium binding to the class I alpha-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif; Lipari F et al.; Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution . These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues . The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M) . Ca2+ protected the enzyme from thermal denaturation . Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding . In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme . The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively . The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM) . The E503Q mutant is completely inactive . Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.

Biochem Mol Biol Int, 1998 Dec, 46(6), 1109 - 16
High-level expression of human interleukin-17 in the yeast Pichia pastoris; Zhou L et al.; Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer . The monomer of rhIL-17 stimulated mouse fibroblast 3T3 cells to secrete IL-6 and was specifically bound to its receptors on 3T3 cells.

Biotechnol Appl Biochem, 1999 Feb, 29 ( Pt 1), 79 - 86
Secretory expression and characterization of insulin in Pichia pastoris; Kjeldsen T et al.; The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin . The S . cerevisiae mating factor alpha (alpha-factor) prepro-leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P . pastoris . Synthetic prepro-leaders developed for the secretory expression of the insulin precursor in S . cerevisiae also facilitated the secretion of the insulin precursor expressed in P . pastoris . In contrast with S . cerevisiae, only insulin precursor and no unprocessed hyperglycosylated alpha-factor pro-leader/insulin precursor fusion protein was secreted from P . pastoris . A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P . pastoris . A synthetic prepro-leader, but not an alpha-factor prepro-leader lacking N-glycosylation sites, facilitated the secretion of the insulin precursor in P . pastoris . P . pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S . cerevisiae . Peptide mapping and MS indicated a structure of the insulin precursor expressed in P . pastoris identical with that of human insulin.

J Hypertens, 1998 Dec, 16(12 Pt 2), 1971 - 8
Purification and characterization of a neutral endopeptidase-like enzyme from human urine; Di Marco GS et al.; OBJECTIVE: The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine . METHODS: Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column . NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp . The Km was determined using Abz-FDQ-EDDnp as a substrate . The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC) . The mol . wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast . RESULTS: The NEP-like was purified from human urine until homogeneity and presented a mol . wt of 94000 . The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP . For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 microM . The enzymatic activity was inhibited by thiorphan and phosphoramidon . In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP . CONCLUSION: A NEP-like enzyme was purified from human urine . Based on the mol . wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney . In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron . An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

J Pharm Biomed Anal, 1998 Sep 1, 17(6-7), 1111 - 28
Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection; Liden H et al.; An enzyme based amperometric biosensor used as a selective and sensitive detection unit in column liquid chromatography for the determination of ethanol and methanol in biological fluids such as plasma and urine is described . The reagentless enzyme electrode is based on the co-immobilisation of alcohol oxidase and horseradish peroxidase in carbon paste . The selectivity of the biosensor was found to vary when four various alcohol oxidase enzyme preparations from Candida boidinii, Pichia pastoris, and Hansenula polymorpha were used in the biosensors described . High sensitivity could be obtained for a number of alcohols, organic acids, and aldehydes . Optimisation regarding the sensitivity and selectivity of the four alcohol oxidase co-immobilised biosensors are outlined . A fast and reliable liquid chromatographic separation system with a PLRP-S polymer based separation column used with a phosphate buffer as the mobile phase was optimised using the best biosensor which was based on alcohol oxidase from P . pastoris and which showed the highest turnover rate for alcohols, as the detector for the determination of ethanol and methanol in human urine and plasma samples . The selectivity and stability of the biosensor were retained by working at an applied potential of -50 mV versus Ag/AgCl, the optimal operational potential, and by the casting of a protective membrane on the electrode surface . High selectivity of the enzyme electrode was also found towards other easily oxidisable interfering species normally present in biological fluids . It was found that stable and reliable determinations of ethanol and methanol in plasma and urine could be performed with only a simple dilution and centrifugation step prior to injection into the liquid chromatographic system . An analysis time of 4 min was required for the assay, with a sample throughput of 13 samples h(-1).

Protein Expr Purif, 1998 Dec, 14(3), 425 - 33
Production of recombinant human bile salt stimulated lipase and its variant in Pichia pastoris; Sahasrabudhe AV et al.; hBSSL and its truncated variant hBSSL-C cDNA clones were expressed in Pichia pastoris using two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium . Both recombinant proteins were secreted into the culture medium to a level of 45-50 mg/liter in shake flask cultures . Native signal peptide of hBSSL was recognized in P . pastoris and was cleaved at the same site as in humans . The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome . The multicopy transformant clone was found to be very stable . When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter . The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile .

Protein Expr Purif, 1998 Dec, 14(3), 327 - 34
Cloning, expression, purification, and immunocharacterization of placental protein-14; Dutta B et al.; Human placental protein-14 (PP-14), a member of the lipocalin superfamily, shares homology at the level of the primary and secondary structures with bovine beta-lactoglobulin . It is the most prominent endometrial protein synthesized by the glandular cells of endometrium under estrogen priming and progesterone stimulation . The temporal and spatial expression of PP-14 in the female reproductive tract combined with its biological activities ex vivo suggest that this glycoprotein probably plays an essential physiological role in the regulation of fertilization, implantation, and maintenance of pregnancy . We proposed to elucidate the molecular mechanisms involved in the function of this protein . A prerequisite to such investigations on any protein is the availability of sufficient amounts of the same in a homogenous form . Therefore, recombinant DNA technology was employed . The PP-14 cDNA was obtained from the first-trimester endometrial tissue RNA by RT-PCR using unique primers . After confirming the identity of the gene, the protein was expressed in Escherichia coli and purified to homogeneity . The gene was also cloned and expressed in Pichia pastoris to obtain the protein product in a glycosylated form . The recombinant proteins were immunocharacterized using a cross-reactive antibody raised to bovine beta-lactoglobulin . Polyclonal antiserum raised to the E coli expressed PP-14 also bound to the native PP-14 from amniotic fluid suggesting that recombinant PP-14 may be exploited to elucidate functional aspects of the protein .

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 227 - 37
Overview of N- and O-linked oligosaccharide structures found in various yeast species; Gemmill TR et al.; Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides . Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue . Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2) . In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S . pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues . Charged groups are also found in S . cerevisiae and P . pastoris N-glycans in the form of mannose phosphate diesters . Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence . O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins . Once transported to the Golgi, sugar transferases add one (C . albicans) or more (P . pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S . cerevisiae) . S . pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.

FEBS Lett, 1998 Dec 11, 441(1), 25 - 8
Selective inhibition of human type 1 11beta-hydroxysteroid dehydrogenase by synthetic steroids and xenobiotics; Hult M et al.; Functional analyses were performed with microsomal human 11beta-hydroxysteroid dehydrogenase type 1 overexpressed in the yeast Pichia pastoris . Cell extracts or microsomes from transformed strains displayed dehydrogenase and reductase activities, which were up to 10 times higher than in human liver microsomes, while for whole cells cortisone reduction but no dehydrogenase activity was observed . The synthetic glucocorticoids prednisolone and prednisone were efficiently metabolized by subcellular fractions, whereas no activity was observed with dexamethasone, budesonide and deflazacort . Inhibitors found to be effective towards the recombinant 11beta-hydroxysteroid dehydrogenase include synthetic steroids and xenobiotic compounds, revealing selective inhibition of the reaction direction, useful for development of specific inhibitors.

FEBS Lett, 1998 Dec 4, 440(3), 356 - 60
Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris; Hochstrasser U et al.; The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZ alphaC, containing the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway . Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.

J Biotechnol, 1998 Dec 11, 66(2-3), 147 - 56
Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties; Minning S et al.; The mature lipase of the fungus Rhizopus oryzae (ROL) was functionally expressed and secreted in the methylotrophic yeast Pichia pastoris . In a batch cultivation, where methanol feeding was linked to the dissolved oxygen content in the cultivation solution, a lipase activity of 500,000 units per liter (60 mg active lipase per liter) of culture was achieved after initial glycerol feeding of the culture . Recombinant ROL lipase was purified to homogeneity by a simple two-step purification procedure and had a specific activity of 8571 U mg-1 (triolein, 30 degrees C, pH 8.1) which is comparable with the purified native enzyme . The properties of the recombinant lipase were similar to those reported both for the native lipase and for the enzyme expressed in Escherichia coli and refolded from inactive inclusion bodies.

J Biotechnol, 1998 Dec 11, 66(2-3), 137 - 46
The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation; Biemans R et al.; A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris . By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site . Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule . Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies . N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products . Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1 . In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals . Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T . gondii tachyzoites.

Infect Immun, 1999 Jan, 67(1), 43 - 9
High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris: strong immunogenicity in Macaca mulatta immunized with P . vivax AMA-1 and adjuvant SBAS2; Kocken CH et al.; The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria . Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated . Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris . To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Deltaglyc) lacking N-glycosylation sites was also developed . Expression of the PV66Deltaglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies . Recombinant PV66Deltaglyc43-487 was reactive with conformation-dependent monoclonal antibodies . With the SBAS2 adjuvant, Pichia-expressed PV66Deltaglyc43-487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000 . This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

Protein Eng, 1998 Oct, 11(10), 847 - 53
NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotein for superior characteristics in NMR experiments; McAlister MS et al.; CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells . The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily for which the three-dimensional polypeptide fold is as yet unknown . Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris . Recombinant CD5d1 expressed in this manner was shown to be correctly folded by binding to anti-CD5 L17F12/Leu1 monoclonal antibody . Preliminary nuclear magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had spectral dispersion typical of a folded protein, but otherwise of such poor quality that NMR structural studies were not feasible . The analysis of glycoproteins by NMR is frustrated by sample heterogeneity and poor spectral quality associated with glycan resonance overlap and the potential for increased line-widths due to the large hydrodynamic volume . In order to pursue NMR structural studies of CD5d1 it was necessary to optimize the quality of NMR spectra of CD5d1 . A range of constructs of varying length and carbohydrate content were expressed in CHO cells and in P . pastoris . In addition the P . pastoris CD5d1 proved susceptible to N-glycan cleavage with endoglycosidase H . The protein products were characterised using size exclusion chromatography, NMR measurement of translational self-diffusion coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy experiments . Removal of an eight residue O-glycosylated C-terminal peptide, in particular, resulted in significant improvements in the quality of the CD5d1 NMR data, while retaining native protein structure . Two-dimensional heteronuclear NMR spectroscopy of nitrogen-15 isotope labelled deglycosylated CD5d1 (residues 1-110) prepared from P . pastoris suggests that this protein product is now amenable to solution structure determination.

Anticancer Res, 1998 Sep-Oct, 18(5A), 3193 - 201
Expression, purification, and characterization of a two domain carcinoembryonic antigen minigene (N-A3) in pichia pastoris . The essential role of the N-domain; You YH et al.; Carcinoembryonic antigen (CEA) is a 180 kDa glycoprotein expressed on the surface of normal and malignant human colon . The structure of CEA has seven predicted Ig-like domains (N-A1-B1-A2-B2-A3-B3) that are encoded by separate exons and contain independent epitopes that are recognized by monoclonal antibodies . The N-domain mediates homotypic cell adhesion as shown by deletion expression analysis, and may also interact with the A3 domain . Although we have been unsuccessful in expressing these domains in high yields of active protein in either bacterial or mammalian expression systems, we now report high yield expression in Pichia pastoris of a mini-gene (N-A3) comprising the N and A3 domains of CEA, and containing epitopes for the monoclonal antibodies T84.1 and T84.66 . N-A3 was constructed by splice overlap PCR from the CEA gene and fused to the yeast alpha-mating factor leader sequence and an N-terminal His6 tag . The secreted protein gave high level expression (20 micrograms/mL) and was purified in two steps using Ni(NTA) affinity chromatography followed by reversed phase HPLC . The purified protein (yield 6 mg from 600 mL of supernatant) had a single N-terminal sequence, the expected amino acid composition, and retained full reactivity to both T84.1 and T84.66 compared to native CEA . BIAcore analysis gave a Kaff of 4.4 x 10(10) M-1 for the binding of N-A3 to T84.1 and 2.2 x 10(10) M-1 for the binding of N-A3 to T84.66 . The molecular weight of N-A3 was 37 kDa before and 24 kDa after enzymatic deglycosylation as determined by SDS gel electrophoresis . The average N-glycosyl unit was calculated at 1850 Da (for 7 N-linked sites) suggesting a GN2Man9 oligosaccharide structure . N-A3 migrated as a dimer at 80 kDa and a monomer at 40 kDa on gel filtration analysis performed at pH 7.5, and 4.0, respectively . CEA exhibited the same conversion of dimers to monomers when analyzed by gel filtration at neutral and acid pH . The availability of this highly active CEA mini-gene should enable further structure-function studies including epitope analysis and investigation of monomer-dimer interactions.

Glycobiology, 1998 Dec, 8(12), 1183 - 94
Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi; Vandersall-Nairn AS et al.; The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole . We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library . The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani . The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures . The recombinant alpha-mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes . The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration . A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits . A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.

J Clin Microbiol, 1999 Jan, 37(1), 202 - 5
Rapid identification of Candida glabrata by using a dipstick to detect trehalase-generated glucose; Peltroche-Llacsahuanga H et al.; Candida glabrata is a yeast frequently isolated from human specimens . Based upon its well-known ability to rapidly hydrolyze trehalose, we have developed a novel and cost-effective test incubating one yeast colony emulsified in 50 microl of citrate buffer (0.1 M {pH 5 . 0}) containing 4% (wt/vol) trehalose for 3 h at 37 degrees C . Trehalase-generated glucose is detected with a commercially available dipstick (range, 1.0 to 50 g/liter) . For evaluation, consecutive clinical isolates and several reference strains of C . glabrata (n = 160), C . albicans (n = 120), and other yeast species with potential ability for utilization of trehalose (C . dubliniensis, n = 11; C . famata, n = 15; C . guilliermondii, n = 5; C . lusitaniae, n = 16; C . parapsilosis, n = 20; C . tropicalis, n = 34; C . viswanathii, n = 5; Pichia angusta, n = 2; C . zeylanoides, n = 2; Saccharomyces cerevisiae, n = 16; C . neoformans, n = 7) were tested . Identification of C . glabrata is achieved within 3 h, with a specificity of 99.1% and a sensitivity of 98.8% when grown on Sabouraud dextrose agar supplemented with 4% glucose.

J Mol Endocrinol, 1998 Dec, 21(3), 327 - 36
Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris; Fidler AE et al.; The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta subunit coding sequences . Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51.0 ng/ml and 17.5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the alpha and one copy of the beta subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay) . It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive . Measurements of secreted roFSH alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta subunits were stably dimerized . The receptor binding properties of the roFSH resemble those of native oFSH . In summary this paper reports the production, by P . pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

Biochemistry, 1998 Nov 24, 37(47), 16711 - 8
Characterization of the extracellular ligand-binding domain of the type II activin receptor; Greenwald J et al.; The binding of a ligand to cell surface receptors initiates a cascade of intracellular signals that generate responses to the external stimuli . Thus, this event plays a pivotal role in the mechanism of transmembrane signaling . Activin is a member of a cytokine family that is involved in diverse biological processes . To study the structural basis that underlies the transmembrane signaling mechanism, we have overexpressed the soluble extracellular domain of the type II activin receptor from mouse (ActRII-ECD) . We used the methylotrophic yeast Pichia pastoris as an expression host to produce a large quantity of ActRII-ECD . Expression was carried out in a fermentor with a typical yield of 10 mg of pure ActRII-ECD from a liter of growth media . Biological function was confirmed by the ability to decrease the activin-stimulated release of FSH from cultured rat pituitary cells in addition to several activin-binding assays, including native gel shift and chemical cross-linking . The glycosylation on ActRII-ECD was shown to be dispensable for high-affinity activin binding, and nonnatural sugars from the yeast expression host did not interfere with binding, indicating that the binding of activin is not sensitive to the environment near the two positions of N-linked glycosylation . Analytical ultracentrifugation of the complex between activin A and ActRII-ECD reveals that two receptors associate with one activin A dimer, consistent with results from chemical cross-linking experiments.

Eur J Biochem, 1998 Nov 1, 257(3), 599 - 606
The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance--distinct enzymic characteristics and alternative expression during pupal development; Houard X et al.; Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity . We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance . Purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors . Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and {Leu5}enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from {Leu5}enkephalinamide . However, Acer hydrolyses this substrate at a slightly faster rate than {Leu5}enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a kcat 15-fold higher than {Leu5}enkephalinamide . In addition, Acer did not cleave angiotensin I . In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration . Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis . Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent . We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4-P7, whereas the ACE activity expressed during stages P9-P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.

Biochim Biophys Acta, 1998 Nov 27, 1425(3), 587 - 98
Oligosaccharides of recombinant mouse gelatinase B variants; Van den Steen P et al.; Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars . Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC . This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants . The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2) . Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses . These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.

Appl Environ Microbiol, 1998 Dec, 64(12), 4809 - 15
Characterization of the prolyl dipeptidyl peptidase gene (dppIV) from the koji mold Aspergillus oryzae; Doumas A et al.; The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG) . We cloned and sequenced the DPPIV gene from an A . oryzae library by using the A . fumigatus dppIV gene as a probe . Reverse transcriptase PCR experiments showed that the A . oryzae dppIV gene consists of two exons, the first of which is only 6 bp long . The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein . Introduction of this gene into A . oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG . The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential . This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth . These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris . Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.

J Biotechnol, 1998 Oct 27, 65(2-3), 225 - 8
Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay; Luo D et al.; We constructed two fusion proteins of scFv linked to biotin mimetic sequence (BMS) via different linkers, and expressed them in the Pichia pastoris expression/secretion system . We found that both bi-functional scFv proteins exhibited their intrinsic binding activities to antigen CA125 determined in competitive radioimmunoassay experiments, but the fusion protein with a spacer between the scFv and BMS (scFv-spacer-BMS) showed higher binding activity of streptavidin than the one with c-Myc peptide as a linker.

J Chromatogr B Biomed Sci Appl, 1998 Sep 25, 716(1-2), 209 - 19
Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris; Tleugabulova D et al.; The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly . This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material . As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process . To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually . As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles . It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.

J Biol Chem, 1998 Nov 27, 273(48), 32000 - 8
Human cathepsin F . Molecular cloning, functional expression, tissue localization, and enzymatic characterization; Wang B et al.; A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library . The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein . Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B . Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup . Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus . The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin . Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L . The protease had a broad pH optimum between 5.2 and 6.8 . Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min . This may suggest a function in an acidic cellular compartment . Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells . However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.

Biochem Biophys Res Commun, 1998 Nov 9, 252(1), 190 - 4
Zinc-binding of endostatin is essential for its antiangiogenic activity; Boehm T et al.; Endostatin is a potent angiogenesis inhibitor in vitro and in vivo . We used the yeast Pichia pastoris to express and purify soluble endostatin . It was discovered that metal chelating agents can induce N-terminal degradation of endostatin . We theorized that a metal was removed from endostatin which changed the conformation and allowed a contaminating protease to degrade the N-terminus . Atomic absorption and amino acid analysis of endostatin purified from Pichia pastoris and mammalian cells showed a 1:1 molar ratio of Zn2+ to protein . Ding et al . have shown that histidines 1, 3, 11, and aspartic acid 76 coordinate the Zn2+ atom (1) . An H1/3A double, an H11A, and a D76A single mutant of endostatin were not able to regress Lewis lung carcinoma . We conclude that the ability of endostatin to bind Zn2+ is essential for its antiangiogenic activity .

Blood, 1998 Nov 15, 92(10), 3669 - 74
The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets; Applegate D et al.; The extended (E) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (EC) . A recombinant form of EC (rEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography . Purified rEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies . Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in E . Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs E613/644 and E780/793 . In the presence of 5 mmol/L EDTA, rEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rEC resistant . No protective effect from plasmic degradation was conferred to rEC by the peptides GPRPamide or GHRP, nor did rEC bind to a GPR peptide column . These results suggest that the EC domain contains a calcium-binding site, but lacks a polymerization pocket . By analogy with the site elucidated in the gammaC domain, we predict that the EC calcium binding site involves residues E772-778: DADQWEE.

Appl Microbiol Biotechnol, 1998 Sep, 50(3), 339 - 45
Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae; Shi NQ et al.; Respiratory and fermentative pathways coexist to support growth and product formation in Pichia stipitis . This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions . Expression of Saccharomyces cerevisiae URA1 (ScURA1) in P . stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present . ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth . Initial P . stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose . Cells produced even more ethanol faster following two anaerobic serial subcultures . Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation . P . stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose . These data imply that P . stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism . This is the first report of genetic engineering to enable anaerobic growth of a eukaryote.

Int J Food Microbiol, 1998 Sep 8, 43(3), 205 - 13
Detection and identification of wild yeasts in lager breweries; van der Aa Kuhle A et al.; Wild yeasts were detected in 41 out of 101 brewery yeast samples investigated using six different selective principles . Malt extract, yeast extract, glucose, peptone (MYGP) agar supplemented with 195 ppm CuSO4 was found to be the most effective selective principle, detecting wild yeasts in 80% of the contaminated samples . Both Saccharomyces and non-Saccharomyces wild yeasts were detected on this medium . Lysine medium, crystal violet medium and incubation of non-selective media at 37 degrees C detected wild yeasts in 46-56% of the contaminated samples . On using actidione medium, only 20% of the wild yeasts were detected . The combined use of MYGP supplemented with 195 ppm CuSO4 and one of the other selective principles did not improve the recovery of the wild yeasts . The wild yeasts found consisted of Saccharomyces cerevisiae (57%), Pichia spp . (28%) and Candida spp . (15%) . Using the API ID 32 C kit, 35 different assimilation profiles were obtained for the 124 wild yeast isolates investigated . All isolates were capable of glucose assimilation, whereas only 79% of the isolates assimilated saccharose, 75% maltose, 70% galactose, 65% raffinose and 65% lactate . Lactose, inositol, rhamnose and glucuronate were not assimilated by any of the isolates . The differences in assimilation pattern did not reflect any differences in recovery by the selective principles investigated . The majority of the wild yeast isolates investigated were capable of growth in wort and beer, indicating their possible role as spoilage organisms . The Sacch . cerevisiae isolates were found to be the most hazardous, with some isolates being capable of extensive growth in bottled beer within seventeen days at ambient temperature.

Eur J Biochem, 1998 Oct 1, 257(1), 131 - 41
Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1; McAlister MS et al.; CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells . The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained . Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris . CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul . Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content . CD5d1 from both CHO cells and P . pastoris have very similar properties . The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure . Observations have been made of the role of glycosylation of CD5 . P . pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies . The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5 . Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2 . Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.

Biochim Biophys Acta, 1998 Oct 23, 1425(2), 419 - 24
A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris; Bom IJ et al.; The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides . Its analysis is seriously hampered by the lack of easily available hydrolytic enzymes in a pure form . Here we describe a simple and efficient purification procedure of a recombinant beta-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris . Transformed cells efficiently secreted the enzyme into the induction medium . We purified the enzyme using a one-step method based on hydrophobic interaction chromatography . The yield was 80% . SDS-PAGE of the purified enzyme revealed a single band with an apparent molecular mass of 43 kDa . The isoelectric point of the enzyme was 5.8, and it showed maximal enzyme activity and stability at pH 5.0 . As beta-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure beta-(1-6)-glucanase will highly facilitate studies of the molecular organization of the fungal cell wall.

