Pichia

Curr Opin Chem Biol, 2003 Feb, 7(1), 39 - 43
Protein expression systems for structural genomics and proteomics; Yokoyama S; One of the key steps of structural genomics and proteomics is high-throughput expression of many target proteins . Gene cloning, especially by ligation-independent cloning techniques, and recombinant protein expression using microbial hosts such as Escherichia coli and the yeast Pichia pastoris are well optimized and further robotized . Cell-free protein synthesis systems have been developed for large-scale production of protein samples for NMR (stable-isotope labeling) and X-ray crystallography (selenomethionine substitution) . Protein folding is still a major bottleneck in protein expression . Cell-based and cell-free methods for screening of suitable samples for structure determination have been developed for achieving a high success rate.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 154 - 60
{Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris}; Xiong AS et al.; Pichia pastoris has been developed to be a very efficient expression host for the heterogeneous proteins since its alcohol promoter was isolated and cloned, and its transformation technique was established . For further improving the secretion expression of heterogeneous proteins, in this research, the signal sequences were studied . At first, the Saccharomyces cerevisiae mating factor alpha prepro-leader sequence was synthesized using successive PCR and designated as MF4I . Then, ten different signal sequences were constructed by adding the N-terminal residues of Pichia pastoris Aox1 protein to the N-terminal of the MF4I . These ten signal sequences were used for directing phytase gene secretion in Pichia pastoris, the secretion of phytase were increased in Pichia pastoris strains containing new signal sequence . Among these strains, the phytase secretion was highest in strain contain signal sequence added with A, I, P three Aox1 N-terminal residues; the phytase secretion of Pichia pastoris was 90 mg/L in flake . The secretion was six-fold of that with original Saccharomyces cerevisiae mating factor alpha prepro-leader sequence . In addition, insert of ten residues E E A E A E A E P K can further increase the phytase secretion by 35%, the secretion reach 120 mg/L.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 127 - 32
{Enhancement of the production of SAM by overexpression of SAM synthetase in Pichia pastoris}; Yu ZL et al.; S-Adenosyl-L-methionine(SAM) is an important metabolic intermediate in the metabolic flux of sulphur . SAM is involved in three key metabolic pathways: transmethylation, transsulfuration and polyamine synthesis . As a potential therapeutic agent, SAM is being used as over the counter drug and nutrient supplement . An expression vector, harboring SAM synthetase 2 gene from S . cerevisiae and regulated by the glyceraldehyde-3-phosphate dehydrogenase gene promoter P(GAP), was transformed into GS115 strain of P . pastoris . Through zeocin resistance and expression screening, a recombinant strain was obtained that had high SAM yield and the fermentation conditions were optimized . The results showed that carbon source, nitrogen source, pH and dissolved oxygen had significant effects on the accumulation of SAM . The SAM production of the recombinant cells reached 2.49 g/L after fermentation for three days under the optimized conditions . The present studies show that fermentation of recombinant P . pastoris strain, expressing heterologous SAM synthetase gene, may be a promising approach for the production of SAM.

Acta Biochim Pol, 2002, 49(4), 959 - 68
The effect of vanadate on Pichia pastoris growth, protein kinase A activity and ribosomal protein phosphorylation; Frajnt M et al.; It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium . Moreover, four vanadate-resistant P . pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected . Growth of P . pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium . In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium . Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains . It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase . From the presented results it can be concluded that a protein kinase A signalling pathway(s) might be involved in the mechanism of P . pastoris vanadate resistance . The results also indicate a possible role of the 40 kDa protein in protection of P . pastoris against vanadate toxicity.

Biochim Biophys Acta, 2003 Jan 31, 1645(1), 1 - 5
Change in maltose- and soluble starch-hydrolyzing activities of chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae; Sugimoto M et al.; The chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes . The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium . The two chimeric M . javanicus alpha-glucosidases, of which the N- and C-terminal regions are substituted for those of A . oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M . javanicus alpha-glucosidase, respectively . The chimeric enzymes changed on the V(max) values for maltose significantly, whereas the K(m) values were similar to that of the native enzyme.

