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),