Protein Expr Purif . 1998 Nov;14(2):IV.
Papers to appear in forthcoming issues
Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity . Sequence homology to two families of serine proteinases.
Department of Internal Medicine, Asthma and Allergic Diseases Center, University of Virginia, Charlottesville, Virginia 22908, USA . jaw4m@virginia.edu

The dermatophyte fungus Trichophyton exhibits unique immunologic properties by its ability to cause both immediate and delayed type hypersensitivity . An 83-kDa Trichophyton tonsurans allergen (Tri t 4) was previously shown to elicit distinct T lymphocyte cytokine profiles in vitro . The homologous protein, Tri r 4, was cloned from a Trichophyton rubrum cDNA library, and the recombinant protein was expressed in Pichia pastoris . This 726-amino acid protein contained an arrangement of catalytic triad residues characteristic of the prolyl oligopeptidase family of serine proteinases (Ser-Asp-His) . In addition, a novel Trichophyton allergen, encoding 412 amino acids, was identified by its human IgE antibody-binding activity . Sequence similarity searches showed that this allergen, designated Tri r 2, contained all of the conserved residues characteristic of the class D subtilase subfamily (41-58% overall sequence identity) . Forty-two percent of subjects with immediate hypersensitivity skin test reactions to a Trichophyton extract exhibited IgE antibody binding to a recombinant glutathione S-transferase fusion protein containing the carboxyl-terminal 289 amino acids of Tri r 2 . Furthermore, this antigen was capable of inducing delayed type hypersensitivity skin test reactions . Our results define two distinct antigens derived from the dermatophyte Trichophyton that serve as targets for diverse immune responses in humans.

FEMS Immunol Med Microbiol, 1998 Sep, 22(1-2), 151 - 61
A transphyletic anti-infectious control strategy based on the killer phenomenon; Conti S et al.; A strategy for the prevention and control of candidiasis, pneumocystosis, and tuberculosis, based on the idiotypic network of the yeast killer effect has been envisaged . Anti-idiotypic antibodies representing the internal image of a candidacidal, pneumocysticidal, and mycobactericidal killer toxin from Pichia anomala and idiotypes of killer toxin-neutralizing monoclonal antibodies mimicking the specific cell wall receptor of sensitive microorganisms might provide a unique approach for engineering innovative antibiotics and vaccines active against taxonomically unrelated pathogenic microorganisms . The rationale of the strategy relies on a phenomenon of microbial competition which has been mutated by the immune system in the response to natural infections.

FEMS Immunol Med Microbiol, 1998 Sep, 22(1-2), 145 - 9
Effect of a killer toxin of Pichia anomala to Pneumocystis . Perspectives in the control of pneumocystosis; Seguy N et al.; Despite the development of drugs in the prophylaxis of pneumocystosis, Pneumocystis carinii remains a major opportunistic microorganism in immunosuppressed individuals, especially in human immunodeficiency virus-infected patients . Since side effects were frequently observed after administration of trimethoprim-sulfamethoxazole or pentamidine, the drugs which are mainly used in treating human P . carinii pneumonia (PCP), new therapeutic strategies should be developed . Over the last years, the inhibitory effect of a Pichia anomala killer toxin (PaKT), a molecule with a wide spectrum of antimicrobial activity, was characterized on P . carinii . The susceptibility of mouse and rat-derived Pneumocystis to PaKT has been demonstrated by in vitro attachment tests and in vivo infectivity assays . Nevertheless, PaKT is strongly antigenic, toxic and could not be used directly as a therapeutic agent . Then, a new strategy using killer toxin-like anti-idiotypic antibodies (KT-antiIds) mimicking the fungal toxin activity has been developed . Different KT-antiIds were obtained by idiotypic immunization with a monoclonal antibody (mabKT4) . This mabKT4 neutralized the killer properties of the PaKT . KT-antiIds were produced by immunization against the variable domain (idiotype) of mAbKT4 (internal image of the killer toxin receptor), or they were obtained directly from vaginal fluid of patients affected by recurrent vaginal candidiosis . In this last case, such natural KT-antiIds were immunopurified by affinity-chromatography with mAbKT4 and their anti-P . carinii activity was then evaluated . Our results showed that both the in vitro attachment of rat-derived parasites and their infectivity to nude rats were inhibited by the KT-antiIds . With regard to KT-antiIds obtained by immunization, the antimicrobial activity of a monoclonal KT-antiIds (mAbK10) has been evaluated by using a PCP experimental nude rat model treated by mAbK10 administered by aerosol . The pneumocystosis extension was significantly reduced in this model . The monoclonal KT-antiIds were effective against P . carinii in reducing parasite proliferation in lungs of nude rats . Further experiments are in progress to study the in vivo anti-P . carinii activity of KT-antiIds by using recombinant single-chain of the variable fragment of KT-antiIds . Yeast killer toxin-like recombinant molecules could provide the basis for a new therapeutic strategy towards the control of pneumocystosis.

Protein Expr Purif, 1998 Nov, 14(2), 197 - 207
Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris; Montesino R et al.; We report the characterization of N-linked oligosaccharides on six foreign glycoproteins secreted from the methylotrophic yeast Pichia pastoris . These proteins included: a bacterial enzyme, Bacillus licheniformis alpha-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase, and Mucor pusillus aspartic protease; and two higher eukaryotic proteins, Boophilus microplus (tick) gut antigen and bovine enterokinase catalytic subunit . The carbohydrates on these proteins were observed to vary in size, with Man8GlcNAc2 and Man9GlcNAc2 structures being the most frequently observed species . Substantial amounts of shorter oligomannoside structures were present only on invertase, and longer structures (up to Man18GlcNAc2) were common on aspartic protease and enterokinase . Phosphorylated oligosaccharides were observed on one protein, aspartic protease . Unlike oligosaccharides on glycoproteins secreted from S . cerevisiae, no terminal alpha1,3-linked mannosylation was observed on any of the six P . pastoris-secreted proteins . Changing the growth and induction medium from a minimal salt-based medium to a molasses-based medium had little effect on the size of the oligomannosides . From these results, it is apparent that most foreign proteins secreted from P . pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs in proteins secreted from S . cerevisiae .

Indian J Exp Biol, 1998 Jul, 36(7), 728 - 31
Comparative behaviour of yeast strains for ethanolic fermentation of culled apple juice; Modi DR et al.; The culled apple juice contained (% w/v): nitrogen, 0.036; total sugars, 11.6 and was of pH 3.9 . Saccharomyces cerevisiae NCIM 3284, Pichia kluyeri and Candida krusei produced more ethanol from culled apple juice at its optimum initial pH 4.5, whereas S . cerevisiae NCIM 3316 did so at pH 5.0 . An increase in sugar concentration of apple juice from natural 11.6% to 20% exhibited enhanced ethanol production and improved fermentation efficiency of both the S . cerevisiae strains, whereas P . kluyveri and C . krusei produced high ethanol at 11.6% and 16.0% sugar levels, respectively . Urea was stimulatory for ethanol production as well as fermentation efficiency of the yeast strains under study.

Syst Appl Microbiol, 1998 Aug, 21(3), 353 - 9
Screening of yeasts from Brazilian Amazon rain forest for extracellular proteinases production; Braga AA et al.; Eighty seven yeast strains representing 34 species isolated from Parahancornia amapa fruit and associated Drosophila flies collected in the Brazilian Amazon rain forest, were screened for proteinase production . Proteolytic activity was tested through casein hydrolysis in solid medium supplemented with 0.5% casein and glucose . Among 23 strains, 18 from genus Candida and 5 from Pichia were caseinolytic and produced proteinases in yeast carbon base liquid medium supplemented with casein 0.01% . The proteolytic activity was tested on pH ranging from 2.0 to 9.0 in correspondence to the pH of the cultures media in which the yeasts were grown . Six highly proteolytic strains: Candida parapsilosis AP153A, C . krusei AP176, C . sorbosa DR215, C . sorbosa AP259, C . valida AP209A and C . sorboxylosa AP287 were selected and the pH optima of production and the proteolytic activity were determined . In general the secretion of proteinase was maximum throughout the exponential and the stationary phases . Greater production occurred in acidic culture and high activity was observed at pH 3.0, 4.0 and 5.0.

Biochemistry, 1998 Oct 20, 37(42), 14958 - 65
Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris: immunodetection of alpha-, beta-, and gamma-secretase products; Le Brocque D et al.; betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP) . Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide . APP default secretion involves cleavage within the betaA4 domain by alpha-secretase . To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA . In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium . Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase . The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage) . Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages . Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide . These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P . pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.

Med Mycol, 1998 Aug, 36(4), 199 - 204
Effect of Pichia anomala killer toxin on Candida albicans; Mathews HL et al.; The effect of a Pichia anomala killer toxin upon a Candida albicans-sensitive strain was studied . Yeast and hyphae, after treatment with the toxin, were less capable of uptaking either {3H}-uridine or {35S}-methionine . In addition, the hyphal form of the fungus appeared to be less capable of DNA synthesis after toxin treatment . No effect of the killer toxin was shown upon a natural resistant mutant of the source strain . These data suggest that, similar to other killer yeast toxins, the toxin of P . anomala can produce a number of quantifiable effects upon sensitive C . albicans cells.

J Biol Chem, 1998 Oct 23, 273(43), 28091 - 7
Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris; Sowka S et al.; Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals . We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients . Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined . We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends . The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids . We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium . The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity . IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins . Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains . Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.

Protein Expr Purif . 1998 Oct;14(1):IV.
Papers to appear in forthcoming issues
Purification and characterization of a soybean root nodule phosphatase expressed in Pichia pastoris.
Department of Biochemistry, University of Nebraska-Lincoln, Nebraska, Lincoln, 68588-0664, USASoybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5'-nucleoside monophosphates . The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications . In order to perform directed mutagenesis and more extensive biochemical characterization, a means of producing recombinant ACP was needed . Several attempts were made to express ACP in Escherichia coli, but all conditions employed resulted in protein that was found entirely in inclusion bodies, and resolubilization experiments were unsuccessful . Therefore, the methyltrophic yeast Pichia pastoris was chosen as a eukaryotic expression host . The coding sequence of ACP was cloned into the pPIC9 vector to create a fusion with the yeast alpha mating factor secretion signal . The ACP:pPIC9 construct was integrated into P . pastoris strain GS115 . Expression of ACP was under the control of an alcohol oxidase methanol-inducible promoter . Methanol induction resulted in secretion of ACP to a level of 10 mg/L . The recombinant ACP was purified 550-fold to homogeneity by phenyl-Sepharose, hydroxyapatite, and MonoS chromatography . The purified enzyme had Km values of 0.08 and 0.12 for 5'-AMP and 5'-GMP . These values were similar to those obtained for the native ACP heterodimer purified from soybean (0.08 and 0.15 mM for 5'-AMP and 5'-GMP) . The specific activity of the recombinant enzyme for all substrates tested was 1.6- to 1.8-fold higher than the values for the purified soybean heterodimer .

Protein Expr Purif, 1998 Oct, 14(1), 97 - 103
Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris; Denton H et al.; beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment . In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy . The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments . At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet .

Biotechnol Appl Biochem, 1998 Oct, 28 ( Pt 2), 125 - 31
Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme; Niles AL et al.; Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin-like serine protease that is stored in and released from mast cell granules . This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome . Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies . Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium . Increasing levels of enzyme were detected in the medium for up to 3 days . Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two-step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin . Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE . These multiple forms were due to differences in post-translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa . A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration . Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 531 - 5
Use of the glyceraldehyde-3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris; Doring F et al.; The promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) was employed to produce the mammalian peptide transporters hPEPT1 and rPEPT2 as models for polytopic transmembrane proteins in the methylotrophic yeast Prichia pastoris . Cells of a recombinant renal peptide transporter (rPEPT2) clone produced constitutively the functional carrier protein . The level of functional expression of rPEPT2 with PGAP varied depending on the carbon source used for cell growth, but was up to five times higher than that obtained with the commonly employed inducible alcohol oxidase 1 promoter (PAOX1) . Similar results were obtained for the expression level of the human intestinal peptide transporter hPEPT1 controlled by either PGAP or PAOX1 . Therefore, the PGAP seems to be an attractive alternative to PAOX1 for generation of transgenic P . pastoris cells expressing functional mammalian membrane transport proteins at high levels.

Biochemistry, 1998 Sep 29, 37(39), 13696 - 703
Iron release is reduced by mutations of lysines 206 and 296 in recombinant N-terminal half-transferrin; Steinlein LM et al.; Human serum transferrin consists of two iron-binding lobes connected by a short peptide linker . While the high homology and structural similarity between the two halves of the molecule would suggest similar characteristics, it has been shown that the pH-dependent rate of release of iron from the N-terminal lobe is quite different from that of its C-terminal counterpart . This suggests that the N-lobe of human serum transferrin has a specific, pH-dependent, molecular mechanism for releasing iron . Sacchettini and co-workers using structural information have hypothesized that two lysines in the N-terminal lobe of ovotransferrin create a dilysine interaction and suggest that this is the trigger for pH-dependent iron release . To investigate this hypothesis, we used a Pichia pastoris expression system to produce large amounts of wild-type nTf, the single point mutants, nTfK206A (Lys 206 to alanine) and nTfK296A (Lys 296 to alanine), and the double mutant, nTfK206/296A . The purified recombinant proteins were then used to measure rates of iron release to pyrophosphate . It was found that the rate of iron release from all three mutant proteins at pH 5.7 (the pH at which nTf would normally release iron) was too slow to measure . Only when the pH was reduced to 5.0 could the rates of iron release from the mutant proteins be reliably determined . Although this precludes a direct comparison to wild-type nTf (the rate of iron release from nTf at pH 5.0 is too fast to measure), it implicates lysines 206 and 296 in the pH-dependent release of iron from nTf.

Plant Mol Biol, 1998 Oct, 38(3), 379 - 91
Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue; Tibbot BK et al.; An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli . The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9 . Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol . The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose . Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase . The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose . The recombinant alpha-glucosidase expressed in E . coli was used to generate polyclonal antibodies . The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination . Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent . Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.

Plant Physiol, 1998 Sep, 118(1), 237 - 47
Characterization of LeMir, a root-knot nematode-induced gene in tomato with an encoded product secreted from the root; Brenner ED et al.; A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet . This gene was therefore named LeMir (L . esculentum miraculin) . Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz) . LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root . Rapid induction of expression upon nematode infection is localized to root tips . In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis . The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies . Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding . LeMir is also expressed in tomato callus tissue . Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction . Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 591 - 6
Phylogenetic heterogeneity of the genus Williopsis as revealed by 18S rRNA gene sequences; James SA et al.; A phylogenetic investigation of the ascomycetous yeast genus Williopsis was performed by using 18S rRNA gene sequence analysis . Comparative sequence analysis revealed the genus to be phylogenetically heterogeneous . The five varieties of Williopsis saturnus {var . mrakii, var . sargentensis, var . saturnus (type), var . suaveolens and var . subsufficiens} were found to have identical 18S rRNA gene sequences and formed a distinct group, quite separate from all other Williopsis and non-Williopsis species examined . Williopsis mucosa was found to be the closet phylogenetic relative to the Williopsis saturnus group, however a sequence divergence of approximately 2.3% suggests this species may belong to a separate genus . The recently described species Williopsis salicorniae was found to exhibit a relatively close association with Ogataea minuta (identical to Pichia minuta), the type species of the genus Ogataea . The remaining two members of the genus, Williopsis californica and Williopsis pratensis, were found to form distinct lineages, displaying no specific association with any other Williopsis or non-Williopsis species . Based on comparative analysis of 18S rRNA genes it is apparent that the genus Williopsis as presently constituted is not monophyletic, and that the five currently recognized species form separate sublines each potentially worthy of separate generic status . The genus Williopsis should be restricted to the type species Williopsis saturnus and its five varieties . Despite the five varieties of Williopsis saturnus being genealogically indistinguishable at the 18S rRNA gene level, sequence analysis of the Internal transcribed spacer (ITS) region revealed that the five varieties could be differentiated on both their ITS1 and their ITS2 sequences, providing further evidence of the value of ITS sequences for discrimination of yeasts at the subspecies level.

Biochemistry, 1998 Sep 8, 37(36), 12631 - 9
A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms; Johnsen LB et al.; Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically . The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments . We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process . The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio . Only the minor form was able to bind and activate plasminogen . Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms . Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted . The same mechanism could be applied to human tPA as well . The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

J Biomol NMR, 1998 Jul, 12(1), 89 - 107
Complete assignment of 1H, 13C and 15N chemical shifts for bovine beta-lactoglobulin: secondary structure and topology of the native state is retained in a partially unfolded form; Uhrinova S et al.; Although beta-lactoglobulin (beta-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure . For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine beta-LG which differ by just two residues have different aggregation properties during milk processing . We have conducted solution-state NMR studies on a recombinant form of the A variant of beta-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer . Using a 13C, 15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances . Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence . From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric beta-LG A . It includes eight antiparallel beta-strands arranged in a barrel, flanked by an alpha-helix, which is typical of a member of the lipocalin family . A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology . Both forms have a ninth beta-strand which, at the higher pH, forms part of the dimer interface . These studies represent the first full NMR assignment of beta-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.

Mol Membr Biol, 1998 Apr-Jun, 15(2), 79 - 88
Expression of the mammalian renal peptide transporter PEPT2 in the yeast Pichia pastoris and applications of the yeast system for functional analysis; Doring F et al.; It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex . PEPT2 represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics . Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter . Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa . Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells . Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app . K0.5 of 0.143 +/- 0.016 mM . Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes . A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed . Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established . In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast . The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.

Carbohydr Res, 1998 Jun, 309(1), 77 - 87
The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the phosphorylated side chains; Parolis LA et al.; The exopolysaccharide produced by Pichia (Hansenula) holstii NRRL Y-2448 is composed of a phosphomannan core to which oligosaccharide diester phosphate side chains are appended . The oligosaccharides of the side chains were released as oligosaccharide phosphates and neutral oligosaccharides by mild hydrolysis with aqueous acetic acid and aqueous hydrogen fluoride, respectively . The liberated oligosaccharide phosphates were studied by NMR spectroscopy and by electrospray and fast atom bombardment mass spectrometry . The structures of the neutral oligosaccharides were determined by 1D and 2D NMR spectroscopic experiments . Further insight into the length of the side chains was obtained from a matrix assisted laser desorption ionisation-time of flight mass spectrometric study of high and low molecular weight fragments obtained from partial acid hydrolysis of the native polysaccharide.

Microbiology, 1998 Aug, 144 ( Pt 8), 2323 - 30
Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha; Pignocchi C et al.; Hansenula polymorpha (syn . Pichia angusta) is able to grow on nitrate as sole nitrogen source . Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity . NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine . Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate . Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate . Increases but not decreases in NR activity were dependent on protein synthesis . Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated . Some of these mutants showed reduced or absent NR activity . Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes . These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.

Yeast, 1998 Jul, 14(10), 895 - 903
Chromosomal DNA patterns and gene stability of Pichia pastoris; Ohi H et al.; We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis . The size of the P . pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains . Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains . P . pastoris is frequently used as an efficient host for heterologous gene expressions . We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth . No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation.

Gene, 1998 Aug 17, 216(1), 93 - 102
A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris; Shen S et al.; In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources . We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris . The gene contains a single short intron with typical yeast-splicing signals near its 5' end, the first intron to be demonstrated in this yeast . The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx . 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61-65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes . Using beta-lactamase as a reporter, we show that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source) . Furthermore, with either methanol or methylamine induction, levels of beta-lactamase produced under control of PFLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1) . Thus, PFLD1 is an attractive alternative to PAOX1 for expression of foreign genes in P . pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.

Scand J Immunol, 1998 Jul, 48(1), 26 - 36
Cloning and expression of ragweed allergen Amb a 6; Hiller KM et al.; We have cloned the protein coding region of an isoform of short ragweed allergen Amb a 6 (Ra6) and expressed the secreted product in Pichia pastoris at mg/l levels . 5' RACE was performed using sequence obtained from a partial Amb a 6 clone . This yielded a product whose deduced protein sequence has a characteristic signal sequence motif at the N-terminus followed by sequence consistent with that previously published for highly purified Amb a 6 {Roebber et al . J Immunol 1983;131:706-11} . The region encoding the secreted product was amplified by PCR and cloned into pPICZ alpha a, an expression vector for the yeast Pichia pastoris . Yeast transformed with this vector secrete a protein which migrates near Amb a 6 in SDS-PAGE . This secreted protein reacts with polyclonal anti-Amb a 6 antisera as well as an anti-Amb a 6 monoclonal antibody, and has the N-terminal sequence of Amb a 6 . By time-of-flight mass spectrometry, recombinant Amb a 6 has a molecular weight of 9884 +/- 0.2% . In addition to the deduced amino acid sequence of an Amb a 6 clone, the amino acid sequence of Amb a 6 protein is reported for comparison . The amino acid sequence was obtained by aligning overlapping tryptic and chymotryptic peptides from enzymatic digests of extensively reduced and alkylated Amb a 6 . Sequences from at least three closely related Amb a 6 isoforms are present among these peptides . The amino acid sequence closely matches the deduced amino acid sequence of the Amb a 6 clone.

Protein Expr Purif . 1998 Aug;13(3):IV.
Papers to appear in forthcoming issues
O-Mannosylation of Pichia pastoris cellular and recombinant proteins.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USAO-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells . Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose . The recombinant kringle 1-4 domain of human plasminogen expressed in P . pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides . Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site . The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components . These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P . pastoris cells and expressed proteins.

Biochemistry, 1998 Aug 4, 37(31), 11033 - 8
Deletion of the conserved first 18 N-terminal amino acid residues in rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA sensitivity and binding; Shi J et al.; To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues . The deletion mutants were expressed in the yeast Pichia pastoris . We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants . The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM) . In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI . Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding . By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein . To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.

Eur J Biochem, 1998 Jul 1, 255(1), 213 - 9
cDNA cloning of the 43-kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichia pastoris; Sowka S et al.; IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years . We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences . A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library . The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa . The deduced amino acid sequence had 39-42% identity to patatins from Solanum tuberosum . The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S . tuberosum, esterase activity . Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals . In contrast to patatins from S . tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated . These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S . tuberosum patatins.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9099 - 104
Crystal structure of a recombinant alphaEC domain from human fibrinogen-420; Spraggon G et al.; The crystal structure of a recombinant alphaEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 A . The protein, which corresponds to the carboxyl domain of the alphaE chain, was expressed in and purified from Pichia pastoris cells . Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize . An x-ray structure was determined by molecular replacement . The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group) . Virtually all of an asparagine-linked sugar cluster is present . Comparison with structures of the beta- and gamma-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains . Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.

Amyloid, 1998 Jun, 5(2), 79 - 89
Subcellular localization of the Alzheimer's disease amyloid precursor protein and derived polypeptides expressed in a recombinant yeast system; Culvenor JG et al.; Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris . The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies . The site of intracellular expression could be readily identified with specific antibodies . Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum . Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles . A construct encoding two copies of beta A4{1-42} linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested . A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP . The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia) . This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms . These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.

Vaccine, 1998 May-Jun, 16(9-10), 1053 - 5
Adjuvant and immunostimulating properties of the recombinant Bm86 protein expressed in Pichia pastoris; Garcia-Garcia JC et al.; The cattle tick Boophilus microplus has remained a latent problem to the cattle industry . The recombinant vaccine GAVAC against the cattle tick has proved its efficacy and, conveniently, combined with the use of chemicals could be the solution to this problem . As this vaccine is based in the recombinant concealed antigen Bm86, it has to be given periodically to the animal to maintain an adequate level of antibodies . Some other commercially available vaccines for cattle also have to be given periodically, which creates the possibility of combining vaccines for cattle . In an attempt to evaluate the possible interactions of the Bm86 with other vaccine antigens, a potent stimulatory effect was demonstrated of the recombinant Bm86 on the humoral immune response to the recombinant Hepatitis B surface antigen in mice, and to the inactivated Infectious Bovine Rhinothraqueitis virus in cattle . These results make the Bm86 antigen expressed in Pichia pastoris a good candidate for combining vaccines for cattle because of its dual role, immunogen and adjuvant.