Ai Zheng, 2002 Nov, 21(11), 1197 - 202
{Inhibitory effects of recombinant human endostatin on growth and metastasis of lung adenocarcinoma LA795 in mice}; Xia H et al.; BACKGROUND & OBJECTIVE: Generally, growth and metastasis of tumor are critically dependent on angiogenesis . Endostatin can specifically inhibits tumor angiogenesis . The current study was designed to evaluate the inhibitory effect of recombinant human endostatin (rhES) secreted by pichia, pastoris, GS115 on the growth and metastasis of mice lung adenocarcinoma LA795 in mice T739 . METHODS: To select a strain that could highly express recombinant human endostatin, then induce the clone to express rhES by adding methanol . Purification of rhES was performed with heparin affinity chromatography . LA795 tumor cells were inoculated subcutaneously into the dorsa of T739 mice, and the mice were randomized into two groups . The first group was given rhES(20 mg/kg/d), and the second group was given equal volume of PBS, for 14 consecutive days . The volume of tumors were measured . And the tumor metastasis in the lungs of the mice was observed . RESULTS: The selected clone was induced to secrete enough soluble rhES . The purified protein could strongly inhibit growth and metastasis of mice lung adenocarcinoma LA795 in T739 mice (P < 0.001) . CONCLUSION: The rhES secreted by pichia, pastoris, GS115 has good biological activities and greatly inhibit growth and metastasis of mice lung adenocarcinoma LA795 in mice T739.

J Biol Chem, 2003 Apr 18, 278(16), 14249 - 56 Epub 2003 Jan 13.
Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein; Pons JL et al.; The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR . The (15)N-labeled protein was produced in Pichia pastoris . Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality . This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A . The structure is composed of five helices forming a right superhelix . The protein presents an inner cavity, which has been measured at 341 A(3) . All of the helices display hydrophobic side chains oriented toward the cavity . The phospholipid is found in this cavity . Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein . The superhelix structure of the protein is coiled around the fatty acid chain . The overall structure shows similarities with ns-LTP1 . Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix . The lipid is orthogonal to the orientation observed in ns-LTP1 . The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues.

Indian J Med Res, 2002 Jul, 116, 5 - 12
Change in distribution & antifungal susceptibility of Candida species isolated from candidaemia cases in a tertiary care centre during 1996-2000; Chakrabarti A et al.; BACKGROUND & OBJECTIVES: As a marked increase in the number of patients with candidaemia was reported in the first half (1991-1995) of the last decade at the Postgraduate Institute of Medical Education & Research, Chandigarh, India, the present study was aimed at determining further change if any, in the incidence and distribution of Candida species and their antifungal resistance pattern during the second half (1996-2000) of the same decade . METHODS: The patients with candidaemia were studied to determine the frequency of candidaemia and Candida species isolated during 1996-2000 . One hundred Candida strains other than Pichia anomala (C . pelliculosa) were randomly selected from those isolates to evaluate antifungal susceptibility pattern against amphotericin B, 5-fluorocytosine, ketoconazole, fluconazole and itraconazole . The results were compared with our previous study . RESULTS: An increase in the number of patients with candidaemia was observed during 1996 (538) and 1997 (421) compared to 1998-2000 due to P . anomala outbreak . With the control of the outbreak, a substantial decrease in the incidence of candidaemia was observed from 1998 (251 in 1998, 122 in 1999 and 165 in 2000) . A higher isolation of non-C . albicans Candida species (89.8%) was observed, with C . tropicalis being the most common (541, 36.1%) agent . No major change in the isolation rate of other non-C . albicans Candida species (C . guilliermondii, C . krusei, C . glabrata and C . parapsilosis) was observed . An emergence of resistance to amphotericin B in 15.4 per cent C . albicans, 8.1 per cent C . tropicalis and 33.3 per cent C . krusei strains was observed . An increase in resistance to ketoconazole (from 0% to 13%) and 5-fluorocytosine (from 1% to 8%) and a decrease to fluconazole (from 13% to 6%) were observed . Resistance to itraconazole was observed in 17 per cent of Candida strains by broth macro-dilution method . INTERPRETATION & CONCLUSION: A change in the isolation of Candida species was observed i.e . in the incidence and isolation of non-C . albicans Candida species . Emergence of resistance to amphotericin B and increase of resistance to most other antifungals are cause for concern.

J Biol Chem, 2003 Mar 7, 278(10), 8257 - 60 Epub 2003 Jan 03.
Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase . Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms; Rozanov DV et al.; An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies . A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces . The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway . To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris . Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography . Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis . This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites . Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112) . The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)) . These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation . Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.

Appl Environ Microbiol, 2003 Jan, 69(1), 495 - 503
Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity; Jin YS et al.; D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts . However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S . cerevisiae on xylose . We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S . cerevisiae . The S . cerevisiae genetic background included single integrated copies of P . stipitis XYL1 and XYL2 driven by the S . cerevisiae TDH1 promoter . Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P . stipitis promoter were introduced into the recombinant S . cerevisiae . In vitro enzymatic activity of XK increased with copy number and promoter strength . Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells . Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity . Uncontrolled XK expression in recombinant S . cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S . cerevisiae tps1 mutant during glucose metabolism . To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose . The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity . It converted xylose into ethanol efficiently.

Clin Chem, 2003 Jan, 49(1), 77 - 86
Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay; Kapadia C et al.; BACKGROUND: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids . METHODS: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system . The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies . A sandwich-type immunoassay was developed with these antibodies . The assay was used to measure hK13 in various biological fluids and tissue extracts . Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections . RESULTS: The hK13 immunoassay had a detection limit of 0.05 micro g/L and showed no cross-reactivity with homologous kallikreins . The assay was linear at 0-20 micro g/L, and within-and between-run CVs were <10% (n = 12) . hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate . hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues . hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate . hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues . Recovery of active enzyme added to milk or amniotic fluid was 70-98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors . In fluids and tissue extracts, hK13 was found in its free (approximately 30 kDa) form . CONCLUSIONS: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2297 - 305
Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening; Kawakami A et al.; We isolated two cDNAs of winter wheat (Triticum aestivum L.), designated wft1 and wft2, which encoded sucrose:fructan 6-fructosyltransferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), respectively, which are involved in the synthesis of fructan in wheat . wft1 and wft2 were cloned by screening of a cDNA library with probed-cDNA fragments corresponding to plant fructosyltransferase and invertase . The identity of the clones was verified by functional characterization of recombinant proteins expressed in methylotrophic yeast, Pichiapastoris . Northern blotting showed that the level of wft2 transcripts increased from autumn to early winter in the crown tissues of all field-grown wheat cultivars examined . Higher levels of wft1 and wft2 transcripts were found in leaf tissues of snow mold-resistant cultivars, which accumulated more fructan than other cultivars . Our results showed that Wft1 and Wft2 were important in fructan accumulation during cold hardening of winter wheat.

J Gen Appl Microbiol, 1999 Oct, 45(5), 239 - 246
A phylogenetic study of ubiquinone Q-8 species of the genera Candida, Pichia, and Citeromyces based on 18S ribosomal DNA sequence divergence; Suzuki M et al.; To clarify phylogenetic relationships among species of the anamorphic ascomycetous genus Candida with ubiquinone Q-8, we determined complete sequences of 18S ribosomal RNA genes (18S rDNAs) from the type strains of 20 species of the genus Candida and 7 of the teleomorphic ascomycetous genera Pichia and Citeromyces, which have Q-8 as the major ubiquinone . Q-8-forming Candida species were divided into six clusters and were phylogenetically distant from a group of Candida species that included the type species of the genus . One Q-8-forming species from each of the genera Pichia, Citeromyces, or Clavispora was included in five of six clusters . Cluster 1 comprised C . ishiwadae, C . ernobii, C . karawaiewii, C . anatomiae, C . populi, and Pichia holstii . Cluster 2 comprised C . globosa and its teleomorph, Citeromyces matritensis . Cluster 3 comprised C . molischiana and Pichia capsulata . Cluster 4 comprised C . silvanorum, C . sequanensis, C . fennica, C . entomophila, C . homilentoma, C . rhagii, C . gotoi, and Pichia burtonii . Cluster 5 comprised C . fructus, C . musae, and C . lusitaniae (anamorph of Clavispora lusitaniae) . Cluster 6 comprised C . stellata, C . lactiscondensi, C . galacta, and C . incommunis and was a heterogeneous group with large interspecific divergence . Pichia pastoris was quite divergent and phylogenetically distant from other Pichia species examined . Pichia methanolica and its synonym, P . cellobiosa, which have both Q-7 and Q-8 as major ubiquinones, were closely associated with Q-7-forming Williopsis salicorniae . Based on this comparative analysis of 18S rDNA sequences, it is evident that Q-8 Candida species and Q-8 Pichia species are polyphyletic.