Yeast, 1998 Jun 15, 14(8), 783 - 90
A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris; Sears IB et al.; The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors . Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P . pastoris . A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene . The utility of these vectors was illustrated by expressing the bacterial beta-glucuronidase (GUS) gene . Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter . The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells . GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF0279959; pIB3, AF027960; pIB4, AF027961.

Yeast, 1998 Jun 15, 14(8), 759 - 71
The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol; Luers GH et al.; Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts . We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol . An open reading frame of 1824 bp was identified that encodes a 65.3 kDa protein with high homology to DAK from Saccharomyces cerevisiae . Although DAK from P . pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic . The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P . pastoris dak delta mutant . Peroxisomes, which are essential for growth of P . pastoris on methanol, were present in the dak delta mutant and the import of peroxisomal proteins was not disturbed . The dak delta mutant grew at normal rates on glycerol and oleate media . However, unlike the wild-type cells, the dak delta mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate . The metabolic pathway explaining the reduced growth rate of the dak delta mutant on dihydroxyacetone is discussed . The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198.

Glycobiology, 1998 Sep, 8(9), 919 - 25
Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris; Gallet PF et al.; A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361) . Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l) . Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst . Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters . The two forms (cell-enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.

Can J Microbiol, 1998 Apr, 44(4), 364 - 72
Cloning of clustered Streptomyces viridosporus T7A lignocellulose catabolism genes encoding peroxidase and endoglucanase and their extracellular expression in Pichia pastoris; Thomas L et al.; A 4.1-kb fragment of chromosomal DNA from the lignocellulose-decomposing actinomycete Streptomyces viridosporus T7A was previously found to encode a lignin peroxidase gene . However, when cloned into Escherichia coli in pBSKS+, peroxidase activity was not expressed . When cloned in pIJ702 in Streptomyces lividans, the gene was expressed in a peroxidase positive background, owing to the production by S . lividans of its own extracellular peroxidase . To circumvent these problems, the DNA was cloned into the commercial expression vector pIC9 for extracellular expression in the yeast Pichia pastoris . Yeast transformants, however, expressed two activities, extracellular peroxidase and an extracellular endoglucanase . The enzymes were not expressed by the yeast cells alone or by yeast cells with pIC9 without the insert . Expression of the enzymes by only those transformants expressing the 4.1-kb DNA was confirmed by Western blot analyses, by nondenaturing activity gel staining, and by spectrophotometric enzyme assays of extracellular culture filtrates . Activity gel staining showed that the two activities resided in different proteins and the peroxidase expressed was similar to ALip-P3, one of the isoenzymes of lignin peroxidase of the S . viridosporus T7A wildtype . Other evidence indicated that in the transformants, the peroxidase and endoglucanase genes in the 4.1-kb insert were controlled by the methanol-inducible AOX1 yeast promoter in pIC9, since their expression was induced by methanol . In the best transformants, extracellular production of peroxidase by recombinant P . pastoris cultures was significantly higher than typically observed in S . viridosporus . The results also indicate that lignocellulose catabolism genes may be clustered on the S . viridosporus chromosome.

Biochemistry, 1998 Jul 14, 37(28), 9983 - 90
Chromophore incorporation, Pr to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins; Remberg A et al.; N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (PthetaB; native chromophore) and phycocyanobilin (PCB) . The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E . coli showed only a slow monoexponential assembly . The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics . With all apoproteins, PthetaB was incorporated faster than PCB . The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr is rapidly converted into Pr . Thus, neither the C-terminal domain nor homodimer formation regulates this property . Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots . The Pr to Pfr kinetics of the N-terminal phyA65 and phyB66 are different . The primary photoproduct I700 of phyA65-PCB decayed monoexponentially and the PthetaB analogue biexponentially, whereas the phyB66 I700 decayed monoexponentially irrespective of the chromophore incorporated . The formation of Pfr from Pr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.

Plasmid, 1998 Jul, 40(1), 58 - 60
Identification of linear DNA plasmids of the yeast Pichia pastoris; Banerjee H et al.; Two DNA plasmids, approximately 11 and 8 kb in size, have been identified in a strain of the yeast Pichia pastoris (Northern Regional Research Laboratories No . Y4290) . The plasmids are resistant to RNase A and lambda exonuclease, but are sensitive to digestion by DNase I, suggesting that they are linear and double-stranded DNA with 5'-protected ends . A restriction map has been constructed for the 11-kb plasmid, confirming that it is linear .

Protein Sci, 1998 Jun, 7(6), 1415 - 22
Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase; Brocca S et al.; The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host . Commercial samples of C . rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent . In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae . As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation . The synthetic gene was functionally expressed in both hosts S . cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated . In particular, using P . pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium . The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C . rugosa lipase preparation containing lipase isoforms.

Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 746 - 50
Molecular and crystal properties of Bos d 2, an allergenic protein of the lipocalin family; Rautiainen J et al.; The relationship between the molecular structure of allergenic proteins and the allergenic determinants is one of the central issues in allergology . We report here that the natural preparation of Bos d 2, a mammalian lipocalin allergen, comprises three molecular variant proteins of 17,829, 17,781, and 17,800 Da . When cDNA of Bos d 2 (Genome Sequence Data Base No . L42867) was recloned and expressed in Pichia pastoris, two proteins were produced . One of the proteins (17,831 Da) and the proteins in the natural preparation had pyroglutamate as the N-terminal residue; in the other (17,849 Da) the N-terminal residue was glutamine . Recombinant Bos d 2 protein was crystallized and the native data set was collected at 1.8 A resolution.

Yeast, 1998 May, 14(7), 647 - 54
Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype; Menendez J et al.; We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris . Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source . Growth was possible with aspartate or glutamate as nitrogen source . The gene PpPYC1 expressed from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase . In a P . pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions . In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed . The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions.

J Clin Invest, 1998 Jun 15, 101(12), 2761 - 7
Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications; Doring F et al.; Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors . For this purpose, ALA is given topically, systemically, or orally . When administered by the oral route, it shows excellent intestinal absorption . ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration . However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown . Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells . Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2 . By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport . Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA . This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Biotechnology (N Y), 1996 Jan, 14(1), 77 - 81
Production of a recombinant bovine enterokinase catalytic subunit in the methylotrophic yeast Pichia pastoris; Vozza LA et al.; We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris . A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL . The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae . The secreted EKL was easily purified from the few native proteins found in the P . pastoris fermentation supernatant, using ion exchange and affinity chromatography . The yield of the purified EKL was 6.3 mg per liter of fermentation culture . This is significantly higher than previous reports of expressions in E . coli and COS cells . The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins . Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.

Biochemistry, 1998 Jun 23, 37(25), 9073 - 82
Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity; Beaudet L et al.; The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity . Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif . Within this segment, we identified two sets of residues {ERGA --> DKGT (522-525) and T578C} that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein {Beaudet, L., and Gros, P . (1995) J . Biol . Chem . 270, 17159-17170} . We wished to gain insight into the molecular basis of this defect . For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris . P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes . P-gp proteoliposomes from P . pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells . Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion . Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity . Photolabeling studies with {125I}iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants . The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.

Biotechnology (N Y), 1995 Mar, 13(3), 255 - 60
Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris; Ridder R et al.; We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF) . The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains . Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen . We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment . More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P . pastoris culture supernatant by one-step affinity chromatography.

Mol Microbiol, 1998 May, 28(3), 543 - 54
The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages; Borg-von Zepelin M et al.; Medically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors . Six closely related gene sequences, SAP1 to SAP6, for secreted proteinases are present in Candida albicans . The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme . Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C . albicans cultures in vitro, were produced as active recombinant enzymes . Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme-linked immunosorbent assay (ELISA) against each recombinant Sap . Two antisera recognized only Sap4 to Sap6 . Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C . albicans cells after phagocytosis by murine peritoneal macrophages . Furthermore, a delta sap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild-type strain . These results support a role for Sap4 to Sap6 in pathogenicity.

Protein Expr Purif, 1998 Jun, 13(1), 136 - 42
Production of human tissue factor using the Pichia pastoris expression system; Austin AJ et al.; Tissue factor plays an important role in the initiation of the blood coagulation cascade resulting in the formation of a fibrin clot . The extracellular domain of human tissue factor has been expressed in the protease-deficient strain of the methylotrophic yeast Pichia pastoris, SMD1168 . Tissue factor was expressed with a human influenza hemagglutinin tag fused at the C-terminus under control of the regulatory sequences from the Pichia AOX1 gene . Expressed protein was secreted in a soluble form at levels of up to 10 mg L-1 and correct processing of the PHO1 signal sequence was confirmed by N-terminal amino acid sequence analysis . Tissue factor was produced in Pichia as three discrete forms which appeared as three bands in the range 37-45 kDa by SDS-PAGE . These were all recognized by an anti-tissue factor monoclonal antibody . Deglycosylation studies using Endo H showed that the three forms were the result of differences in glycosylation of the protein . The low levels of secreted proteins produced by P . pastoris make this an efficient host for producing biologically active recombinant tissue factor requiring little purification.

Protein Expr Purif, 1998 Jun, 13(1), 73 - 82
High-level secretion of a wheat lipid transfer protein in Pichia pastoris; Klein C et al.; Plant nonspecific lipid transfer proteins are small basic proteins with eight cysteine residues, all engaged in disulfide bonds . The sequence encoding the wheat 9-kDa LTP was cloned into the secretion vector pYAM7SP8 giving rise to pYTdltp4.90 . Production in shake-flasks and a fermentor led to the synthesis of two major species of LTP: a larger than expected species of 14 kDa and a species of 10 kDa, close to the expected size of wheat LTP . When production was carried out in a fermentor with regulation of pH, oxygen level, and feed rate of carbon source, the 10-kDa species was the main protein at the end-point of culture . The recombinant wheat LTP (rLTP), secreted at a level of 720 mg/liter into the culture medium, is soluble . The rLTP was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography, with a recovery yield of 36% . However, the molecular mass of rLTP, determined by mass spectrometry, is 9996 Da, while its naturally occurring counterpart has a molecular mass of 9607 Da . This discrepancy in size corresponds to a protein carrying three extra amino acids (DKR) at its N-terminal end, and this was confirmed by sequencing . In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein . These data indicate that Pichia pastoris is an efficient system for production of large quantities of soluble and biologically active rLTP for structure/function analysis.

Protein Expr Purif, 1998 Jun, 13(1), 36 - 40
Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization; Yang Y et al.; A cDNA clone encoding human pancreatic triglyceride lipase was cloned into a yeast expression vector so that the yeast PHO1 signal peptide replaced the native signal peptide . Pichia pastoris cells were transfected with the vector, and clones expressing human pancreatic triglyceride lipase were isolated . Recombinant human pancreatic lipase was expressed in broth cultures and was purified from the medium by DEAE blue Sepharose and hydroxyapatite chromatography . The highly purified lipase had specific activities for various triglyceride substrates identical to those of tissue-purified human pancreatic triglyceride lipase; it was inhibited by bile salts, required colipase for activity, and demonstrated interfacial activation . This expression system is suitable for the rapid, efficient production of human pancreatic triglyceride lipase in amounts adequate for biophysical studies.

Chin J Biotechnol, 1997, 13(4), 253 - 61
Study on the hydrolyzate of sugarcane bagasse to ethanol by fermentation; Yang B et al.; Study of glucose and xylose utilization by Pichia stipitis in a limited oxygen supply condition revealed that the rate of glucose utilization decreased rapidly while that of xylose decreased slowly until the time that glucose and xylose were shown to level out, at which point the rate of xylose utilization increased rapidly . Based on the results, ethanol fermentation technology in continuous connective tower fermenters was advanced, e.g., fermentation by P . stipitis in an airlift loop tower focusing on xylose utilization and then residue glucose utilization by Saccharomyces cerevisiae in an overflow tower . When the fed hydrolyzate of bagasse was concentrated in five folds and the dilution rate was 0.1 h-1, the total utilization ratio of reducing sugar was 97.2%; the concentration of ethanol was 46.4 g/L.h.

Chin J Biotechnol, 1997, 13(4), 225 - 31
Effect on product formation in recombinant Saccharomyces cerevisiae strains expressing different levels of xylose metabolic genes; Bao X et al.; The XYL1 and XYL2 genes from Pichia stipitis encoding xylose reductase (XR) and xylilitol dehydrogenase (XDH), respectively, were transformed into Saccharomyces cerevisiae . These two genes were placed in different directions under the control of the alcohol dehydrogenase I (ADHI) and phosphoglycerate kinase (PGK) promoters and inserted into the E . coli-yeast shuttle plasmid YEp24 . Different recombinant S . cerevisiae strains were constructed with different specific activities of XR and XDH . The highest XR or XDH activities were obtained when the expressed gene was controlled by the PGK promoter and located downstream after the ADHI promoter-gene-terminator sequence . The XR/XDH ratio (ratio of specific enzyme activities of XR and XDH) in these recombinant S . cerevisiae strains varied from 17.5 to 0.06 . In order to enhance xylose utilization, in the XYL1, XYL2 containing S . cerevisiae strains, the native TKL1 gene encoding transketolase and the TALI gene encoding transaldolase were also overexpressed, which showed considerably good growth on the xylose plate . Fermentation of the recombinant S . cerevisiae strains containing XYL1, XYL2, TKL1, and TAL1 were studied with mixtures of glucose and xylose . The strain with XR/XDH ratio of 0.06 consumed 3.25 g/L xylose and formed no xylitol and less glycerol and acetic acid, but more ethanol compared with the strains with a higher XR/XDH ratio.

J Cell Biol, 1998 May 4, 141(3), 625 - 36
Peroxisome degradation by microautophagy in Pichia pastoris: identification of specific steps and morphological intermediates; Sakai Y et al.; We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl ) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy . Structures corresponding to the intermediates were also identified by electron microscopy . The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy . Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version {GFP})-SKL via both autophagic pathways . P . pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates . These mutants, comprising 6 complementation groups, support the model for microautophagy . Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

Glycobiology, 1998 Jun, 8(6), 585 - 95
Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human alpha1,2-mannosidase gene involved in N-glycan maturation; Tremblay LO et al.; Class I alpha1,2-mannosidases play an essential role in the elaboration of complex and hybrid N -glycans in mammalian cells . Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs . One of these was related to the previously cloned human and murine alpha1, 2-mannosidase IA whereas the other was very similar to murine alpha1, 2-mannosidase IB . Northern blot analysis of human tissues with these two alpha1,2-mannosidase probes revealed very different patterns of tissue-specific expression . Similar tissue-specific expression of alpha1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues . A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human alpha1,2-mannosidase IB cDNA . This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine alpha1,2-mannosidase IB (Herscovics et al., 1994, J . Biol . Chem., 269, 9864-9871) . A truncated soluble form of the human alpha1,2-mannosidase IB lacking its N -terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris . The recombinant enzyme was incubated with {3H}Man9GlcNAc and {3H}Man8GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that {3H}Man9GlcNAc was readily converted to {3H}Man6GlcNAc and much more slowly to {3H}Man5GlcNAc, whereas {3H}Man8GlcNAc was rapidly trimmed to {3H}Man5GlcNAc . The human alpha1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons . The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers . These results show that there are at least two Class I alpha1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues.

J Clin Microbiol, 1998 Jun, 36(6), 1634 - 41
Rapid identification of Candida albicans and other human pathogenic yeasts by using short oligonucleotides in a PCR; Mannarelli BM et al.; A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed . The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a new Candida sp., C . sojae, C . tropicalis, C . maltosa, C . viswanathii, C . albicans, and C . dubliniensis and between another five more divergent species, Pichia guilliermondii, C . glabrata, C . zeylanoides, C . haemulonii, and C . haemulonii type II . A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described . The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers . Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5' end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3' end . Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene . In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present . The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.

Planta Med, 1998 May, 64(4), 387 - 8
Recombinant expression of alliin lyase from garlic (Allium sativum) in bacteria and yeasts; Weik R et al.; Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris . A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors . The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm . After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution . Specific alliin lyase activity could be recovered by this procedure.

Appl Microbiol Biotechnol, 1998 Apr, 49(4), 399 - 404
Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris; Handumrongkul C et al.; A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized . The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa . The derived amino acid sequence of C . guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis . The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris . Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems . The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo . The different cofactor specificity between P . pastoris and C . guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins . The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions.

Vaccine, 1998 Feb, 16(4), 374 - 80
Effect of particulation on the immunogenic and protective properties of the recombinant Bm86 antigen expressed in Pichia pastoris; Garcia-Garcia JC et al.; The recombinant Bm86 tick antigen expressed in Pichia pastoris is obtained in a highly particulated form, as a distinguish feature of this expression system . This particulated protein, the active principle of the recombinant vaccine Gavac against the cattle tick, have shown high immunogenic and protective properties, probably associated with its own characteristics . To evaluate the effects of particulation on the properties of Bm86, three groups of calves were immunized with particulated or non-particulated recombinant Bm86 and the anti-Bm86 antibody response determined . Animals were challenged with a controlled tick infestation and the protective capacities of both proteins assessed . Humoral immune response and protection in cattle vaccinated with the particulated antigen were higher . These experiments suggested that particulation of the Bm86 expressed in P . pastoris is an important feature for the protective properties of the antigen in vaccine preparations.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1137 - 47
Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris; Weiss HM et al.; Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes . Here, we demonstrate that the P . pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor . Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P . pastoris alcohol oxidase 1 (AOX1) gene . In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors . After transformation into P . pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using {N-methyl-3H}lysergic acid diethylamide and {5,7-3H}(-)CGP-12177 respectively . The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide . Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors . The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor . For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect . Binding assays revealed that approx . 25 pmol of beta2-adrenergic receptor and approx . 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced . The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems . Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence . Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly . Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing . In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase . The Kex2-processed beta2-adrenergic receptor was not glycosylated . In conclusion, the high-level production of the two receptors in P . pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.

Transfusion, 1998 Apr, 38(4), 332 - 6
Hemagglutination inhibition of Cromer blood group antibodies with soluble recombinant decay-accelerating factor; Daniels GL et al.; BACKGROUND: Cromer blood group antigens are located on decay-accelerating factor (DAF, CD55), which contains four short consensus repeats (SCRs) . Cromer system antibodies may be of clinical significance in blood transfusion . STUDY DESIGN AND METHODS: Soluble recombinant DAF (srDAF) constructs, consisting of all four SCRs or of only two SCRs, were expressed in the yeast Pichia pastoris . They are used in hemagglutination-inhibition tests with Cromer system antibodies and with DAF-specific monoclonal antibodies . RESULTS: The srDAF inhibited hemagglutination by all Cromer system alloantibodies in undiluted serum . Antibodies to antigens of other blood group systems were not inhibited by the srDAF . Hemagglutination-inhibition tests with domain-deleted srDAF showed that UMC is on SCR-4 and confirmed that Tca, TcaTcb, and WESb are on SCR-1; Dra is on SCR-3; and Cra is on SCR-4 . CONCLUSIONS: Hemagglutination inhibition with srDAF is useful in the recognition of antibodies that belong to the Cromer blood group system and facilitates pretransfusion testing . This use of domain-deleted srDAF provides an easy method of determining epitope location on DAF and is an aid to more precise identification of Cromer system antibodies.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 847 - 52
High-level secretion of biologically active recombinant porcine follicle-stimulating hormone by the methylotrophic yeast Pichia pastoris; Richard F et al.; An active recombinant glycoprotein hormone, porcine follicle-stimulating hormone (recFSH), has been produced for the first time in the methylotrophic yeast, Pichia pastoris . The yield of secreted recFSH (10 mg/l) was the highest ever reached . RecFSH displayed an apparent molecular mass of 41 kDa by SDS-PAGE and was found to bear only N-linked carbohydrates of the high-mannose type . Its in vitro binding and cell-stimulating activities were identical to those of pituitary porcine FSH . The large availability and the noncharged N-glycans of FSHrec should render it highly valuable for structural studies.

Biochimie, 1998 Jan, 80(1), 19 - 31
Artificial fibrous proteins: a review; Heslot H; Several kinds of natural fibrous proteins have been chosen as models: silk fibroin from Bombyx mori, silks from various species of spiders and collagens . The dragline silk of the spider Nephila clavipes is able to stretch by 30% before breaking and has a high tensile strength . It is stronger per unit weight than high tensile steel . Although the partial sequence of the two components of dragline silk is known, its molecular structure is still far from being clearly established . It is however demonstrated that it contains beta-sheet crystals composed of polyalanine residues . Artificial fibrous proteins have been prepared in vivo using either Escherichia coli or the yeast Pichia pastoris . As these proteins contain repetitive sequences, there is a risk of deletion at the DNA level . This difficulty has been solved by making use of the genetic code degeneracy . One group has successfully synthesized silk-like polymers; prolastin polymers containing both silk-like and elastin-like blocks; proNectin polymers containing the RGD triplet coming from fibronectin and able to fix numerous mammalian cell types; and synthetic collagen analogs . Some of these polymers have been spun into fibers that, up-to-now, do not display any measurable molecular orientation . Another group has studied artificial fibrous proteins able to form beta-sheet crystals of defined thickness and bearing functional groups at their surface, for instance Glu residues, selenomethionine or p-fluorophenylalanine . Apart from university laboratories, a venture capital society, an industrial research center and a US army research center are quite active in this field . A number of patents has been deposited.

Biochemistry, 1998 Apr 7, 37(14), 4712 - 21
The solution structure of type II antifreeze protein reveals a new member of the lectin family; Gronwald W et al.; A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system . The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein . The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H- inverted question mark15N inverted question mark heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints . The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets . The structure confirms the proposed existence of five disulfide bonds . The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.

Protein Expr Purif, 1998 Apr, 12(3), 361 - 70
Expression and characterization of human tissue kallikrein variants; Chan H et al.; Human tissue kallikrein is a serine protease implicated in the pathology of various inflammatory disorders . As one of the two principal enzymes that generate proinflammatory kinin peptides in vivo, tissue kallikrein represents an attractive target for therapeutic intervention in diseases such as asthma, pancreatitis, and rheumatoid arthritis . Three distinct human tissue kallikrein variants, differing in one or two amino acid substitutions, are predicted to exist based on genomic or cDNA nucleotide sequences derived from different tissues . The effects of these substitutions on the biochemical properties of tissue kallikrein are unknown but could, in principle, confer tissue-specific functions on the enzyme or affect the clinical utility of specific kallikrein inhibitors . All three variants, as well as a deglycosylated derivative, were expressed in high yield as recombinant proteins in Pichia pastoris . The recombinant kallikrein variants and natural urinary kallikrein all hydrolyzed synthetic peptides with similar specificity and efficiency and released kallidin from kininogen at comparable rates . Similarly, no significant differences were observed in the interactions between kallikrein variants and protein inhibitors such as SBTI, alpha1-PI, and aprotinin . We conclude that the known tissue kallikrein variants represent allelic variants and are not likely to have tissue-specific activity related to the amino acid substitutions .

Protein Expr Purif, 1998 Apr, 12(3), 315 - 22
Secretion of recombinant pro- and mature fungal alpha-sarcin ribotoxin by the methylotrophic yeast Pichia pastoris: the Lys-Arg motif is required for maturation; Martinez-Ruiz A et al.; alpha-Sarcin is a ribosome-inactivating protein from the mold Aspergillus giganteus . The methylotrophic yeast Pichia pastoris has been transformed with two plasmids (pHILD2prealphaS and pHILS1prealphaS), which contain the complete alpha-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter . The second one is indeed fused to the signal sequence of P . pastoris acid phosphatase . The transformed yeasts secreted both mature and pro-alpha-sarcin . The presence of this pro-alpha-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase . A third plasmid accounting for a single mutation of the alpha-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif . This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide . This recombinant alpha-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria . In addition, pro-alpha-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does . Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum .

Appl Environ Microbiol, 1998 May, 64(5), 1852 - 9
Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose; Ho NW et al.; Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose . However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose . We developed recombinant plasmids that can transform Saccharomyces spp . into xylose-fermenting yeasts . These plasmids, designated pLNH31, -32, -33, and -34, are 2 microns-based high-copy-number yeast-E . coli shuttle plasmids . In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae) . These xylose-metabolizing genes were also fused to signals controlling gene expression from S . cerevisiae glycolytic genes . Transformation of Saccharomyces sp . strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth . Furthermore, the resulting recombinant yeasts also have additional extraordinary properties . For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium . These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

Arch Biochem Biophys, 1998 Apr 1, 352(1), 1 - 8
Structural studies of alpha-N-acetylgalactosaminidase: effect of glycosylation on the level of expression, secretion efficiency, and enzyme activity; Zhu A et al.; alpha-N-Acetylgalactosaminidase (alphaNAGAL, EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine from oligosaccharide chains . After cloning of its cDNA, the recombinant alphaNAGAL (ralphaNAGAL) was produced in Pichia pastoris, a methylotrophic yeast strain . The enzyme was hyperglycosylated by the host cells, resulting in a protein with a molecular mass of approximately 50 kDa, which was 7 kDa larger than that of its native counterpart . When deglycosylated with endoglycosidase H under nondenaturing conditions, ralphaNAGAL remained fully active, suggesting that the glycosylation is not required for enzyme activity . Data derived from mass spectrometry indicated that all three putative N-glycosylation sites {Asn residues at positions 161 (N1), 185 (N2), and 369 (N3)} in the enzyme were glycosylated, and a high-mannose structure, which was possibly phosphorylated, was attached to the sites N1 and N2 . In order to examine the effect of individual N-linked oligosaccharide chains on the expression of ralphaNAGAL in P . pastoris, we mutated each of the N-glycosylation sites, as well as all three sites in the same protein molecule, by substituting the Asn with a Gln residue . The results indicate that ralphaNAGAL mutations in any of the three glycosylation sites, N2 being the most profound, impaired the expression level, altered subcellular distribution, and decreased the efficiency of secretion . Our data suggest that the N-glycosylation of ralphaNAGAL expressed in P . pastoris may be important in protein folding and resistance to protease degradation during protein synthesis, although it is apparently not required for enzyme activity .

Diagn Microbiol Infect Dis, 1998 Mar, 30(3), 229 - 31
Pichia ohmeri fungemia; Bergman MM et al.; A patient with a history of diabetes, coronary artery disease, stroke, previous renal transplantation, and multiple hospital admissions for recurrent pancreatitis was transferred to the hospital from a chronic care facility because of fever and severe epigastric discomfort . At the time of admission, she was receiving hyperalimentation through a central venous TPN catheter . Multiple blood cultures obtained on the first and second hospital days yielded pure cultures of the yeast, Pichia ohmeri . The patient developed acute renal failure, and despite high-dose amphotericin B therapy, ultimately expired.

J Biotechnol, 1998 Feb 5, 60(1-2), 3 - 14
The bovine IFN-omega 1 is biologically active and secreted at high levels in the yeast Pichia pastoris; Rodriguez M et al.; The gene coding for bIFN-omega 1 was isolated from bovine genomic DNA by polymerase chain reaction (PCR) . Recombinant bIFN-omega 1 was expressed in the yeast Pichia pastoris and high levels of the recombinant protein (0.4 mg ml-1) were secreted to the culture media . The obtained bIFN-omega 1 showed a cross-species antiviral activity on four mammalian cell lines of calf, pig, hamster and human origin, but this activity was absent on Madin-Darby canine kidney (MDCK) cells . A delivery carrier was developed to permit a better release of bIFN-omega 1 . When compared with a control group, an increase in 6 days in the corpus luteum lifespan was obtained in cyclic ewes following three interferon (IFN) intrauterine administrations on days 9, 10 and 11 post-estrus . In summary, these results demonstrated for the first time that biologically active recombinant bIFN-omega 1 was highly secreted by P . pastoris showing antiviral activity in different cell lines and an antiluteolytic effect in cyclic ewes, with no detrimental effects on the animals.

Protein Sci, 1998 Apr, 7(4), 875 - 85
Crystal structure of recombinant human tissue kallikrein at 2.0 A resolution; Katz BA et al.; Human tissue kallikrein, a trypsin-like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris, purified, and crystallized . The crystal structure at 2.0 A resolution is described and compared with that of porcine kallikrein and of other trypsin-like proteases . The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar to those of porcine kallikrein . Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis-Pro 219 . The replacement Tyr 228 --> Ala further enlarges the S1 pocket . However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between O delta1Asp189 and O gammaSer226 . These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin . The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin-like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys peptide bond in its natural protein substrate, kininogen . A Zn+2-dependent, small-molecule competitive inhibitor of kallikrein (Ki = 3.3 microM) has been identified and the bound structure modeled to guide drug design.

Biochemistry, 1998 Mar 31, 37(13), 4592 - 602
Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites; Urbatsch IL et al.; Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3) . Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography . This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P . pastoris is highly functional . Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites . The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae . Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P . pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized . Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3) . Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation . No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C . Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity) . These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site . These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule . In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

Biochim Biophys Acta, 1998 Mar 13, 1396(3), 306 - 19
Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli; Heim J et al.; The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein . Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag . With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB) . However, some activity was found in the periplasmic space . The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE . The refolded AChE was partially purified (approx . 300-fold) by affinity chromatography with a specific activity of approx . 250 U/mg . Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding . Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5 . Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris . By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced . In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time . The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE . The properties of the yeast expressed AChE and the native AChE were similar, whereas the E . coli expressed enzyme was found to be less stable and had different inhibition properties.

Appl Microbiol Biotechnol, 1998 Feb, 49(2), 141 - 6
Cloning and disruption of the beta-isopropylmalate dehydrogenase gene (LEU2) of Pichia stipitis with URA3 and recovery of the double auxotroph; Lu P et al.; Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast . To this end, we used P . stipitis URA3 (PsURA3) to disrupt P . stipitis LEU2 in a P . stipitis ura3 mutant . A highly fermentative P . stipitis mutant (FPL-DX26) was selected for resistance to 5'-fluoroorotic acid to obtain P . stipitis FPL-UC7 (ura3-3) . A URA3:lacZ "pop-out" cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame . The P . stipitis LEU2 gene (PsLEU2) was cloned from a P . stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette . FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu- Ura+ phenotype . The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mutants . Excision of the flanked PsURA3 gene resulted in the Leu- Ura- phenotype . The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.

Protein Expr Purif, 1998 Mar, 12(2), 208 - 14
Expression of human interleukin-17 in Pichia pastoris: purification and characterization; Murphy KP Jr et al.; A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17) . Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells . The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms . Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution . A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation . These results show that P . pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.

Protein Expr Purif, 1998 Mar, 12(2), 201 - 7
Expression of recombinant human soluble type II transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli: two powerful systems to express a potent inhibitor of transforming growth factor-beta; Glansbeek HL et al.; Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation . To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required . Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli . Expression of the soluble TGF beta sRII using P . pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium . Although expression of TGF beta sRII in E . coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein . Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose . The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3 . In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2 . In conclusion, biologically active TGF beta sRII can be produced using P . pastoris and E . coli expression systems . The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.

Protein Expr Purif, 1998 Mar, 12(2), 145 - 50
Expression of human monocyte chemoattractant protein-1 in the yeast Pichia pastoris; Beall CJ et al.; The human monocyte chemoattractant protein-1 (MCP-1) was expressed at high levels in Pichia pastoris with the alcohol oxidase promoter . It was secreted from the yeast when either its natural signal sequence or the Saccharomyces cerevisiae alpha-factor signal peptide was used . SDS-PAGE and Western blot revealed two immunoreactive MCP-1 species at 15 and 8.5 kDa designated MCP-1H and MCP-1L, respectively; both were purified by cation-exchange chromatography . MCP-1H could be converted to MCP-1L by treatment with peptide N-glycosidase F, showing that the former is an N-glycosylated form of the latter . Laser desorption mass spectrometry showed that MCP-1L actually consisted of a mixture of three polypeptides of 8449, 8614, and 8780 Da and MCP-1H showed a broad peak at 11,134 Da . N-terminal peptide sequencing indicated that nearly half of MCP-1L lacked the two N-terminal amino acids found in the native protein . Both MCP-1H and MCP-1L could induce monocyte migration and calcium influx in THP-1 monocytic leukemia cells, although these activities were about 10- to 100-fold lower than those of MCP-1 produced in insect cells.

Protein Eng, 1997 Nov, 10(11), 1339 - 45
High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties; Kim TR et al.; Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae . P . pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus . In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h . After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l . The secreted protein could be purified to homogeneity by ion-exchange chromatography . Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present . The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy . By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.

J Virol Methods, 1997 Dec, 69(1-2), 159 - 69
The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris; Sugrue RJ et al.; The dengue virus envelope protein was expressed as a GST fusion protein using E . coli and P . pastoris as expression hosts . In E . coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein . Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein . The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein . This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture . In contrast, expression of this GST fusion protein in P . pastoris is associated with extensive proteolytic degradation of the recombinant E protein . However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed . One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium.

J Allergy Clin Immunol, 1998 Feb, 101(2 Pt 1), 274 - 80
High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris; Vailes LD et al.; Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach-sensitive inner-city children . Bla g 4 is a ligand-binding protein (or calycin) that causes IgE antibody responses in 40% to 60% of patients allergic to cockroaches . Recombinant Bla g 4 was expressed in Escherichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/L culture) . To improve yields, Bla g 4 was expressed in the Pichia pastoris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L . By cross-inhibition radioimmunoassay, Bla g 4 expressed in E . coli or P . pastoris provided overlapping inhibition curves . Both allergen preparations bound comparable levels of serum IgE antibody and showed similar skin test reactivity in individuals allergic to cockroaches (10{-1} to 10{-3} microg/ml) . Deglycosylation of Pichia-expressed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet, and liquid chromatography-mass spectrometry results suggested that the 20 kd band contained residual sugar residues . Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE antibody binding in radioimmunoassay . Pichia-produced Bla g 4 had the same antigenic reactivity as that produced in E . coli, and glycosylation had no effect on IgE antibody binding . The high yield of Bla g 4 obtained in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma patients to cockroach allergens.

J Cell Biol, 1998 Feb 23, 140(4), 807 - 20
A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris; Elgersma Y et al.; Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2) . Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor . The pex7Delta mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism . In contrast, pex7Delta cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1 . Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal . This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes . In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata . The corresponding PpPex7p mutant proteins were stably expressed in P . pastoris, but they failed to complement the pex7Delta mutant and were impaired in binding to the PTS2 sequence.

J Biol Chem, 1998 Feb 20, 273(8), 4478 - 84
Autocatalytic processing of recombinant human procathepsin L . Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro; Menard R et al.; The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris . Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L . The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible . The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L . Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme . A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L . Processing is initiated by decreasing the pH from 8.0 to 5.3 . The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing . The nature of the unimolecular event that triggers processing remains elusive . Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH . However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.

Int J Food Microbiol, 1997 Sep 16, 38(2-3), 143 - 55
Fatty acid profiling: a feasible typing system to trace yeast contamination in wine bottling plants; Malfeito-Ferreira M et al.; The long-chain fatty acid composition of yeast strains was determined for several species associated with the wine industry . The Saccharomyces cerevisiae, Zygosaccharomyces bailii, Saccharomycodes ludwigii, Schizosaccharomyces pombe, Brettanomyces/Dekkera spp., Pichia anomala, Pichia membranaefaciens and Lodderomyces elongisporus species presented distinct fatty acid profiles after multivariate statistical analysis . The Zygosaccharomyces rouxii species showed profiles similar to Zygosaccharomyces bailii . The use of fatty acid profiling in wine bottling plants and wines makes it possible to trace the origin of the strains responsible for spoiling the final product . In one case the origin was found at the outlet of the finishing filter and identified as Zygosaccharomyces bailii . In the other case the source of contamination was discovered in the heads of the filling machine and assigned to the Pichia membranaefaciens species . The results point out the discriminating power and the industrial applicability of the technique described in this work to analyse yeast long-chain fatty acid compositions.

J Infect Dis, 1998 Mar, 177(3), 807 - 11
Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies; Conti S et al.; Human natural (KTAb), murine monoclonal (KTMAb), and single-chain recombinant (KTScFv) candidacidal antibodies representing the internal image of a killer toxin from the yeast Pichia anomala (KT), characterized by a wide spectrum of antibiotic activity, exerted a lethal effect against a KT-sensitive multidrug-resistant isolate of Mycobacterium tuberculosis . KTMAb and KTScFv were produced by the hybridoma and DNA technologies, respectively, from the spleen lymphocytes of animals immunized with the idiotype of a KT-neutralizing MAb (MAb KT4), while KTAb were purified against MAb KT4 from the vaginal fluid of women infected with Candida albicans cells bearing an idiotype-like KT cell wall receptor . Mycobactericidal activity was related to the binding of KTAb, KTMAb, and KTScFv to the cell surface of KT-sensitive bacterial cells and was prevented by specific absorption of KT-like antibodies onto MAb KT4 . These data identify a novel potentially useful immunotherapeutic approach to tuberculosis.

Protein Expr Purif, 1998 Feb, 12(1), 85 - 92
Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme; Heimo H et al.; The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized . Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter . yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP . Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca . 62 and 65 kDa . This difference was due to heterogenous N-glycosylation . Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2 . The expression system is discussed in comparison to previously reported expression systems .

Biotechniques, 1998 Feb, 24(2), 266 - 8, 270-1
Protein expression both in mammalian cell lines and in yeast Pichia pastoris using a single expression plasmid; Liu Z et al.; We have designed and constructed a novel expression vector capable of producing recombinant proteins in both mammalian cell lines and the yeast strain Pichia pastoris . In this vector, a yeast promoter is placed inside an intron of the mammalian transcription unit . A yeast transcription termination sequence is placed immediately downstream of the mammalian polyadenylation site . In mammalian cells, transcription is driven by a mammalian promoter . The yeast promoter within the intron is removed by RNA processing . However, protein expression in yeast cells can be achieved utilizing the yeast promoter immediately upstream of the 3' splice site and the target genes . Our data indicate that this vector can express beta-galactosidase efficiently in both mammalian cell lines and the yeast strain P . pastoris.

J Biol Chem, 1998 Feb 6, 273(6), 3535 - 41
Overexpression of human procarboxypeptidase A2 in Pichia pastoris and detailed characterization of its activation pathway; Reverter D et al.; The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeast Pichia pastoris and secreted into the culture medium by means of the alpha-mating factor signal sequence, yielding a major protein of identical size and N-terminal sequence as the wild-type form . Two other forms containing the proenzyme have also been overexpressed: one of them resulted from an incomplete processing of the signal peptide, whereas the other was a glycosylated derivative . Recombinant procarboxypeptidase A2 was purified to homogeneity, and it was shown that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas . The overall yield was approximately 250 mg of proenzyme or 180 mg of mature enzyme/liter of cell culture . The proteolysis-promoted activation process of the recombinant proenzyme has been studied in detail . During maturation by trypsin, the increase in activity of the enzyme is a rapid and monotonic event, which reflects the rate of the proteolytic release of the inhibitory pro-segment and the weaker nature of its interactions with the enzyme moiety compared with procarboxypeptidases of the A1 type . Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a single mature carboxypeptidase A2 are produced during this process . No further proteolysis of these pro-segments by the generated carboxypeptidase A2 occurs, in contrast with observations made in other procarboxypeptidases (A1 and B) . This differential behavior is a result of the extreme specificity of carboxypeptidase A2.

Protein Eng, 1997 Oct, 10(10), 1221 - 5
Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris; FitzGerald K et al.; Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains . To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer . A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a crosslinked bispecific diabody against CEA and the T-cell co-receptor CD3 were expressed from Pichia pastoris and Escherichia coli by secretion . From Pichia (but not E.coli) the chains were almost quantitatively crosslinked . Compared with the parent diabodies both crosslinked diabodies were more stable to heat (by >7 degrees C) and the crosslinked bivalent diabody showed improved localization to CEA+ human tumour xenografts in nude mice.

FEMS Microbiol Lett, 1998 Feb 1, 159(1), 107 - 12
Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase; Kostrzynska M et al.; Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts . To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis . The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme . The apparent affinity of the variant for NADPH was decreased 5-16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged . This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate . The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde . The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5'-phosphate . Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and D-xylose binding in the P . stipitis xylose reductase.

Yeast, 1998 Jan 15, 14(1), 11 - 23
Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase; Raymond CK et al.; We describe a protein expression system in the methylotrophic yeast, Pichia methanolica . Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene . A vacuolar protease-deficient strain was constructed . Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations . The promoter from this gene was used to drive expression . An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled . A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l . Identical amounts were made in Pichia pastoris . The recombinant GAD65 was purified to greater than 90% purity.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 170 - 6
Human IFN gamma receptor cytoplasmic domain: expression and interaction with HuIFN gamma; Green MM et al.; To investigate the structural basis for human interferon gamma (huIFN gamma) binding to intracellular regions of the human IFN gamma receptor (huIFN gamma R), we have subcloned and expressed the huIFN gamma R free of fusion proteins in the yeast strain Pichia pastoris . HuIFN gamma bound to the cytoplasmic domain of the receptor via the IFN gamma C-terminus . Binding was inhibited by both human and mouse C-terminus peptides . N-terminus peptides failed to inhibit cytoplasmic binding . Thus, while extracellular receptor domain binding is species specific, binding to the cytoplasmic domain of the receptor is species non-specific . In solid-phase binding assays, IFN gamma had a Kd of 3.7 x 10(-8) M for the newly expressed cytoplasmic domain . Peptide competitions showed that IFN gamma bound to a receptor site corresponding to the membrane proximal residues 253-287, which is adjacent to the site of binding of the tyrosine kinase JAK2 . The cytoplasmic binding affinity and binding site specificity suggest that the huIFN gamma R cytoplasmic domain can function independent of the extracellular domain to bind huIFN gamma and induce the biological activity previously associated with internalized huIFN gamma.

FEBS Lett, 1998 Jan 23, 422(1), 23 - 6
Secretion, purification and activity of two recombinant pepper endo-beta-1,4-glucanases expressed in the yeast Pichia pastoris; Ferrarese L et al.; Two pepper endo-beta-1,4-glucanases, involved in fruit softening (cCel1) and leaf and flower abscission (cCel2), have been expressed in Pichia pastoris . Secretion was obtained by using either the mouse alpha-factor signal (cCel1) or the native signal sequence (cCel2) . Times for optimal expression of the two proteins were different and cCel2 appeared very sensitive to proteolytic degradation . A one-step purification protocol yielded cCel2 in a pure form, while an additional chromatography step was necessary to purify cCel1 . The two recombinant proteins are highly active and able to degrade carboxymethylcellulose in viscometric assays . Moreover, they have both a molecular mass (54 kDa) and an isoelectric point (7.2 for cCel2 and 8.5 for cCel1) equal to those of the native proteins, thus suggesting that post-translational modifications have properly occurred.

Anal Biochem, 1998 Feb 1, 256(1), 56 - 62
Physicochemical and immunochemical properties of recombinant human serum albumin from Pichia pastoris; Ohtani W et al.; We analyzed and compared the physicochemical and immunochemical properties of recombinant human serum albumin (rHSA) from Pichia pastoris with those of plasma-derived human serum albumin (pHSA) . The second virial coefficient of rHSA, obtained from colloid osmotic pressure measurements at pH 6.7 +/- 0.1 was not significantly different from that of pHSA (P > 0.05) . A 25% rHSA solution exhibited Newtonian flow, and the viscosity of 25% rHSA at 20 +/- 0.02 degrees C was not significantly different from that of 25% pHSA (P > 0.05) . We analyzed the long- and medium-chain fatty acid composition of rHSA by reverse-phase HPLC using 9-anthryldiazomethane as the fluorescent labeling reagent . The total amount of fatty acid was higher for pHSA than for rHSA . The fatty acid composition of the rHSA preparation was the same as that of the pHSA preparation . However, the amounts of palmitic acid (C16:0) and stearic acid (C18:0) in rHSA were much lower than those in pHSA . Interestingly, we found that P . pastoris produced linolenic acid (C18:3) because it was detected in rHSA . The immunochemical properties of rHSA were analyzed by a parallel line assay method using anti-pHSA polyclonal antibody, and were identical to those of pHSA (P > 0.05).

Biochem J, 1998 Jan 1, 329 ( Pt 1), 55 - 63
Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor; Rodenburg KW et al.; The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR) . To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations . The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding . Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively . alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1 . Binding of the mutant complexes to the uPA receptor was not affected . These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar . The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure . Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.

J Cell Biol, 1997 Dec 15, 139(6), 1419 - 31
Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris; Waterham HR et al.; Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein . In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates . The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import . To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS) . We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF) . To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes . To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX-HA) . Coexpression of AOX-HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX-HA present in octameric active AOX, suggesting that newly synthesized AOX-HA cannot oligomerize with AOX-WT in the cytoplasm . Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.

J Gen Virol, 1997 Oct, 78 ( Pt 10), 2639 - 45
Altered antigenicity of 'a' determinant variants of hepatitis B virus; Chiou HL et al.; The 'a' determinant of hepatitis B virus (HBV) surface antigen (HBsAg) is the most important target for diagnosis and immunoprophylaxis . Several HBV variants with point mutations within the 'a' determinant have been identified among fully vaccinated children in Taiwan . We investigated the effect of each of these mutations on the antigenic nature of the S protein by cloning and expression of the mutant S antigens in Pichia pastoris . Four variants, Ser-126, His-129, Arg-129 and Arg-145, all exhibited various degrees of altered binding of HBsAg to several monoclonal antibodies . Arg-145, a well-characterized immune escape mutant, and Arg-129 had the lowest binding capacities to all monoclonal antibodies as compared with other variants . Similar to Arg-145, the Arg-129 variant could be isolated from both vaccinated children and unvaccinated adults, thus representing a naturally occurring mutant with an altered 'a' determinant . Whether these 'a' determinant variants with altered antigenicity might gradually become major circulating strains, as a consequence of the immune pressure against the wild-type HBV created by vaccination, remains to be monitored.

Ukr Biokhim Zh, 1997 Jan-Feb, 69(1), 21 - 5
{Thermostable riboflavin kinase in the yeast Pichia guilliermondii}; Faiura LR et al.; It is found out when studying the process of riboflavin kinase (E.C.2.7.1.26) thermoinactivation in cell-free extracts of yeast, that the reaction of riboflavin phosphorylation in the cells of this organism are catalyzed by two enzymes one of which is distinguished by high thermal stability . The inactivation rate constant of the process of thermoinactivation (K(in)) at 90 degrees C is 11.2 x 10(-4) sec-1 for thermostable enzyme and 4.0 x 10(-2) sec-1 for another one . The enzymes are considerably distinguished by the values of activation thermodynamical parameters (delta H*, delta S*, delta P*, Eact*) of the process of thermoinactivation in the temperature range 75-90 degrees C . Thermostable and thermolabile forms of riboflavin kinases are separated under electrophoresis in polyacrylamide gel.

FEBS Lett, 1997 Dec 22, 420(1), 7 - 10
Characterisation and preliminary X-ray diffraction analysis of human pancreatic procarboxypeptidase A2; Reverter D et al.; Human procarboxypeptidase A2 has been expressed in a Pichia pastoris heterologous system and purified by hydrophobic interaction and anion exchange chromatographies . The hydrolytic action of carboxypeptidase A2 on peptide substrates with different lengths and residues at the C-terminus was analysed, and a preference towards long substrates with aromatic amino acids in their C-terminal end, particularly tryptophan, was found; with such substrates its activity is similar or higher than that of bovine carboxypeptidase A1 . Procarboxypeptidase A2 has been crystallised using a vapour diffusion approach; the crystals obtained belong to the monoclinic system, spacegroup P2(1), and present one procarboxypeptidase A2 molecule per asymmetric unit . The crystals diffract beyond 1.8 A resolution and are suitable for detailed X-ray analysis.

Anal Chem, 1998 Jan 15, 70(2), 425 - 9
Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection; Ohtani W et al.; We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins . rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial) . We had to consider not only proteins from host cells as impurities but also mannan, which exhibits harmful effects on humans . However, the analysis of mannan in biopharmaceuticals produced from yeast has not been reported . Contaminating mannans in the final product were one important index to assess the clinical safety of rHSA . We have developed a highly sensitive enzyme immunoassay (EIA), utilizing an avidin-biotin system, for the detection of either the protein or mannan polysaccharide components from P . pastoris components (PPC) in rHSA . In addition, we used anion exchange chromatography with pulsed amperometric detection (AE-PAD) for monosaccharide analysis of glycoconjugates for the detection of mannan from PPC in rHSA . The detection limits of the EIA for PPC (PPC EIA) and the AE-PAD were 1 ng of protein/250 mg of rHSA and 180 ng of mannose/mg of rHSA, respectively . The mannan content in partially purified rHSA as determined by the AE-PAD was about same as the PPC content as determined by the PPC EIA . We showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.

Mol Cell Biol, 1998 Feb, 18(2), 936 - 43
Two AAA family peroxins, PpPex1p and PpPex6p, interact with each other in an ATP-dependent manner and are associated with different subcellular membranous structures distinct from peroxisomes; Faber KN et al.; Two peroxins of the AAA family, PpPex1p and PpPex6p, are required for peroxisome biogenesis in the yeast Pichia pastoris . Cells from the corresponding deletion strains (Pp delta pex1 and Pp delta pex6) contain only small vesicular remnants of peroxisomes, the bulk of peroxisomal matrix proteins is mislocalized to the cytosol, and these cells cannot grow in peroxisome-requiring media (J . A . Heyman, E . Monosov, and S . Subramani, J . Cell Biol . 127:1259-1273, 1994; A . P . Spong and S . Subramani, J . Cell Biol . 123:535-548, 1993) . We demonstrate that PpPex1p and PpPex6p interact in an ATP-dependent manner . Genetically, the interaction was observed in a suppressor screen with a strain harboring a temperature-sensitive allele of PpPEX1 and in the yeast two-hybrid system . Biochemially, these proteins were coimmunoprecipitated with antibodies raised against either of the proteins, but only in the presence of ATP . The protein complex formed under these conditions was 320 to 400 kDa in size, consistent with the formation of a heterodimeric PpPex1p-PpPex6p complex . Subcellular fractionation revealed PpPex1p and PpPex6p to be predominantly associated with membranous subcellular structures distinct from peroxisomes . Based on their behavior in subcellular fractionation experiments including flotation gradients and on the fact that these structures are also present in a Pp delta pex3 strain in which no morphologically detectable peroxisomal remnants have been observed, we propose that these structures are small vesicles . The identification of vesicle-associated peroxins is novel and implies a role for these vesicles in peroxisome biogenesis . We discuss the possible role of the ATP-dependent interaction between PpPex1p and PpPex6p in regulating peroxisome biogenesis events.

Biochem Biophys Res Commun, 1998 Jan 14, 242(2), 277 - 81
Nucleotide sequence of a cDNA clone encoding a Caenorhabditis elegans homolog of mammalian alkyl-dihydroxyacetonephosphate synthase: evolutionary switching of peroxisomal targeting signals; de Vet EC et al.; The nucleotide sequence is reported of a cDNA clone encoding a Caenorhabditis elegans homolog of guinea pig and human alkyl-dihydroxyacetonephosphate synthase . The open reading frame encodes a protein of 597 amino acids which shows extensive homology with the mammalian enzymes (52% identical and about 76% similar in the overlapping region) . In contrast to the mammalian enzymes, which carry a consensus peroxisomal targeting signal type 2 in a cleavable N-terminal presequence, this Caenorhabditis elegans homolog carries a consensus peroxisomal targeting signal type 1 (CKL) at its C-terminus . Expression of this protein in an in vitro transcription/translation system yielded a 65 kDa protein . Recombinant aenorhabditis elegans alkyl-DHAP synthase expressed in the yeast Pichia pastoris was enzymatically active.

Genetika, 1997 Oct, 33(10), 1345 - 53
{Genetic control of purine biosynthesis in the yeast Pichia methanolica . "Roman's effect" in red adenine-dependent pur6 and pur7 mutants}; Dutova TA et al.; Most ade1 and ade2 mutants of the yeast Pichia methanolica generate white and pink secondary colonies (SCs) on the surface of red colonies in complete medium . The formation of SCs was shown to be caused by "Roman's effect" described in Saccharomyces cerevisiae . This effect is known to result from the preferential growth in a colony of spontaneous mutations for genes controlling the first five steps of purine biosynthesis . It has been found that the ADE3, ADE5, ADE4, 7, and ADE8 loci correspond to these genes . In studies on twelve red adenine-dependent mutants, the expression of Roman's effect was shown to vary in different cultures, dependent on the original mutant ade1 and ade2 alleles . It was assumed that spontaneous mutability of each gene of purine biosynthesis ADE ade does not depend on mutations in other genes of this biosynthetic pathway . On the basis of this assumption and data from genetic analysis of mutants, it is concluded that the ade1 and ade2 mutant alleles do not determine the mutability level but affect the level of selective advantage of double mutants . This conclusion was used to estimate the lowest frequencies of spontaneous mutability of early purine genes in P . methanolica (with respect to the identified mutants) . As calculated for one red mutant, frequencies of spontaneous mutations in the most mutable ADE5 gene and in the least mutable ADE8 gene of this group were 2.5 x x 10(-8) and 0.4 x 10(-8), respectively.

J Biol Chem, 1997 Dec 26, 272(52), 33045 - 55
Cloning and expression of acetylcholinesterase from Electrophorus . Splicing pattern of the 3' exons in vivo and in transfected mammalian cells; Simon S et al.; We cloned and expressed a cDNA encoding acetylcholinesterase (AChE) of type T from Electrophorus electricus organs . When expressed in COS, HEK, and Chinese hamster ovary cells, the AChET subunits generated dimers and tetramers . The cells produced more activity at 27 than at 37 degrees C . The kinetic parameters of a recombinant enzyme, produced in the yeast Pichia pastoris, were close to those of the natural AChE . Analysis of genomic clones showed that the coding sequence is interrupted by an intron that does not exist in Torpedo and differs in its location from that observed in the mouse . This intron is preceded by a sequence encoding a non-conserved 29-amino acid peptide, which does not exist in Torpedo or mammalian AChEs . According to a three-dimensional model, this non-conserved peptide is located at the surface of the protein, opposite from the entry of the catalytic gorge; its deletion did not modify the catalytic parameters . Sequence analyses and expression of various constructs showed that the gene does not contain any H exon . We also found that splicing of transcripts in mammalian cells reveals cryptic donor sites in exons and acceptor sites in introns, which do not appear to be used in vivo.

Anal Biochem, 1997 Dec 1, 254(1), 41 - 8
Fluorometric assay of binding specificity of plant lectins to yeast cells by biotin-avidin system and its application to the classification of yeast cells; Oda Y et al.; A fluorometric assay of lectin binding to yeast cells is reported . The relative amount of biotinylated lectins bound to the yeast cells was estimated by enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate for the lectin-bound beta-galactosidase through biotin-avidin interaction . Binding properties of 4 mannose-specific and 3 glucose/mannose-specific lectins to 22 different species of yeast cells were studied . The binding reaction of biotinylated lectins to the yeast cells was rapid and became constant within 10 min . Each lectin showed its characteristic binding specificity to each yeast species . The relative fluorescent intensities observed for 4-methylumbelliferone released by the action of bound beta-galactosidase were good indicators for the classification of yeast cells in quantitative base . We found that the yeast cells of the Saccharomyces genus can be classified into three groups, and those of Pichia were grouped into two groups . The present method can examine many samples simultaneously and be completed within 3 h .

Curr Genet, 1997 Dec, 32(6), 425 - 30
Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris; Jonsson LJ et al.; A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR . The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide . The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter . Transformants were found to secrete active recombinant enzyme after induction with methanol . The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures . The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae alpha-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9 . The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the alpha-factor secretion signal . Utilisation of the P . pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P . pastoris GS115.

Cancer Immunol Immunother, 1997 Nov-Dec, 45(3-4), 156 - 8
Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein; Gerstmayer B et al.; T cells require at least two signals for activation and clonal expansion . The first signal conferring specificity is initiated by interaction of the T cell receptor with peptide-bearing MHC molecules . The second, costimulatory signal can be provided by cell-surface molecules on antigen-presenting cells such as B7-1 (CD80) and B7-2 (CD86), which interact with CD28 on T cells . To direct the costimulatory B7-2 molecule to the surface of tumor cells we have constructed a chimeric fusion protein, which consists of the extracellular domain of human B7-2 fused to a single-chain antibody domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas . This B7-2(225)-scFv(FRP5) molecule, expressed in the yeast Pichia pastoris and purified from culture supernatants, binds to B7 counter-receptors and to ErbB2 . B7-2(225)-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal, which results in enhanced proliferation of syngeneic T cells.

Photochem Photobiol, 1997 Nov, 66(5), 710 - 5
Large-scale generation of affinity-purified recombinant phytochrome chromopeptide; Mozley D et al.; Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-terminal portion of oat phytochrome A (phyA, spanning amino acids 1-595) . The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material . In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein . From identical volumes of cell culture, H . polymorpha synthesized between 8- and 10-fold more functional protein than P . pastoris . Following the observation by Wu and Lagarias (Proc . Natl . Acad . Sci . USA 93, 8989-8994, 1996) that P . pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (P phi B) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded . A His6-tag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides . Fully reconstituted chromoproteins showed an A660/A280 ratio of > 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure . The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (tau obs) of 35 s . The lambda max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with P phi B are nearly identical to those of native full-length oat phytochrome . The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr-->I700--> -->Ptr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome . Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure . The decay of the primary intermediate (I700) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 microseconds, respectively) . The formation of the Ptr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms) . This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C-terminus that in the His-tagged form exhibits increased polarity.

EMBO J, 1997 Nov 17, 16(22), 6702 - 12
Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase; Vuorela A et al.; Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI) . Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer . A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI . Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits . The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum . The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein . The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression . This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

Biochem J, 1997 Nov 15, 328 ( Pt 1), 121 - 9
Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris; Morel N et al.; In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain) . We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast . The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P . pastoris can correctly process a mammalian C-terminal GPI-addition signal . Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity . In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide . We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide . Surprisingly, the highest yield was obtained with the endogenous signal peptide . For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides . The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P . pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.

Mol Cell Biol, 1998 Jan, 18(1), 388 - 99
Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants; Otera H et al.; To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105 . These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins . Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139 . PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells . PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region . Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively . A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS) . Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene . Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139 . PTS2 import in ZP105 was rescued only by PTS1RL . This finding strongly suggests that PTS1RL is also involved in the transport of PTS2 . Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139 . Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation . The implications of these mutations are discussed.

Biochemistry, 1997 Dec 2, 36(48), 14874 - 82
Expression, purification, and inhibitory properties of human proteinase inhibitor; Dahlen JR et al.; In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells . Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin {Sprecher, C . A., et al . (1995) J . Biol Chem . 270, 29854-29861} . In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases . PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C . PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM . The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM . A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A . PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM . PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM . The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.

Biochemistry, 1997 Dec 2, 36(48), 14874 - 82
Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8
Dahlen JR, Foster DC, Kisiel W.
In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells . Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin {Sprecher, C . A., et al . (1995) J . Biol Chem . 270, 29854-29861} . In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases . PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C . PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM . The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM . A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A . PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM . PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM . The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.

FEBS Lett, 1997 Nov 3, 417(1), 33 - 7
Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools; Linder S et al.; We expressed the alpha2- and beta2-tubulin isoforms of the giant freshwater amoeba Reticulomyxa filosa in the methylotrophic yeast Pichia pastoris . Single expression lead to little or no detectable material . Coexpression of both tubulins, however, resulted in a significant increase of expressed proteins . At the same time, the detectable internal tubulins of the host yeast cell were downregulated . This finding indicates the functionality of the expressed amoeba tubulins . Further regulation phenomena were observed on the level of equilibrium between the two R . filosa tubulin isoforms and on the level of the total tubulin pool . The P . pastoris/R . filosa system therefore seems to be an accessible system for the simultaneous study of the various mechanisms involved in tubulin regulation.

Infect Immun, 1997 Dec, 65(12), 4984 - 8
Production of Vibrio cholerae accessory cholera enterotoxin (Ace) in the yeast Pichia pastoris; Trucksis M et al.; Accessory cholera enterotoxin (Ace) is a recently identified toxin of Vibrio cholerae . Preliminary studies using crude toxin extracts in animal models indicate that Ace increases transcellular ion transport, which is proposed to contribute to diarrhea in cholera . The lack of purified toxin has hindered elucidation of the mechanism of action of Ace . In this study, ace was cloned and was expressed in and secreted by the methylotrophic yeast Pichia pastoris . Secreted toxin constituted 50% of the total supernatant protein from Pichia pastoris . Presumed monomer and dimer forms with molecular masses of 9 and 18 kDa, respectively, were observed . The 18-kDa form predominated . Biological activity was assayed by studying ion fluxes across epithelial membranes in Ussing chambers . Among the characteristics of Ace was the unusual property of staining with silver but not Coomassie blue stain . To our knowledge this is the first report of a biologically active bacterial toxin produced with the P . pastoris system . The purified protein may now be used in studies of the mechanism of action of Ace in physiologic systems.

Appl Microbiol Biotechnol, 1997 Oct, 48(4), 480 - 6
Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris; Loewen MC et al.; Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date . These proteins are known to bind to the surface of ice and inhibit its growth . To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed . Several different expression systems have been tested including baculovirus, Escherichia coli and yeast . The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive . In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l . The recombinant protein has an identical activity to SRAFP from sea raven serum . In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut(s) clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones . The mixed-feeding/Mut(s) strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.

Plant Physiol, 1997 Nov, 115(3), 1135 - 43
Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris; Su W et al.; Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6.1) was produced in the methylotrophic yeast Pichia pastoris and purified to near-electrophoretic homogeneity . Purified enzyme had the spectral and kinetic properties typical of highly purified NR from natural plant sources . Site-directed mutagenesis altering several key residues and regions was carried out, and the mutant enzyme forms were expressed in P . pastoris . When the invariant cysteine residue, cysteine-191, in the molybdo-pterin region of the A . thaliana NIA2 protein was replaced with serine or alanine, the NR protein was still produced but was inactive, showing that this residue is essential for enzyme activity . Deletions or substitutions of the conserved N terminus of NR retained activity and the ability to be inactivated in vitro when incubated with ATP . Enzyme with a histidine sequence appended to the N terminus was still active and was easily purified using metal-chelate affinity chromatography . These results demonstrate that P . pastoris is a useful and reliable system for producing recombinant holo-NR from plants.

J Virol, 1997 Dec, 71(12), 9306 - 12
Interaction between echovirus 7 and its receptor, decay-accelerating factor (CD55): evidence for a secondary cellular factor in A-particle formation; Powell RM et al.; Soluble forms of decay-accelerating factor (DAF) (CD55), the receptor for echovirus 7, were synthesized in the yeast Pichia pastoris . Purified recombinant protein containing SCR domains 2, 3, and 4, but lacking the serine/threonine rich region, was shown to block infection of susceptible cells by echovirus 7 . In contrast to the situation with poliovirus and its receptor, the neutralization of echovirus 7 by soluble DAF was completely reversible and did not lead to the formation of 135S A-particles . Binding of virus to susceptible cells, by contrast, did lead to the formation of A particles, mainly from virus that had been internalized . The data suggest that a secondary factor(s) may contribute to A-particle formation and uncoating of echovirus 7.

Clin Exp Immunol, 1997 Nov, 110(2), 257 - 64
Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera; Harmsen MC et al.; The open reading frame of human proteinase 3 (PR3) without the prepro-peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3) . The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography . Purified rpPR3 migrated as a single 32-kD band on SDS-PAGE and harboured protease activity that could be inhibited with inhibitors specific for serine-proteases . By indirect antigen-capture ELISA using rpPR3, 60% of sera from patients with Wegener's granulomatosis bound to the recombinant product, although it was not recognized in ELISA with directly coated rpPR3.

FEBS Lett, 1997 Oct 6, 415(3), 303 - 7
Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease; Mok SS et al.; Deletion mutagenesis studies have suggested that there are two domains within APP which bind heparan sulphate . These domains have been cloned and expressed in the yeast Pichia pastoris . Both recombinant proteins bound to heparin . One domain (APP316-447) was further characterised by binding studies with peptides encompassing this region . Peptides homologous to APP316-346 and APP416-447 were found to bind heparin . Circular dichroism studies show that APP416-447 shifted towards an alpha-helical conformation in the presence of heparin . This study suggests that heparin-binding domains may lie within regions high in alpha-helical structure.

Biotechnol Appl Biochem, 1997 Oct, 26 ( Pt 2), 79 - 83
Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator; Miele RG et al.; The N-linked glycans assembled in Pichia pastoris on the recombinant kringle 2 domain of human tissue-type plasminogen activator (r-{K2tPA}) are composed of approx . 80% neutral and 20% charged species . After peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase-catalysed liberation of the oligosaccharides from the purified glycopeptide, the glycan mixture was resolved by HPLC on amino-silica-based resin . Oligosaccharide mapping of the resulting mixture by HPLC, gel filtration and time-of-flight matrix-assisted laser-desorption-ionization-with-delayed-extraction mass spectrometry (TOF-MALDI DE-MS) revealed that > 90% of the charged species consisted of a series of oligosaccharides possessing molecular masses that were consistent with a range of saccharides comprising phospho-Man10GlcNAc2-phospho-Man14GlcNAc2, with phospho-Man11GlcNAc2 representing the major species . The remaining material in the charged fraction contained identifiable phosphorylated glycans that were one or two mannose units shorter, and one to four mannose units longer, than those present in the above range of oligosaccharides . Treatment of the native charged glycan pool with alkaline phosphatase did not result in molecular-size alterations, showing that phosphomonoesters are not present . Mild acid hydrolysis of the glycans led to a decrease in the size of all charged glycans by one mannose residue, providing phospho-Man9GlcNAc2-phospho-Man13GlcNAc2 . Following this procedure, treatment with alkaline phosphatase resulted in size decreases that were equivalent to the loss of one phosphate group from each glycan . This demonstrates that all charged glycans isolated contained phosphate in phosphodiester bonds to two mannose units . The present study shows that P . pastoris cells possess the capability of assembling phosphorylated glycans having the phosphate moiety present in phosphodiester linkages with two mannose units . These saccharides, like the neutral oligosaccharides, contain considerably smaller amounts of mannose than glycans present in other strains of yeast.

J Struct Biol, 1997 Oct, 120(1), 69 - 72
Crystallization and preliminary X-ray analysis of the class 1 alpha 1,2-mannosidase from Saccharomyces cerevisiae; Dole K et al.; The alpha 1,2-mannosidase from Saccharomyces cerevisiae catalyzes the conversion of Man9GlcNAc2 to Man8GlcNAc2 during the formation of N-linked oligosaccharides and is a member of the Class 1 alpha 1,2-mannosidases conserved from yeast to mammals . The enzyme is a type II membrane protein and a recombinant form of the alpha 1,2-mannosidase from S . cerevisiae, lacking the transmembrane domain, has been expressed in Pichia pastoris and crystallized using the hanging drop vapor diffusion technique . The crystals grow as flat plates, with unit cell dimensions a = 57.5 A, b = 84.1 A, c = 107.1 A, alpha = beta = gamma = 90 degrees . The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and diffract to a minimum d-spacing of 3.5 A resolution . On the basis of density calculations one monomer is estimated to be present in the asymmetric unit (Vm = 2.08 A3 Da-1) . This is the first report of the crystallization of any glycosidase involved in N-glycan biosynthesis.

Curr Opin Biotechnol, 1997 Oct, 8(5), 554 - 60
Production of recombinant proteins by methylotrophic yeasts; Hollenberg CP et al.; The methylotrophic yeasts Hansenula polymorpha, Pichia pastoris and Candida boidinii have been developed as production systems for recombinant proteins . The favourable and most advantageous characteristics of these species have resulted in an increasing number off biotechnological applications . As a consequence, these species--especially H . polymorpha and P . pastoris--are rapidly becoming the systems of choice for heterologous gene expression in yeast . Recent advances in the development of these yeasts as hosts for the production of heterologous proteins have provided a catalogue of new applications, methods and system components.

Infect Immun, 1997 Nov, 65(11), 4718 - 24
Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus; Calera JA et al.; Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility) . The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients . The antigenic catalase has been purified . The enzyme is a tetrameric protein composed of 90-kDa subunits . The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide . A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase . Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively . cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain . In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis.

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 498 - 502
Reconstitution of highly expressed human heart muscle carnitine palmitoyltransferase I; Zhu H et al.; The human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene was expressed at high levels from a strain of the methylotrophic yeast Pichia pastoris containing approximately 24 copies of the expression vector . Levels of M-CPTI were more than ten-fold higher than previously reported by our group with a single-copy strain (Arch . Biochem . Biophys., in press) and were sufficient to perform reconstitution studies on the membrane protein, a key step in purification and structural analysis of the enzyme . Solubilization of yeast mitochondria containing M-CPTI in 5% Triton X-100 abolished M-CPTI activity . The detergent-inactivated M-CPTI was then reconstituted by removal of the detergent in the presence of phospholipids . The reconstituted proteoliposomes exhibited M-CPTI activity of 2.4 nmol palmitoylcarnitine formed/mg protein/min, a recovery of 23% of the activity present in the starting mitochondrial preparation . The malonyl-CoA sensitivity of the reconstituted reactivated M-CPTI was 88% . This is the first demonstration of direct reactivation of malonyl-CoA-sensitive M-CPTI activity from solubilized materials from any organism . Previously, M-CPTI was presumed to be irreversibly inactivated by detergents .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 53 - 61
Functional studies of yeast-expressed human heart muscle carnitine palmitoyltransferase I; Zhu H et al.; Long-chain fatty acids are the primary source of energy production in the heart . Carnitine palmitoyltransferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in beta-oxidation . In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris . Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone . The clone has an open reading frame of 2316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untranslated region with the poly(A)+ addition sequence AATAAA . The predicted protein has 772 amino acids and a molecular mass of 88 kDa . Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of approximately 3.1 kb that was only present in human heart and skeletal muscle tissue . Expression of the human M-CPT-I cDNA in P . pastoris, a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria . Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile . The I50 for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nM, and the Kms for carnitine and palmitoyl-CoA were 666 and 42 microM, respectively . The I50 for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and the Km for carnitine is more than 20 times higher than that of the liver CPT-I . This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II .

Protein Expr Purif, 1997 Oct, 11(1), 104 - 10
High-level expression of recombinant Aplysia ADP-ribosyl cyclase in offhia pastoris by fermentation; Munshi C et al.; Cyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2(+)-induced Ca2+ release mechanisms in various cellular systems . ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR . In this study we have utilized the methylotrophic yeast Pichia pastoris to express high levels of this enzyme . The cyclase construct consisted of the soluble domain, with isoleucine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast alpha-factor mating signal sequence . Cyclase yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% active transformation . All active clones comprised the methanol utilization slow (Muts) phenotype . The protein was expressed using the tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae alpha-factor mating secretion signal . Using high biomass fermentations, up to 300 mg/liter of cyclase was achieved . SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly . The enzyme characteristics of the recombinant cyclase compared favorably with those of the native enzyme . The yeast expression system can thus produce gram quantities of this novel protein.

Protein Expr Purif, 1997 Oct, 11(1), 61 - 71
High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts; Hasslacher M et al.; (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones . This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals . Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris . Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained . Highest yields were obtained in a high cell density batch fermentation of a P . pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein . At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained . Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful . Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.

Protein Expr Purif, 1997 Oct, 11(1), 35 - 40
High-level production of recombinant Geotrichum candidum lipases in yeast Pichia pastoris; Holmquist M et al.; We describe the heterologous high-level expression of the two Geotrichum candidum lipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeast Pichia pastoris . The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter . The lipases expressed in P . pastoris were fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and were secreted into the culture medium . Cultures of P . pastoris expressing lipase accumulated active recombinant enzyme in the supernatant to levels of approximately 60 mg/L virtually free from contaminating proteins . This yield exceeds that previously reported with S . cerevisiae by a factor of more than 60 . Recombinant GCL I and GCL II had molecular masses of approximately 63 and approximately 66 kDa, respectively, as determined by SDS-PAGE . The result of endoglucosidase H digestion followed by Western blot analysis of the lipases suggested that the enzymes expressed in P . pastoris received N-linked high-mannose-type glycosylation to an extent, 6-8% (w/w), similar to that in G . candidum . The specific activities and substrate specificities of both recombinant lipases were determined and were found to agree with what has been reported for the enzymes isolated from the native source.

Biochem Biophys Res Commun, 1997 Sep 29, 238(3), 920 - 4
Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme; Sun J et al.; Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis . Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease . Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography . The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 microM) but not YVAD-AFC . It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1beta . It is inhibited by the peptide DEVD-CHO (Ki 2.2 nM), far less efficiently by YVAD-CMK (Ki 0.3 microM), and not detectably by CrmA . By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts . Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate . However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3 in vivo is not likely to occur unless very high concentrations are achieved.

Mol Med, 1997 Aug, 3(8), 544 - 52
Inhibitory effect of human natural yeast killer toxin-like candidacidal antibodies on Pneumocystis carinii; Seguy N et al.; BACKGROUND: Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT) . Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms . Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P . carinii . MATERIALS AND METHODS: Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P . carinii attachment to epithelial lung cells as well as infectivity to nude rats . Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P . carinii organisms . RESULTS: Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P . carinii organisms that are susceptible to PaKT itself . The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4 . Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P . carinii organisms . CONCLUSIONS: The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.

Biochim Biophys Acta, 1997 Aug 29, 1336(2), 132 - 46
Cloning, expression, purification, and characterization of the murine lysosomal acid alpha-mannosidase; Merkle RK et al.; Catabolism of alpha-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal alpha-mannosidase (EC 3.2.1.24) . Based on regions of protein sequence conservation between the lysosomal alpha-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing alpha 1,3/1,6-mannosidase, alpha-mannosidase II, we have cloned a cDNA encoding the murine lysosomal alpha-mannosidase . The longest of the clones was 3.1 kb in length and encoded a polypeptide of 992 amino acids containing a putative NH2-terminal signal sequence and 11 potential N-glycosylation sites . The deduced amino acid sequence was 76.5% identical to the human lysosomal alpha-mannosidase and 38.1% identical to the lysosomal alpha-mannosidase from D . discoideum . Expression of the cDNA in Pichia pastoris resulted in the secretion of an alpha-mannosidase activity into the culture medium . This recombinant expression product was purified and was shown to have enzymatic characteristics highly similar to the enzyme purified from mammalian sources and to the human lysosomal alpha-mannosidase cDNA expressed in Pichia . These characteristics include a similar pH optimum, Km, Vmax, inhibition by swainsonine, and activity toward natural substrates . Northern blots identified a major 3.5 kb RNA transcript in all murine tissues tested . A minor transcript of 5.4 kb was also detected in some murine tissues similar to the alternatively spliced transcripts that have been previously identified in human tissues.

FEBS Lett, 1997 Sep 1, 414(1), 23 - 6
Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes; Kneip C et al.; The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PphiB) and phycocyanobilin (PCB), respectively . The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C-terminal half of phyA does not alter the structure of the chromophore site within the N-terminal half . Most of the RR spectral changes observed upon replacing PphiB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs . ethyl), implying that the chromophore structures are largely the same for PphiB- and PCB-assembled phytochromes . Only in the Pr state may the RR spectral changes also reflect subtle differences of the PphiB and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.

Biosci Biotechnol Biochem, 1997 Aug, 61(8), 1391 - 3
Ester synthesis by NAD(+)-dependent dehydrogenation of hemiacetal: production of methyl formate by cells of methylotrophic yeasts; Murdanoto AP et al.; A water-soluble ester, methyl formate, was detected as a metabolite in the culture medium of methylotrophic yeasts . Methyl formate synthase, which catalyses NAD(+)-dependent dehydrogenation of the hemiacetal adduct of methanol and formaldehyde, catalyses the ester synthesis . The enzyme activity was induced on a methanol medium and was increased further by the addition of formaldehyde . In the reaction system using intact cells of Pichia methanolica AKU 4262, 135 mM (8.1 g/liter) methyl formate was produced from 2 M methanol . This is a new biological process for ester synthesis that couples spontaneous formation of hemiacetal and alcohol dehydrogenase.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 218 - 24
Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation; Walfridsson M et al.; Saccharomyces cerevisiae was transformed with the Pichia stipitis CBS 6054 XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) respectively . The XYL1 and XYL2 genes were placed under the control of the alcohol dehydrogenase 1 (ADH1) and phosphoglycerate kinase (PGK1) promoters in the yeast vector YEp24 . Different vector constructions were made resulting in different specific activities of XR and XDH . The XR:XDH ratio (ratio of specific enzyme activities) of the transformed S . cerevisiae strains varied from 17.5 to 0.06 . In order to enhance xylose utilisation in the XYL1-, XYL2-containing S . cerevisiae strains, the native genes encoding transketolase and transaldolase were also overexpressed . A strain with an XR:XDH ratio of 17.5 formed 0.82 g xylitol/g consumed xylose, whereas a strain with an XR:XDH ratio of 5.0 formed 0.58 g xylitol/g xylose . The strain with an XR:XDH ratio of 0.06, on the other hand, formed no xylitol and less glycerol and acetic acid compared with strains with the higher XR:XDH ratios . In addition, the strain with an XR:XDH ratio of 0.06 produced more ethanol than the other strains.

J Mol Biol, 1997 Sep 19, 272(2), 253 - 65
NMR studies of a viral protein that mimics the regulators of complement activation; Wiles AP et al.; Vaccinia virus complement control protein (VCP) is a 243-residue protein that is similar in sequence to the regulators of complement activation; its role is to defend the virus against attack by the host complement system . A fragment of this protein spanning the two complement protein (CP)-modules (residues 126 to 243) which make up the C-terminal half of VCP has been expressed in Pichia pastoris . A 15N-labelled sample was purified for the purposes of structure determination and measurements of dynamics in solution using NMR . Structures were calculated on the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence . The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; average r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons,=1.2 A) but less similar to the fifth CP-module of fH (average r.m.s.d.=2.2 A) . In the VCP fragment, the orientation of one module with respect to the other is clearly defined by the experimental data, and T1 measurements are consistent with only limited flexibility at the module-module interface . The r.m.s.d . over all of the 118 residues (backbone atoms) is 0.73 A . The intermodular orientation is better defined than, and significantly different from, that observed in a CP-module pair from fH (re-calculated using the extended SA protocol) . In VCP the long axis of the second module is tilted by 59(+/-4) degrees with respect to the first module (50(+/-13) degrees in the fH pair), and twisted with respect to the first module by 22(+/-6) degrees (223(+/-17) degrees in fH) . The differences between the human and viral proteins may be rationalised in terms of the lack of hydrogen-bond stabilised secondary structure in the N-terminal portion of fH module 16, and the number and type of amino acid side-chains which make up the interface . A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matrix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimental data .

J Cell Sci, 1997 Aug, 110 ( Pt 16), 1935 - 45
Glucose-induced microautophagy in Pichia pastoris requires the alpha-subunit of phosphofructokinase; Yuan W et al.; We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris . These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells . When P . pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized . During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy . We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes . In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole . In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1 . First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1 . Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115 . Third, a PFK1 knockout (delta pfk1) is unable to degrade alcohol oxidase during glucose adaptation . As observed in gsa1, the peroxisomes in delta pfk1 remain outside the vacuole during adaptation . Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e . signaling, selection, or sequestration) . However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase . The inability of delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation . We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.

J Biol Chem, 1997 Sep 19, 272(38), 23792 - 8
The polymerization pocket "a" within the carboxyl-terminal region of the gamma chain of human fibrinogen is adjacent to but independent from the calcium-binding site; Cote HC et al.; The carboxyl-terminal region of the gamma chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor . Protein fragments encoding amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site . rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation . Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized . rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not . Similarly, rFbggammaC30 and gamma'C30 were protected from plasmin degradation by the presence of Ca2+ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca2+-binding site and polymerization pocket are contained within each of these fragments . The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested . These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.

Enzyme Microb Technol, 1997 Aug 15, 21(3), 182 - 90
Purification and characterization of an extracellular beta-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in beta-glucan degradation; Pitson SM et al.; A beta-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH4)2SO4 precipitation followed by anion-exchange and gel filtration chromatography . SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa . The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5 . Comparison of the N-terminal amino acid sequence revealed similarities between the A . persicinum enzyme and several other extracellular fungal beta-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala . In addition to the hydrolysis of p-nitrophenyl-beta-glucoside, the enzyme was also active against several other aryl-beta-glucosides as well as a range of beta-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose . D-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by N-bromosuccinimide, N-acetylimidazole, dicyclohexyl carbodiimide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4, and some metal ions . Possible roles for this enzyme in the noncellulolytic fungus A . persicinum are discussed in light of the increase in the rate of reducing sugar release from beta-glucans by (1-->3)- and (1-->6)-beta-glucanases when the beta-glucosidase is also present in the reaction mixtures.

Yeast, 1997 Sep 15, 13(11), 1043 - 52
Impairment of peroxisome degradation in Pichia methanolica mutants defective in acetyl-CoA synthetase or isocitrate lyase; Kulachkovsky AR et al.; Single recessive mutations of the methylotrophic yeast Pichia methanolica acs1, acs2, acs3 and icl1 affecting acetyl-CoA synthetase and isocitrate lyase, and growth on ethanol as sole carbon and energy source, caused a defect in autophagic peroxisome degradation during exposure of methanol-grown cells to ethanol . As a control, a mutation in mdd1, which resulted in a defect of the 'malic' enzyme and also prevented ethanol utilization, did not prevent peroxisome degradation . Peroxisome degradation in glucose medium was unimpaired in all strains tested . Addition of ethanol to methanol-grown cells of acs1, acs2, acs3 and icl1 mutants led to an increase in average vacuole size . Thickening of peroxisomal membranes and tight contacts between groups of peroxisomes and vacuoles were rarely observed . These processes proceeded much more slowly than in wild-type or mdd1 mutant cells incubated under similar conditions . No peroxisomal remnants were observed inside vacuoles in the cells of acs1, acs2, acs3 and icl1 mutants after prolonged cultivation in ethanol medium . We hypothesize that the acs and icl mutants are defective in synthesis of the true effector--presumably glyoxylate--of peroxisome degradation in ethanol medium . Lack of the effector suspends peroxisome degradation at an early stage, namely signal transduction or peroxisome/vacuole recognition . Finally, these defects in peroxisome degradation resulted in mutant cells retaining high levels of alcohol oxidase which further led to increased levels of acetaldehyde accumulation upon incubation of mutant cells with ethanol.

J Biol Chem, 1997 Aug 8, 272(32), 20077 - 81
Riboflavin 5'-hydroxymethyl oxidation . Molecular cloning, expression, and glycoprotein nature of the 5'-aldehyde-forming enzyme from Schizophyllum commune; Chen H et al.; Vitamin B2-aldehyde-forming enzyme catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group . We have purified the enzyme from the culture media of Schizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M . (1981) J . Biol . Chem . 256, 6682-6685) for cell-free extract . By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa . The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase . Selected peptides were sequenced from their amino termini . We have isolated a full-length cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences . From the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp . The enzyme was determined to be a glycoprotein; however, O-deglucosylation only slightly affects activity . Computer searches showed that the B2-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins . The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B2-aldehyde-forming enzyme activity.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 693 - 700
Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution; Zhang JG et al.; Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high-affinity, functional, receptor complex . Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor alpha-chain, and this property was used to purify a stable complex of human LIF and mouse LIF receptor alpha-chain from pregnant-mouse serum . Recombinant soluble human gp130 was expressed, with a FLAG(R) epitope (DYKDDDDK) at the N-terminus, in the methylotropic yeast Pichia pastoris and purified using affinity chromatography . The formation of a trimeric complex in solution was established by native gel electrophoresis, gel-filtration chromatography, sedimentation equilibrium analysis, surface plasmon resonance spectroscopy and chemical cross-linking . The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of interleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.

Protein Expr Purif, 1997 Aug, 10(3), 345 - 55
Expression of biologically active beta subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris; Samaddar M et al.; Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an alpha subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific beta subunit . The objective of the present study is to develop a recombinant DNA expression system for the beta subunit of FSH that can be applied to study structure-function relationships while producing large quantities of the hormone subunit for immuno-contraceptive, clinical, and veterinary purposes . We report here the expression of biologically active bovine FSH beta (bFSH beta) in the methylotrophic yeast Pichia pastoris . The Pichia-expressed FSH beta (pFSH beta) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSH beta . Replacement of cognate signal peptide with the yeast alpha mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 micrograms/ml (alpha mating factor signal peptide) of the culture supernatant . pFSH beta His.tag (pFSH beta with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography . The molecular weight of purified pFSH beta His.tag was approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSH beta . pFSH beta His.tag could assemble with the alpha subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response . These data show that pFSH beta His.tag is properly folded and biologically active.

J Gen Virol, 1997 Aug, 78 ( Pt 8), 1861 - 6
Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles; Sugrue RJ et al.; We have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME) . The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the expected molecular mass for correctly processed and glycosylated E protein . Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains . The E protein migrated with a buoyant density of 1.13 g/cm3 when analysed using sucrose density gradient centrifugation . Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy . Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies . This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.

Protein Sci, 1997 Aug, 6(8), 1643 - 52
Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes; Jerva LF et al.; Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide . Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis . We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris . Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines . Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1 . MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively . We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor . MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM) . The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor . Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor . Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.

Infect Immun, 1997 Aug, 65(8), 3042 - 7
Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans; Beauvais A et al.; A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus . The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate . This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and to a lesser extent Xaa-Ala dipeptides from the N termini of peptides, including bioactive peptides such as neuropeptide Y, {des-Arg1} bradykinin, and glucagon-like peptide 1, activity at neutral pH, and presence in the amino acid sequence of the Gly-X-Ser-X-Gly consensus motif of the serine-hydrolases and the putative catalytic triad (Ser613, Asp690, His725) of the dipeptidyl-peptidases . Moreover, the last 200 amino acids displayed 60 to 65% similarity with the other dipeptidyl-peptidases IV from rat, mouse, human, and yeast . However, unlike the other dipeptidyl-peptidases, the dipeptidyl-peptidase IV of A . fumigatus is a secreted enzyme with a cleavable signal peptide . Expression of a recombinant dipeptidyl-peptidase IV of A . fumigatus has been attained in the yeast Pichia pastoris.

Gene, 1997 Jul 31, 194(2), 179 - 82
Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes; Payne MS et al.; High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp) . In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6 . MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase . This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.

J Biol Chem, 1997 Jul 18, 272(29), 18298 - 303
Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambda1 protein; Bisaillon M et al.; Previous studies have shown that the reovirus lambda1 core protein harbors a putative nucleotide-binding motif and exhibits an affinity for nucleic acids . In addition, a nucleoside triphosphate phosphohydrolase activity present in reovirus cores has been recently assigned to lambda1 using gene reassortment analysis . In this study, it was demonstrated that the recombinant lambda1 protein, expressed in the yeast Pichia pastoris, is able to hydrolyze nucleoside 5'-triphosphates or deoxynucleoside 5'-triphosphates . This activity was absolutely dependent on the presence of a divalent cation, Mg2+ or Mn2+ . The protein can also unwind double-stranded nucleic acid molecules in the presence of a nucleoside 5'-triphosphate or deoxynucleoside 5'-triphosphate . These results provide the first biochemical evidence that the reovirus lambda1 protein is a nucleoside triphosphate phosphohydrolase/helicase and strongly support the idea that lambda1 participates in transcription of the viral genome.

Eur J Biochem, 1997 Jul 1, 247(1), 332 - 8
Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase; Martinet W et al.; The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P . pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence . Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l . Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity . Although P . pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S . cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues . N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment . Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection . The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.

Protein Expr Purif, 1997 Jul, 10(2), 283 - 91
Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization; Henry A et al.; We have examined the use of the yeast Pichia pastoris for expression of the human amyloid precursor protein (APP) . The ectodomains of the isoforms APP695, APP751, and APP770 were expressed in both P . pastoris protease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1 . Expression of recombinant APP in each of these strains produced intact recombinant protein, together with a small number of breakdown products . The levels of these breakdown products were not significantly altered by expression in the protease-deficient strain compared with wild-type GS115 . The effects of induction time and medium composition on recombinant APP stability were also examined . After optimization of expression and culture conditions, baffled shaker flask cultures of clones selected for high expression routinely yielded 13-24 mg/liter recombinant protein following a two-step purification procedure . The recombinant isoforms possessed the heparin binding, metal binding, and Kunitz-type protease inhibitor properties of human brain-derived APP . These data indicate that P . pastoris is an appropriate laboratory-scale expression system for production of sufficient quantities of recombinant APP for use in biological studies.

Biochemistry, 1997 Jun 24, 36(25), 7652 - 63
Role of tryptophan-63 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability, folding, and ligand binding properties; Chang Y et al.; Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2tPA), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties of this isolated protein domain . The variants were expressed in Pichia pastoris cells . The binding constants of epsilon-aminocaproic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrations of the alterations in intrinsic fluorescence of the variant kringles with the ligands . As compared to wild-type r-K2tPA, increases in the Kd (dissociation) values of approximately 15-fold and 20-200-fold were found for the W63F and W63Y mutants, respectively, toward these three ligands . Neither the W63H nor the W63S variant interacted with these same ligands . Differential scanning calorimetric analyses were also performed on each of the peptides to determine whether the alterations affected the conformational stability of wtr-K2tPA . The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (Tm) values that were 12-20 degrees C lower than that of wtr-K2tPA . Addition of EACA resulted in increases (approximately 12 degrees C) in the Tm values of r-{W63F}-K2tPA and r-{W63Y}K2tPA, a result showing that EACA stabilized the native conformations adopted by these kringle domains . As expected from its greatly diminished binding to r-{W63H}K2tPA and r-{W63S}-K2tPA, high concentrations of EACA had little effect on the Tm of thermal denaturation of these latter mutants . 1H-NMR analysis of the two aromatic mutant kringles was employed to assess their overall comparative folding properties . The high upfield chemical shifts (-0.98 ppm) of the CH3(delta') protons of L47, a major signal of proper kringle folding, were slightly lowered to -0.83 to -0.86 ppm in the cases of all of the mutants . This is due to alterations in the W25-L47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distance relationships of these two amino acid side chains . Assignments of nearly all of the protons of the aromatic residues in the W63F and W63Y mutants were accomplished, and few additional differences from their wild-type counterpart were noted . Reactivities of the mutants against four different monoclonal antibodies directed to wtr-K2tPA revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W63 . We conclude that W63 possesses an important direct role in the ligand binding properties of r-K2tPA . This residue also contributes significantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.

Yeast, 1997 Jun 15, 13(7), 673 - 6
Sequence analysis of Candida albicans phosphoribosyl-aminoimidazole carboxylase (ADE2) gene; Tsang WK et al.; The nucleotide sequence of the Candida albicans ADE2 gene, which encodes phosphoribosylaminoimidazole carboxylase, has been determined . The sequence possesses an uninterrupted open reading frame of 1704 nucleotides corresponding to 568 amino acid residues . The deduced amino acid sequence shares a high degree of homology with ADE2 homologues in other fungal species including Saccharomyces cerevisiae, Pichia methanolica, Schwanniomyces occidentalis and Schizosaccharomyces pombe . Three regions of amino acid sequence were highly conserved among all reported ADE2 genes . The hexanucleotide TGACTC characteristic of genes involved in purine and amino acid biosynthesis is located in front of putative TATA boxes in the promoter region.

Biochem Mol Biol Int, 1997 Jun, 42(1), 169 - 72
Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers; Haaning J et al.; Yeast is widely used in molecular biology . Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants . We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris by means of PCR without any prior DNA purification steps . This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature.

Anal Chem, 1997 Jun 1, 69(11), 1986 - 91
Complete determination of disulfide forms of purified recombinant human serum albumin, secreted by the yeast Pichia pastoris; Ikegaya K et al.; In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute . Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable . In this context, disulfide linkages play a significant structural role and their determination is of paramount importance . As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest . The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing . Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined . The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.

Protein Expr Purif, 1997 Jun, 10(1), 70 - 9
Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain; Heimo H et al.; In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc) . Pichia pastoris produced GAc to the level of 0.4 g per liter medium . This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae . The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity . The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein . The propeptide present in the glucoamylase N terminus was found to be removed correctly by P . pastoris . Glucoamylase produced by P . pastoris is N- and O-glycosylated, with 23% carbohydrate content . The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P . pastoris . O-glycosides (studied to our knowledge for the first time in P . pastoris in this report) contribute about half of the total carbohydrate content in GAc . Purified GAc appears as multiple hands on isoelectric focusing with p1 values around 3.5, a value that is little higher than that for GAc produced in S . cerevisiae . GAc could be used as a versatile tool in studying protein expression in P . pastoris: as an affinity handle for other secreted proteins produced in P . pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P . pastoris.

Protein Expr Purif, 1997 Jun, 10(1), 61 - 9
Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris; Abdulaev NG et al.; The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin . An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P . pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal . Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome . The expression level in this transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0) . Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction . Similar to retinal opsin, P . pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation . A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment . Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin . These results show that P . pastoris cells have the capacity to functionally express bovine opsin.

Arch Biochem Biophys, 1997 May 15, 341(2), 347 - 52
Production of the rat complement regulator, Crry, as an active soluble protein in Pichia pastoris; He C et al.; In this report, we describe the use of the methylotrophic yeast Pichia pastoris for the production of the rat complement regulator, Crry . Crry normally exists as an intrinsic membrane protein containing six to seven short consensus repeats (SCRs), a transmembrane region, and a cytoplasmic tail . To produce Crry as a soluble recombinant protein, nucleotides encoding the five N-terminal SCRs from the rat Crry cDNA were amplified by PCR, and cloned into the P . pastoris expression vector, pPIC9 . This vector contains the yeast alpha-factor signal sequence, thereby leading to secretion of recombinant protein . This construct was subsequently integrated into P . pastoris strain GS115 genomic DNA . Secreted soluble Crry was produced by induction of the AOX1 promoter with methanol . Recombinant Crry protein was purified to homogeneity by sequential Mono Q and Mono P chromatography . The protein was highly active toward the alternative and classical pathways of complement, inhibiting the latter by approximately 90% at a concentration of 15 nM . The P . pastoris system offers an efficient method for the production of soluble recombinant Crry . Production of active rat Crry offers opportunities to study long-term models of disease in rats, which has not been possible with available heterologous complement inhibitors.

J Immunol, 1997 May 15, 158(10), 4584 - 90
Costimulation of T cell proliferation by a chimeric B7-2 antibody fusion protein specifically targeted to cells expressing the erbB2 proto-oncogene; Gerstmayer B et al.; T cells require at least two signals for activation and clonal expansion . The first signal conferring specificity is initiated by interaction of the T cell receptor with antigenic peptides in the context of MHC molecules . The second, costimulatory signal can be provided by cell surface molecules on APCs such as B7-1 (CD80) and B7-2 (CD86), which interact with their counter-receptors on T cells . The absence of costimulatory signals presents one possible mechanism for tumor cells to escape immune surveillance . In experimental models transfection of B7 genes into tumor cells can result in T cell-dependent tumor rejection . We have developed a novel approach to direct the costimulatory B7-2 molecule to the surface of target cells . Our approach is based on a chimeric fusion protein that consists of the extracellular domain of human B7-2 fused to a single-chain Ab domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas . This B7-2(225)-scFv(FRP5) molecule expressed in the yeast Pichia pastoris and purified from culture supernatants is functionally active and binds to B7 counter-receptors and to ErbB2 . B7-2(225)-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal that results in enhanced proliferation of syngeneic T cells . Our results suggest that effective tumor vaccines for cancer immunotherapy could be created by targeting such chimeric ligands to the surface of tumor cells.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 68 - 72
The Golgi sialoglycoprotein MG160, expressed in Pichia pastoris, does not require complex carbohydrates and sialic acid for secretion and basic fibroblast growth factor binding; Chen YJ et al.; MG160, a type I membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, shows high homology (over 90%) with CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand of the cell surface of murine myeloid cells . When Chinese Hamster Ovary (CHO) cells were stably transfected with a cDNA lacking the transmembrane and C-terminus cytoplasmic domain of MG160 (delta TMCT), a fully processed protein of 160 kDa apparent molecular mass was recovered in the culture medium . When these cells were treated with tunicymycin, a 130- to 140-kDa protein was immunoprecipitated from the culture medium . A construct lacking the signal sequence, the single transmembrane, and the cytoplasmic domains of MG160 (delta TMCT-) was integrated at the HIS Pichia pastoris genome site using the expression vector pPIC 9 which possesses a yeast compatible signal sequence (Invitrogen) . Recombinant protein accumulated in the medium to approximately 10 mg/L . The yeast recombinant protein lacked complex carbohydrates and sialic acid but bound 125I bFGF . Similarly, rat MG160 subjected to deglycosylation by peptide:N-glycosidase F (PNGase) bound 125I bFGF.

Electrophoresis, 1997 May, 18(5), 819 - 25
Comparison of natural and recombinant isoforms of grass pollen allergens; Petersen A et al.; More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses . We studied the structural variability of the group I allergens in single species and among different grasses . By 2-DE blotting using patients' IgE and monoclonal antibodies, we detected IgE-reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6 . While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively . By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens . The post-translational modifications (one N-glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all . From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross-reactivity among the grasses . In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E . coli and in P . pasteuris . 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI . While the E . coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15% . This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.

J Biochem (Tokyo), 1997 May, 121(5), 831 - 4
An engineered bivalent single-chain antibody fragment that increases antigen binding activity; Luo D et al.; Bivalent single chain Fv (scFv) was constructed by fusing a polypeptide extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment . The scFv protein was expressed and secreted in a recombinant Pichia pastoris system as a dimer with a C-terminal disulfide bridge, as determined by Western blot analysis under non-reducing conditions . We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer ratio than the two cysteine counterpart (scFv-2Cys) . Binding activity measurements performed by means of a competitive radioimmunoassay showed that scFv-1Cys exhibited specific antigen binding activity, which was almost the same as that of the parental MAb, and approximately four- and fortyfold higher than those of the control scFv monomer and scFv-2Cys.

Virus Res, 1997 May, 49(1), 41 - 7
Production of active polyomavirus large T antigen in yeast Pichia pastoris; Peng YC et al.; The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2 . Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter . Large T antigen was purified by immunoaffinity chromatography . We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner . This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.

Appl Environ Microbiol, 1997 May, 63(5), 1715 - 20
Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts; Murdanoto AP et al.; Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells . Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit . The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct {CH2(OH)OCH3} of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate . Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family . The methyl formate synthase activity of C . boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p . These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts . The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.

Gene, 1997 Apr 29, 190(1), 63 - 7
Expression and characterization of the hepatitis E virus ORF3 protein in the methylotrophic yeast, Pichia pastoris; Lal SK et al.; We have used the methylotrophic yeast, Pichia pastoris, to express the open reading frame 3 (ORF3) of the hepatitis E virus (HEV) . The ORF3 gene codes for a 123-amino-acid protein that contains highly immunodominant epitopes and is a potentially useful diagnostic and immunoprophylactic antigen . The expressed protein showed positive on immunoblots probed against antibodies raised in rabbit and infected human patient sera . In order to optimize the ORF3 protein expression, we have examined the regulated expression of this protein and characterized it . Unlike its expression in E . coli, the ORF3 protein was present in both the soluble and insoluble fractions of the cell lysate . The expressed protein is not glycosylated and does not undergo any major processing in the host strain.

Gene, 1997 Apr 29, 190(1), 55 - 62
Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris; Sreekrishna K et al.; Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter . The factors that drastically influence protein production in this system include: copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions, and fermentation parameters . All these factors should be considered in designing an optimal production system . The inherent ability of P . pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms (which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the production of MMP . However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

Gene, 1997 Apr 29, 190(1), 45 - 51
Expression and characterization of Pichia etchellsii beta-glucosidase in Escherichia coli; Pandey M et al.; The beta-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation . Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach) . We present here the purification of the enzyme beta-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone . The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported . The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme . Temperature directed substrate specificity for aryl beta,1-4 linkage, and beta (1-2), beta(1-4), beta(1-6) and beta(2-1) linkages in various natural disaccharides was observed . Glycosylation of the enzyme was found to be unimportant for enzyme activity . With both cellobiose and glucose, oligosaccharide synthesis was detected . The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.

Biochemistry, 1997 Apr 29, 36(17), 5285 - 92
Functional characterization of mitochondrial carnitine palmitoyltransferases I and II expressed in the yeast Pichia pastoris; de Vries Y et al.; The rate-limiting step in beta oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA . The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII . Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII . In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI . To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity . High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains . Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not . Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polypeptide in mammalian mitochondrial membranes . We describe the kinetic characteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII . Yeast-expressed CPTI is inactivated by detergent solubilization . However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment . CPTI is thus reversibly inactivated by detergents.

J Biol Chem, 1997 Apr 25, 272(17), 11581 - 7
Hexose oxidase from the red alga Chondrus crispus . Purification, molecular cloning, and expression in Pichia pastoris; Hansen OC et al.; Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose . The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J . D., and Ikawa, M . (1973) Biochim . Biophys . Acta 309, 11-22) . We report here the purification to homogeneity of hexose oxidase from C . crispus . The purified enzyme was cleaved with cyanogen bromide and endoproteinase Lys-C and the peptide fragments were subjected to amino acid sequence analysis . Oligonucleotides were designed on the basis of the peptide sequences and a cDNA clone encoding C . crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA . The nucleotide sequence of the hexose oxidase cDNA contained an open reading frame of 546 amino acid residues with a predicted relative molecular mass of 61,898 . No significant sequence similarity was found between hexose oxidase and other protein sequences available in data bases . Expression of the hexose oxidase cDNA in Pichia pastoris as an active enzyme confirmed the identity of the DNA sequence . Native hexose oxidase from C . crispus was characterized and compared with purified, recombinant enzyme.

FEBS Lett, 1997 Apr 21, 407(1), 63 - 8
Construction and expression in the yeast Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA-4; Gerstmayer B et al.; We have generated soluble recombinant forms of the costimulatory molecules B7-1 and B7-2, and their counter-receptor CTLA-4 using a yeast Pichia pastoris expression system . Fragments comprising the extracellular domains of human B7-1, B7-2, and CTLA-4 molecules were expressed at high levels and could be purified from culture supernatants following a simple one-step purification protocol . The recombinant proteins retained their functionality and specific binding to their natural counterparts could be demonstrated by FACS analysis . In T cell proliferation assays costimulatory activity of immobilized B7-1 and B7-2 proteins in the presence of an anti-CD3 antibody was observed with the B7-1 protein being more potent than B7-2.

J Biol Chem, 1997 Apr 11, 272(15), 10125 - 34
The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding; Kiselyov VV et al.; To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris . For comparison, other NCAM domains were also produced as fusion proteins . By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M . Furthermore, it was found that the first Ig-like domain binds heparin . It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.

Biochemistry, 1997 Apr 8, 36(14), 4327 - 36
Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family; Wuebbens MW et al.; The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity . Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma {Roush, E . D., & Fierke, C . A . (1992) Biochemistry 31, 12536-12542}, and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family . We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library . Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column . Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved . However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin . pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding . In addition, the antigenic determinants of pICA and the transferrins are not identical . These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.

Mol Cell Endocrinol, 1997 Apr 4, 128(1-2), 39 - 45
Cleavage-secretion of angiotensin I-converting enzyme in yeast; Williams TA et al.; Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post-translational proteolytic cleavage in mammalian cells to release the soluble ectodomain . The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, would facilitate its identification . We have expressed a secreted form of the ACE C domain, lacking the C-terminal membrane anchor (C domain(deltaCOOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor . Immunofluorescent labelling localises the ACE C domain to the periphery of yeast cells but not C domain(deltaCOOH), however, expression of both C domain and C domain(deltaCOOH) produced soluble enzymes in the culture medium . Immunocharacterisation of the two soluble forms of the C domain indicates a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart . Thus ACE undergoes a proteolytic cleavage in yeast.

Vaccine, 1997 Apr-May, 15(6-7), 679 - 88
Yeast-secreted bovine herpesvirus type 1 glycoprotein D has authentic conformational structure and immunogenicity; Zhu X et al.; Bovine herpesvirus-1 (BHV-1) glycoprotein D (gD), an envelope glycoprotein, engenders mucosal and systemic immunity protecting cattle from viral infection . Production of gD with authentic immunogenicity is required for a subunit vaccine . We placed the truncated BHV-1 gD gene, lacking its putative transmembrane and cytoplasmic domains, under the control of the methanol-inducible AOX1 promoter in the yeast Pichia pastoris . Truncated BHV-1 gD (tgD) was efficiently secreted into the culture medium as a 68 kDa protein using either the yeast alpha prepro or native BHV-1 gD signal sequences . The yeast-secreted tgD had N-linked glycosylation and appears to have authentic conformational structure and immunogenicity based on the following observations A panel of monoclonal antibodies recognizing five neutralizing epitopes reacted with yeast tgD . Sera from yeast tgD-immunized mice immunoprecipitated native BHV-1 gD and neutralized BHV-1 infection in vitro . Yeast tgD competitively blocked all reaction between native gD and monospecific gD polyclonal sera from cattle . Based on these data, yeast-derived BHV-1 tgD is an excellent candidate for a subunit vaccine.

Yakugaku Zasshi, 1997 Apr, 117(4), 220 - 32
{Structure of recombinant human serum albumin from Pichia pastoris}; Ohtani W et al.; The structure of recombinant human serum albumin derived from Pichia pastoris (rHSA) was analyzed in detail . Complete amino acid analysis was performed by the phenyl isothiocyanate precolumn labeling method . The amino terminal sequence was determined by the Edman degradation . The carboxyl terminal amino acid was determined by digestion with carboxypeptidase, and the carboxyl terminal peptide fragment was analyzed by electrospray mass spectrometry . The peptide fragments of rHSA digested with Lysylendopeptidase, Endoproteinase Glu-C, or Endoproteinase Asp-N were analyzed by electrospray mass spectrometry . The complete amino acid composition, the terminal sequences and the complete amino acid sequence of rHSA agreed with the primary structure deduced from its cDNA . The elution pattern of reduced and carboxymethylated rHSA digested with Lysylendopeptidase and the elution pattern of intact rHSA digested with pepsin were respectively similar to those of plasma-derived human serum albumin (pHSA) . The pattern of CD spectrum of rHSA was identical in both shape and magnitude to that of pHSA . 1H-NMR spectra of rHSA and pHSA in deuterium oxide showed the same signal patterns in the observed region (delta 10.5-0.5 ppm) . Cross peaks assigned to the alpha proton-beta proton of Asp-1 (delta 4.2/2.8 ppm) and the delta proton-epsilon proton of lysine residues (delta 2.8-3.2/1.4-2.0) showed the same cross peak patterns and chemical shifts in two-dimensional phase sensitive double-quantum filtered 1H-1H correlation spectra of rHSA and pHSA.

Biosci Biotechnol Biochem, 1997 Apr, 61(4), 577 - 82
The phylogeny of species of the genus Issatchenkia Kudriavzev (Saccharomycetaceae) based on the partial sequences of 18S and 26S ribosomal RNAs; Yamada Y et al.; The ten strains of Issatchenkia species were examined for their partial base sequences of 18S and 26S rRNAs . In the 18S rRNA partial base sequences (positions 1451-1618, 168 bases), the strains of the species of the genus Issatchenkia were found to be not uniform phylogenetically . The calculated base differences numbered 5-0 . The strains of Issatchenkia species examined had 3-1 base differences with the type strain of Pichia membranaefaciens . Especially, the type strain of Issatchenkia orientalis, the type species of the genus Issatchenkia was found to be closely related phylogenetically to that of P . membranaefaciens . The calculated number of base differences was only one . The base sequences on the fingerprint segment were comprised of four bases (four kinds of AUAU, CCAU, AUAG, and ACAU), as found in P . membranaefaciens (ACAA) . In the 26S rRNA partial base sequences, the calculated number of base differences was 8-0 (positions 1611-1835, 225 bases), and the calculated percent similarities were 61-80 (positions 493-622, 130 bases), within the genus Issatchenkia . Discussion was made phylogenetically and taxonomically, especially on the phylogenetic relationship between the type species of the genera Issatchenkia and Pichia and on a circumscription of the genus Issatchenkia.

Biotechnol Appl Biochem, 1997 Apr, 25 ( Pt 2), 151 - 7
Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator; Miele RG et al.; The oligosaccharide structures present on Asn5 of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator {(r)-{K2tPA}} have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry . The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)8(N-acetylglucosamine)2 (Man8GN2) to Man18GN2 . The preponderance of these glycans spanned Man9GN2 to Man12GN2, and the major overall product was Man10GN2 . An additional (less than 5%) amount of the polypeptide was hyperglycosylated . In contrast with glycoproteins produced in Saccharomyces cerevisiae, our results with specific mannosidase digestions were consistent with previous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man8GN2 unit . The results show that the N-linked glycosylation pathways in P . pastoris are substantially different from those found in S . cerevisiae, with shorter Man(alpha 1,6) extensions to the core Man8GN2 and the apparent lack of significant Man(alpha 1,3) additions representing the major processing modality of N-linked glycans in P . pastoris.

Biotechniques, 1997 Apr, 22(4), 718 - 29
Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent; Mistry AR et al.; This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris . Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system . Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative . Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus . Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture . Transient expression of a luciferase reporter gene was observed . Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome . HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage . In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method . HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Int J Syst Bacteriol, 1997 Apr, 47(2), 385 - 93
Clinical isolates of Candida guilliermondii include Candida fermentati; San Millan RM et al.; Clinical isolates of Candida guilliermondii that were investigated by isoenzyme and randomly amplified polymorphic DNA analyses represented two distinct species . The two species were distinguished on the basis of delayed fermentation of galactose . The larger group of isolates was closely related to the anamorph C . guilliermondii ATCC 6260T (T = type strain) and its teleomorph, Yamadazyma (= Pichia) guilliermondii ATCC 46036T . The remaining group, whose members fermented galactose, was very similar to Candida fermentati CBS 2022, which had for many years been placed in synonymy with C . guilliermondii . Three additional groups were represented by individual strains; these strains included C . guilliermondii var . soya ATCC 20216, which was found to represent Yamadazyma ohmeri . The type strain of Y . guilliermondii is redefined.

Cancer Res, 1997 Apr 1, 57(7), 1329 - 34
A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer; Sim BK et al.; Endogenous murine angiostatin, identified as an internal fragment of plasminogen, blocks neovascularization and growth of experimental primary and metastatic tumors in vivo . A recombinant protein comprising kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris had physical properties (molecular size, binding to lysine, reactivity with antibody to kringles 1-3) that mimicked native angiostatin . This recombinant Angiostatin protein inhibited the proliferation of bovine capillary endothelial cells in vitro . Systemic administration of recombinant Angiostatin protein at doses of 1.5 mg/kg suppressed the growth of Lewis lung carcinoma-low metastatic phenotype metastases in C57BL/6 mice by greater than 90%; administration of the recombinant protein at doses of 100 mg/kg also suppressed the growth of primary Lewis lung carcinoma-low metastatic phenotype tumors . These findings demonstrate unambiguously that the antiangiogenic and antitumor activity of endogenous angiostatin resides within kringles 1-4 of plasminogen.

Protein Sci, 1997 Apr, 6(4), 919 - 21
Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex; Linnevers CJ et al.; Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling . To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site . We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system . Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate . A 2.2 A X-ray data set has been collected on crystals belonging to space group P2(1)2(1)2(1), with a = 41.66 A, b = 51.41 A, and c = 107.72 A . There is most likely one molecule per asymmetric unit.

Biochem Biophys Res Commun, 1997 Mar 27, 232(3), 656 - 62
Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotropic yeast Pichia pastoris; Doring F et al.; The methylotrophic yeast Pichia pastoris was used for heterologous expression of the rabbit intestinal peptide transporter PepT1 and its functional characterization . PepT1 mediates the electrogenic transmembrane transport of di- and tripeptides and peptido-mimetics such as beta-lactam antibiotics and ACE-inhibitors . Functional expression of PepT1 was determined in different recombinant clones by flux studies employing the radiolabeled dipeptide 3H-(D)-Phe-(L)-Ala . One clone (GS-PepT1) displayed high level functional expression that was pH dependent and saturable with an app . K0.6 of 1.17 +/- 0.18 mM . Inhibition of 3H-(D)-Phe-(L)-Ala uptake into GS-PepT1 by selected dipeptides, tripeptides and peptidomimetics including beta-lactam antibiotics and ACE-inhibitors revealed the same substrate specifity as reported for PepT1 when expressed in mammalian cells or Xenopus laevis oocytes . Pichia cells expressing PepT1 will provide an excellent tool for in vitro bioavailability studies for peptides and peptidomimetics . Moreover, to our knowledge, this is the first demonstration of functional expression of a mammalian membrane transport protein using P . pastoris.

Gene, 1997 Mar 18, 187(2), 193 - 200
Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants; McGrew JT et al.; Pichia pastoris is a yeast capable of expressing large amounts of some proteins . When expression vectors are introduced into P . pastoris, individual transformants typically express widely varying amounts of protein . Because clones expressing the highest level of protein occur infrequently during the transformation process, finding them can be very labor-intensive . We developed an immunological based filter screening method that rapidly detects transformants secreting large amounts of a heterologous protein . We have applied this method to the expression of a soluble trimeric form of CD40L, a molecule that regulates B-cell responses . Using this method, we identified transformants with one to 13 copies of the CD40L expression cassette . Maximum expression was obtained with clones containing eight or more copies of the expression cassette, and a clone with eight copies was selected for further analysis . High cell density fermentation of this clone using a mixed glycerol:methanol feed yielded 255 mg CD40L per liter of supernatant.

J Biol Chem, 1997 Mar 7, 272(10), 6238 - 44
Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus; Beauvais A et al.; A novel dipeptidyl-peptidase (DPP V) was purified from the culture medium of Aspergillus fumigatus . This is the first report of a secreted dipeptidyl-peptidase . The enzyme had a molecular mass of 88 kDa and contained approximately 9 kDa of N-linked carbohydrate . The expression and secretion of dipeptidyl-peptidase varied with the growth conditions; maximal intra- and extracellular levels were detected when the culture medium contained only proteins or protein hydrolysates in the absence of sugars . The gene of DPP V was cloned and showed significant sequence homology to other eukaryotic dipeptidyl-peptidase genes . Unlike the other dipeptidyl-peptidases, which are all intracellular, DPP V contained a signal peptide . Like the genes of other dipeptidyl-peptidases, that of DPP V displayed the consensus sequences of the catalytic site of the nonclassical serine proteases . The biochemical properties of native and recombinant DPP V obtained in Pichia pastoris were unique and were characterized by a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum . In addition, we showed that DPP V is identical to one of the two major antigens used for the diagnosis of aspergillosis.

Vaccine, 1997 Mar, 15(4), 414 - 22
Large-scale production in Pichia pastoris of the recombinant vaccine Gavac against cattle tick; Canales M et al.; A gene coding for the Bm86 tick protein was recently cloned, expressed in Pichia pastoris and shown to induce an inmunological response in cattle against ticks . Moreover, the Gavac vaccine (Heber Biotec S.A., Havana, Cuba), which contains this recombinant protein, has proved to control the Boophilus microplus populations under field conditions . This paper reviews the development and large-scale production of this vaccine, the efficacy of the resulting product and the strategy followed in designing its production plant . The production plant fulfills biosafety requirements and GMP.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 117 - 22
Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae; Parekh RN et al.; The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector . Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion . Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER) . Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels . Optimal specific productivity of a standard laboratory strain of S . cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S . cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion . Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.

Clin Diagn Lab Immunol, 1997 Mar, 4(2), 142 - 6
Monoclonal yeast killer toxin-like candidacidal anti-idiotypic antibodies; Polonelli L et al.; Rat monoclonal yeast killer toxin (KT)-like immunoglobulin M (IgM) anti-idiotypic antibodies (KT-IdAbs) were produced by idiotypic vaccination with a mouse monoclonal antibody (MAb; MAb KT4) that neutralized a Pichia anomala KT characterized by a wide spectrum of antimicrobial activity . The characteristics of the KT-IdAbs were demonstrated by their capacity to compete with the KT to the idiotype of MAb KT4 and to interact with putative KT cell wall receptors (KTRs) of sensitive Candida albicans cells . The internal-image properties of KT-IdAbs were proven by their killer activity against KT-sensitive yeasts . This lethal effect was abolished by prior adsorption of KT-IdAbs with MAb KT4 . These findings stressed the potential importance of antibody-mediated immunoprotection against candidiasis and suggested a feasible experimental approach for producing antimicrobial receptor antibodies without purifying the receptor . KT-IdAbs might represent the basis for producing engineered derivatives with a high potential for effective therapeutic antifungal activity.

Protein Expr Purif, 1997 Mar, 9(2), 159 - 70
Overexpression and characterization of Aspergillus awamori wild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiae and Aspergillus niger as hosts; Fierobe HP et al.; Glucoamylase from Aspergillus niger (identical to Aspergillus awamori glucoamylase) is an industrially important, multidomain N- and O-glycosylated starch-hydrolase . To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeast Pichia pastoris . Using the vector pHIL-D2, the cDNA encoding A . awamori glucoamylase is inserted in the yeast genome downstream of the 5' AOX1 promoter to replace the AOX1 gene . Induction by 0.75% methanol for 48 h led to synthesis of secreted glucoamylase to give around 0.4 g/liter, as directed by the A . awamori signal sequence . Recombinant glucoamylase produced in P . pastoris, Saccharomyces cerevisiae, or A . niger displayed similar catalytic properties, thiol content, and isoelectric point . Glucoamylase from P . pastoris, however, has higher thermostability than the enzymes from S . cerevisiae, A . niger, or a commercial preparation of A . niger glucoamylase . The average Mr determined by matrix-assisted laser desorption ionization mass spectrometry of these enzymes is thus 82,327, 83,869, 82,839, and 80,370, respectively, and neutral sugar analysis shows the differences to be due to variation in the extent of glycosylation . Compared to wild-type, single-residue mutation generally reduced the amount of secreted glucoamylase in S . cerevisiae and A . niger . In P . pastoris, however, the Cys320 --> Ala/Glu400 --> Cys double mutant is produced at 0.3 g/liter, or 75% of wild-type glucoamylase, while the corresponding single mutants have been produced at l and 20% of the wild-type level in S . cerevisiae and A . niger, respectively.

J Immunol, 1997 Mar 1, 158(5), 2303 - 9
Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes; Tanaka M et al.; Fas ligand (FasL) is a type II membrane protein . Binding of FasL to its receptor, Fas, induces apoptosis . Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form . Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris . The purified rFasL was found to be glycosylated and to exist as a trimer . The rFasL was effective in inducing apoptosis in a Fas-expressing T cell or a fibroblast cell line . The ID50 of rFasL for mouse Fas-expressing T cells was about 0.5 ng/ml . The killing process with rFasL was quick . That is, >80% Fas-expressing mouse cells were killed within 1 h by a saturation concentration of human rFasL . Intravenous administration of 500 microg of human rFasL had a lethal effect in mice . When the mice were pretreated with Propionibacterium acnes, the subsequent injection of 30 microg of human rFasL induced hepatic failure and killed the mice within 24 h . These results indicated that the soluble human FasL is active in inducing apoptosis in vitro and in vivo, and its deleterious effect may be strengthened in patients who are suffering from bacterial infection.

Gene, 1997 Feb 20, 186(1), 37 - 44
Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter; Waterham HR et al.; We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris . The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms . Promoter studies in P . pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth . Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells . As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal . P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P . pastoris.

FEMS Microbiol Lett, 1997 Feb 15, 147(2), 227 - 32
Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase; Zhang Y et al.; Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts . We previously showed that a cystein residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies . The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding . We cloned the XYL1 gene encoding xylose reductase from P . stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis . Each of the three cysteine residues (Cys19, Cys27 and Cys130) was individually mutated to serine . All three Cys-->Ser variants remained functional, but with reduced catalytic activity . Sensitivity of the P . stipitis xylose reductase to thiol-specific reagents was attributed to both Cys27 and Cys130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS . The apparent Km values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold . Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.

Eur J Biochem, 1997 Feb 15, 244(1), 21 - 30
Production and characterization of recombinant active mouse gelatinase B from eukaryotic cells and in vivo effects after intravenous administration; Masure S et al.; Gelatinase B is a matrix metalloproteinase involved in tissue remodelling . When mouse cells are triggered in vitro with interleukin-1, bacterial endotoxin, virus-mimicking double-stranded RNA or cytokine inducers, they produce gelatinase B . To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells . Hybrid genomic DNA-cDNA constructs under the control of two constitutive viral promoters were generated by PCR-mediated exon amplification . In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79-kDa protein, and expression in CHO cells resulted in an intact glycosylated 110-kDa gelatinase B which was enzymically active . However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B . Therefore, the enzyme was expressed in the yeast Pichia pastoris . Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/l . Amino-terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin-Sepharose yielded the expected N-glycosylated proenzyme form (110 kDa) in addition to an amino-terminally truncated unglycosylated variant (69 kDa) . Both forms had gelatinolytic activity on zymography . The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo . After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h . In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow . Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.

J Immunol Methods, 1997 Feb 14, 201(1), 67 - 75
High-level secretion of two antibody single chain Fv fragments by Pichia pastoris; Eldin P et al.; The diagnostic and therapeutic applications of antibody single-chain Fv (sFv) fragments often require large amounts of protein that can be problematic and expensive to obtain . Here we report the secretion of two sFv fragments by the yeast Pichia pastoris at levels up to 250 mg/l . Soluble sFv fragments were purified from culture supernatants in one step by affinity or metal-chelating chromatography, and were indistinguishable from their bacterially expressed counterparts in terms of affinity . Secretion of functional sFv fragments by Pichia pastoris provides a low cost, high yield alternative to current sFv expression systems.

Protein Eng, 1997 Feb, 10(2), 99 - 101
Circular dichroism analysis of the interaction between the alpha and beta subunits in a killer toxin produced by a halotolerant yeast, Pichia farinosa; Suzuki C et al.; SMK toxin is a killer toxin produced by a halotolerant yeast, Pichia farinosa . It is a heterodimer consisting of alpha (63 aa) and beta (77 aa) subunits, between which no disulfide bond exists . The two subunits interact tightly with each other below pH 5 . However, the subunits dissociate under neutral conditions, resulting in the aggregation of the alpha subunit and the concomitant loss of killer activity . CD spectral measurements showed that the secondary structure of the SMK toxin changes drastically in the pH range 5.1-5.5 and that after the dissociation of the subunits, the soluble beta subunit alone cannot take any secondary structure . It was also shown that the concentration of NaCl does not affect the secondary structure of the SMK toxin.

Nat Biotechnol, 1997 Feb, 15(2), 155 - 8
Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity; Magliani W et al.; Single chain fragment (ScFv) antiidiotypic antibodies (antilds) of a killer toxin (KT) from the yeast Pichia anomala have been produced by recombinant DNA methodology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a KT-neutralizing monoclonal antibody (Mab KT4) . ScFv KT-like antilds (KTIdAb) react with specific Candida albicans KT cell wall receptors (KTR) exerting a candidacidal activity in vitro could be neutralized by adsorption with Mab KT4 . ScFv KTIdAb displayed an effective therapeutic activity in an experimental model of rat candidal vaginitis.

Biotechnol Appl Biochem, 1997 Feb, 25 ( Pt 1), 63 - 74
High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator; Nilsen SL et al.; The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI 15 and KM71 . This construct contained a hexahistidine sequence at the C-terminus of the kringle to aid in purification by immobilized metalion-affinity chromatography . The exact amino acid sequence of the isolated kringle was EAEAYV-{K2tPA}SR(H)6, where {K2tPA} represents amino acid sequence residues C1-C82 of the kringle domain (residues 180-261 of tPA) . The clones of the yeast transformants provided large amounts of the recombinant (r)-{K2tPA}-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter . Purification of the kringle domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni(+)-affinity chromatography or lysine-Sepharose affinity chromatography . N-linked glycans were present on approx . 30% of this yeast-expressed material, at N5 of the kringle (corresponds to N11 of the particular construct, N184 of full-length tPA) . The expressed recombinant kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 domain of the protein, interacted properly with various omega-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1H-NMR . The results demonstrate that the P . pastoris system can be employed to obtain large amounts of secreted and properly folded kringle domains.

Gene, 1997 Jan 31, 185(1), 147 - 52
Mitochondrial DNA polymerases from yeast to man: a new family of polymerases; Lecrenier N et al.; We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p) . This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p . The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma) . Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.

J Theor Biol, 1997 Jan 21, 184(2), 171 - 86
A theoretical study on the nucleotide changes under a definite functional constraint of forming stable base-pairs in the stem regions of ribosomal RNAs; its application to the phylogeny of eukaryotes; Otsuka J et al.; Homologous alignment of 5 S rRNAs shows the characteristic features that (i) nucleotide changes are more remarkably seen in the stem region than in the loop region and (ii) most of the changes in the former region occur under a definite functional constraint of maintaining the stable base pairs G:C, C:G, A:U and U:A . In order to obtain a better evolutionary measure, we derived a theoretical equation for expressing the changes between the stable base-pairs in the stem region from an elementary process, in which the nucleotides in a pair mutate individually and the mis-matched pairs thus generated are eliminated by selection or return to the stable pairs by successive mutations . This equation leads us to a simple method of estimating the base-pair change rate by formally enumerating the base-pair changes observed in the pairwise comparison of homologous sequences from different species, just like the estimation of the change rate of individual nucleotides . The application of this method to 5 S rRNAs of eukaryotes reveals a new feature, in which the evolutionary distance of yeasts (Saccharomyces, Pichia and Hansenula) from higher plants and animals is much more expanded than that obtained previously by the enumeration of individual nucleotide changes observed in a whole region of 5 S rRNA and many other fungi, protozoans and algae are allocated to the middle positions between the yeasts and higher plants . The base-pair change rate is estimated to be about 2 x 10(-10) year-1, which is less than the mutation rate by one order of magnitude, and is suitable for resolving the phylogeny of species which diverged a few billion years ago . The selective term for eliminating the mis-matched pairs is also evaluated to be stronger than the mutation rate by about one order of magnitude even for the outstanding mis-matched pair of G:U and U:G.

Biochem J, 1997 Jan 15, 321 ( Pt 2), 289 - 95
Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae . Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris; Romero PA et al.; The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied . In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p . Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris . After induction with methanol, the medium contained approx, 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively . Both recombinant proteins were shown to exhibit alpha 1,2-mannosyltransferase activity . The enzymes have an absolute requirement for Mn2+ and a similar K(m) for mannose (280-350 mM), methyl-alpha-mannoside (60-90 mM) and GDP-mannose (50-90 microM), but the Vmax was approx . 10 times higher for Kre2p/Mnt1p than for Ktr1p . The enzymes have similar substrate specificities and utilize mannose, methyl-alpha-mannoside, alpha-1,2-mannobiose and methyl-alpha-1,2-mannobiose, as well as Man15-30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates . The enzymes do not utilize alpha-1,6-mannobiose, alpha-1,6-mannotriose, alpha-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9-10GlcNAc . These results indicate that Kre2p/ Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.

Structure, 1997 Jan 15, 5(1), 81 - 94
The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin; Kashiwagi T et al.; BACKGROUND: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains . The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration . The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions . However, they are easily dissociated under neutral conditions and lose the killer activity . The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability . RESULTS: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions . The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family . The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4 . This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins . Many acidic residues are clustered at the bottom of the SMK toxin molecule . Some of the carboxyl sidechains interact with each other through hydrogen bonds . The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule . CONCLUSIONS: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif . This observation may suggest that these toxins are evolutionally and/or functionally related . The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups . This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range . The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

FEBS Lett, 1997 Jan 13, 401(1), 73 - 7
Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein; Ferrari E et al.; The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system . We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris . Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form . Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one . This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

J Biol Chem, 1997 Jan 10, 272(2), 1197 - 202
Role of the occluding loop in cathepsin B activity; Illy C et al.; Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase . This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft . Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris . A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity . This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site . In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme . These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.

Biochemistry, 1997 Jan 7, 36(1), 103 - 11
Recombinant type A and B phytochromes from potato . Transient absorption spectroscopy; Ruddat A et al.; The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris) . In addition, a deletion mutant of phyB (delta 1-74) was expressed . The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB) . The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB . All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore . The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced . Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time . Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different . Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C . The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms . The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms . Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr . For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available . As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

J Clin Lab Anal, 1997, 11(4), 196 - 201
Enzyme immunoassays for specific IgG and IgE antibodies to Pichia pastoris components in normal humans; Ohtani W et al.; We developed enzyme immunoassays for human anti-Pichia pastoris components (PPC) IgG and anti-PPC IgE antibody titers . Anti-PPC IgG antibody assay were performed using antigen-coated plate and anti-human IgG peroxidase conjugate . The intra- and interassay coefficients of variation (CV) of anti-PPC IgG antibody were 1.83-2.51% and 1.97-2.76%, respectively . The anti-PPC IgE antibody assay was performed using an anti-IgE monoclonal antibody-coated plate, biotin-labeled PPC and avidin-labeled peroxidase, which was not subject to interference by the high titer of anti-PPC IgG antibody . The intra- and interassay CV were 3.83-5.34% and 3.56-5.84%, respectively . We determined and compared anti-PPC IgG antibody titers in the 40 normal individuals . We confirmed that a high titer of anti-PPC IgG antibody is contained in all normal human sera and that these antibodies are directed primarily to mannan by immunoblotting analysis . The ratio of the maximum to minimum anti-PPC IgG antibody titers in normal individuals was > 8,000 . Anti-PPC IgG antibody titers did not correlate with the age . However, we did not detect anti-PPC IgE antibody in normal individuals.

J Biochem (Tokyo), 1997 Jan, 121(1), 128 - 37
Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78; Hagihara Y et al.; In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity . Purified Domain V was found to have the native disulfide bonds . It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea . Also, it bound liposomes containing CL . Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318 . We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78 . The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein . The stability of the nicked Domain V to urea was slightly lower than that of the intact protein . Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL . In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site . These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Can J Microbiol, 1997 Jan, 43(1), 35 - 44
Analysis of population structure of cactophilic yeast from the genus Pichia: P . cactophila and P . norvegensis; Ganter PF et al.; DNAs from 40 strains of Pichia cactophila and Pichia norvegensis, yeasts characteristic of cactus necroses, were compared using randomly amplified polymorphic DNA (RAPD) banding patterns and killer/sensitive phenotypes . Both species belong to the same species complex within the genus . The levels of between-strain RAPD variation were high in both species (higher in the automictic P . cactophila than in the heterothallic P . norvegensis), although there is little variation in physiological abilities within either species . Although each species was a separate lineage, RAPD analysis confirms that the species are related . Within each species, RAPD variation was related to the geographic origin of the strains . Pichia cactophila strains from southern Florida were more related to those from Antigua than to those from northern Florida . These results correlated well with the differences among killer/sensitive phenotypes of strains . Principal component analysis indicated that the phenotypes of each species differ . Here too, strains from southern Florida were more similar to those from Antigua than to those from northern Florida . Previous work had identified differences in the cactophilic yeast communities from southern and northern Florida, and these results indicate that the differences are detectable at the population levels as well.

Appl Microbiol Biotechnol, 1997 Jan, 47(1), 33 - 9
Production of synthetic spider dragline silk protein in Pichia pastoris; Fahnestock SR et al.; The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers . Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter . Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation . Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes . Silk-producing P . pastoris strains were stable without selection for at least 100 doublings.

Biochem J, 1997 Jan 1, 321 ( Pt 1), 21 - 8
Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization; Qin YM et al.; In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II . The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver {Malila, Siivari, Makela, Jalonen, Latipaa, Kunau and Hiltunen (1993) J . Biol . Chem . 268, 21578-21585} was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced . Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu) . The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence . The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation . Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters . The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes . They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Ann N Y Acad Sci, 1996 Dec 27, 804, 516 - 23
From expressed sequence tags to peroxisome biogenesis disorder genes; Dodt G et al.; Isolation of human disease genes is a challenging process and can often only be achieved by labor-intensive positional cloning techniques . Fortunately, there are alternative strategies for isolation of peroxisome biogenesis disorder genes . The first, functional complementation, was established as a viable approach by Fujiki and colleagues, who identified PAF-1, the first known peroxisome biogenesis disorder gene . The second strategy, computer-based homology probing, relies on (1) the fact that peroxisome assembly has been conserved throughout the evolution of eukaryotes, (2) knowledge of the amino acid sequences of an increasing number of yeast peroxisome assembly (PAS) genes, and (3) the existence of sequence data from large numbers of human genes . The recent development of the expressed sequence tag (EST) database (dbEST) is fulfilling the last of these requirements . We have applied the homology probing strategy in the search for candidate genes for the peroxisome biogenesis disorders by routinely screening the database of ESTs for genes with significant sequence similarity to yeast PAS genes . The validity of this approach is demonstrated by its use in identifying PXR1 as the human orthologue of the Pichia pastoris PAS8 gene and PXAAA1 as a human homologue of the Pichia pastoris PAS5 gene . Furthermore, detailed analysis of PXR1 has revealed that mutations in this gene are responsible for complementation group 2 of the peroxisome biogenesis disorders . The demonstration that human homologues of yeast PAS genes exist and that mutations in these genes cause peroxisome biogenesis disorders demonstrates that yeast pas mutants are accurate and useful models for the analysis of these diseases.

Anal Biochem, 1996 Dec 15, 243(2), 203 - 9
Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase; Lipniunas PH et al.; Oligomannosidic branch isomers (structures differing only in the branch location of a single residue) which are biosynthetic intermediates in yeast and higher eukaryotics have been separated using high-performance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon . A mixture of two Man6GlcNAc isomers from IgM, which was determined from 1H NMR analysis, was completely separated by PGC-HPLC . Mixtures of larger yeast oligomannosidic branch isomers were also chromatographically resolved using PGC-HPLC . Man10GlcNAc and Man11GlcNAc species from invertase expressed in Pichia pastoris showed three and five peak fractions, respectively, by PGC-HPLC in agreement with the number of isomeric forms from one- and two-dimensional 1H NMR analyses of the individual sized fractions (Trimble, R . B., Atkinson, P . H., Tschopp, J . R., Townsend, R . R., and Maley, F . (1991) J . Biol . Chem . 266, 22807-22817) . Selected peak fractions were further analyzed to confirm assignments using matrix- assisted laser-desorption mass spectrometry after digestion with an alpha(1 --> 2)-specific mannosidase (Aspergillus saitoi) . PGC-HPLC should prove invaluable for the preparation of singular oligosaccharides to define exoglycosidase and glycosyl transferase branch specificity and for preparing standards to develop more sensitive methods for structural elucidation of oligosaccharides.

FEBS Lett, 1996 Dec 2, 398(2-3), 231 - 4
Identification of the quinone cofactor in a lysyl oxidase from Pichia pastoris; Dove JE et al.; A copper amine oxidase from Pichia pastoris is the only known non-mammalian lysyl oxidase {Tur, S.S . and Lerch, K . (1988) FEBS Lett . 238, 74-76} . Recently, the cofactor in mammalian lysyl oxidase has been identified as a novel lysine tyrosylquinone moiety {Wang, S.X., Mure, M., Medzihradszky, K.F., Burlingame, A.L., Brown, D.E., Dooley, D.M., Smith, A.J., Kagan, H.M . and Klinman, J.P . (1996) Science 273, 1078-1084} . In order to identify the cofactor in P . pastoris lysyl oxidase, we have isolated the phenylhydrazone-derivative of the active-site peptide . This peptide has the active-site sequence conserved among topa quinone containing amine oxidases . The resonance Raman spectra of the phenylhydrazone derivatives of the enzyme, active-site peptide, and a topa quinone model compound are essentially identical . Collectively, these results establish that P . pastoris lysyl oxidase is a topa quinone enzyme.

Int Arch Allergy Immunol, 1996 Dec, 111(4), 385 - 95
Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit; De Vouge MW et al.; Alternaria alternata is recognized as an important source of fungal aeroallergens . Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000 . IgE antibodies to this protein are present in the sera of >90% of A . alternata-sensitive individuals . Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical . We now report the isolation of clones from an A . alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1 . One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp . An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins . The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A . alternata total RNA . The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals . The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions . Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions . Recombinant Alt a 1, like the natural allergen in A . alternata extracts, is also reactive with serum IgE from A . alternata-sensitive individuals.

Protein Expr Purif, 1996 Dec, 8(4), 476 - 82
Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris; Rosenfeld SA et al.; A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leech Hirudo medicinalis, was cloned and expressed in the methylotrophic yeast Pichia pastoris . The HIR gene was inserted into the P . pastoris pPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor signal sequence . A Tn903Kan(r) determinant and His4+ gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene . Following electroporation of the DNA into the P . pastoris strain GS115 (his-4), His+ transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418 . The resulting His+ G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium . Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression . One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter . Using a straightforward two-step chromatography procedure, the rHIR was purified to > 97% with a recovery yield of 63% . The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems . Based on these data, P . pastoris offers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.

Protein Expr Purif, 1996 Dec, 8(4), 456 - 62
Expression, purification, and characterization of recombinant alpha-N-acetylgalactosaminidase produced in the yeast Pichia pastoris; Zhu A et al.; alpha-N-Acetylgalactosaminidase (alpha NAGAL, EC 3.2.1.49) purified from chicken liver has been used in seroconversion of human erythrocytes . Blood group A, defined by the terminal alpha-linked N-acetylgalactosamine, can be cleaved in vitro by alpha NAGAL, resulting in the underlying penultimate blood group H (O) epitope structure . In order to produce sufficient quantities of recombinant alpha NAGAL (r alpha NAGAL) for such studies, we expressed the cDNA encoding chicken liver alpha NAGAL in Pichia pastoris, a methylotrophic yeast strain . The alpha NAGAL coding sequence was cloned into the EcoRI site of the vector pPIC 9 such that the protein was in the same reading frame as the secretion signal of yeast alpha-mating factor derived from the vector . After P . pastoris transformation, colonies were screened for high-level expression of r alpha NAGAL based on enzyme activity . As a result of methanol induction of high-density cell cultures in a fermentor, enzymatically active r alpha NAGAL was produced and secreted into the culture medium . The recombinant enzyme was purified over 150-fold by chromatography on a cation exchange column followed by an affinity column . Its homogeneity was confirmed by Coomassie blue-stained SDS-PAGE, Western blot, and N-terminal sequencing . The purified r alpha NAGAL has a molecular mass of approximately 50 kDa while its native counterpart has a molecular mass of 43 kDa . This discrepancy in size was eliminated by endoglycosidase treatment, suggesting that the recombinant protein was hyperglycosylated by the host P . pastoris cells . r alpha NAGAL was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability by comparing with alpha NAGAL purified from chicken liver . The data presented here suggest that by overexpressing r alpha NAGAL in P . pastoris and purifying with affinity chromatography one can readily obtain the quantity of enzyme needed for seroconversion studies.

J Immunol Methods, 1996 Nov 29, 199(1), 47 - 54
Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor; Vollmer P et al.; The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp 130 . In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp 130 . The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing . We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris . We obtained, however, no detectable expression and secretion of the recombinant protein . When we used only the coding sequence of the cytokine receptor domain of the sIL-6R for the construction of an expression plasmid, this truncated version of the sIL-6R accumulated in the supernatant to 1-5 mg/l . The protein was purified by a single affinity chromatography step using a monoclonal antibody directed against the human IL-6R . Following the same approach, we expressed a truncated splice variant of the sIL-6R . Both, the secreted truncated sIL-6R and the splice variant showed full agonistic biological activity on human hepatoma cells . The described expression strategy will be useful for large scale production of biologically active sIL-6R and might be adapted for the expression of other members of the hematopoietic cytokine receptor family.

J Biol Chem, 1996 Nov 8, 271(45), 28348 - 58
Cloning, expression, purification, and characterization of the human broad specificity lysosomal acid alpha-mannosidase; Liao YF et al.; We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA . This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells . The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from alpha-mannosidosis fibroblasts, and p-nitrophenyl-alpha-D-mannopyranoside substrates . In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, Km, and Vmax characteristic of the enzyme purified previously from human liver . The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing . No alpha-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme . Antiserum raised to the recombinant product of the 3-kb alpha-mannosidase cDNA immunoprecipitated lysosomal alpha-mannosidase activity from human fibroblast extracts . Northern blots identified a 3-kb RNA transcript in all human tissues tested, including alpha-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues . Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19 . Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal alpha-mannosidase (Nebes, V . L., and Schmidt, M . C . (1994) Biochem . Biophys . Res . Commun . 200, 239-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.

Curr Genet, 1996 Nov, 30(5), 417 - 22
Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae; Skory CD et al.; The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins . Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory . Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae . Expression was initially performed in E . coli using either the lacZ or tac promoter . This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented . Secretion and glycosylation of active beta-glucosidase was achieved with several different S . cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids . When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated . Expression in P . pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate . Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P . pastoris . However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.

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