J Gen Appl Microbiol, 1999 Oct, 45(5), 229 - 238
A phylogenetic study on galactose-containing Candida species based on 18S ribosomal DNA sequences; Suzuki M et al.; Phylogenetic relationships of 33 Candida species containing galactose in the cells were investigated by using 18S ribosomal DNA sequence analysis . Galactose-containing Candida species and galactose-containing species from nine ascomycetous genera were a heterogeneous assemblage . They were divided into three clusters (II, III, and IV) which were phylogenetically distant from cluster I, comprising 9 galactose-lacking Candida species, C . glabrata, C . holmii, C . krusei, C . tropicalis (the type species of Candida), C . albicans, C . viswanathii, C . maltosa, C . parapsilosis, C . guilliermondii, and C . lusitaniae, and 17 related ascomycetous yeasts . These three clusters were also phylogenetically distant from Schizosaccharomyces pombe, which contains galactomannan in its cell wall . Cluster II comprised C . magnoliae, C . vaccinii, C . apis, C . gropengiesseri, C . etchellsii, C . floricola, C . lactiscondensi, Wickerhamiella domercqiae, C . versatilis, C . azyma, C . vanderwaltii, C . pararugosa, C . sorbophila, C . spandovensis, C . galacta, C . ingens, C . incommunis, Yarrowia lipolytica, Galactomyces geotrichum, and Dipodascus albidus . Cluster III comprised C . tepae, C . antillancae and its synonym C . bondarzewiae, C . ancudensis, C . petrohuensis, C . santjacobensis, C . ciferrii (anamorph of Stephanoascus ciferrii), Arxula terrestris, C . castrensis, C . valdiviana, C . paludigena, C . blankii, C . salmanticensis, C . auringiensis, C . bertae, and its synonym C . bertae var . chiloensis, C . edax (anamorph of Stephanoascus smithiae), Arxula adeninivorans, and C . steatolytica (synonym of Zygoascus hellenicus) . Cluster IV comprised C . cantarellii, C . vinaria, Dipodascopsis uninucleata, and Lipomyces lipofer . Two galactose-lacking and Q-8-forming species, C . stellata and Pichia pastoris, and 5 galactose-lacking and Q-9-forming species, C . apicola, C . bombi, C . bombicola, C . geochares, and C . insectalens, were included in Cluster II . Two galactose-lacking and Q-9-forming species, C . drimydis and C . chiropterorum, were included in Cluster III.

Genetika, 2002 Nov, 38(11), 1451 - 62
{Formation of ARS-independent miniplasmids upon transformation of yeast Pichia methanolica with DNA molecules containing "transforming" and "nontransforming" genes}; Tarutina MG et al.; In the study of transforming DNA molecules in cells of the methylotrophic yeast Pichia methanolica, it was found that the efficiency of transformation and the possibility of autonomous replication of hybrid plasmids and miniplasmids consisting of only transforming gene (Trg) sequences do not depend on the presence of ARS sequences with the canonical structure, such as 2 microns DNA in the yeast Saccharomyces cerevisiae . Data supported a suggestion about the existence of the relationship between gene transforming activity, its replicative ability, and transcription . The efficiency of vector activity of Trg is not sufficient to provide the transformation of other sequences of transforming DNA (tDNA) . The loss of nonselected sequences of tDNA observed upon transformation is the result of rearrangements and deletions and is associated with the activity of cellular systems involved in nucleic acid metabolism . Miniplasmids similar in structure were shown to differ in their stability in independent transformants . The rate of loss of autonomously replicating plasmids strongly varied (from 1 to 99%) under nonselective conditions and nearly did not depend on plasmid structure.

Zhonghua Nan Ke Xue, 2002, 8(4), 292 - 4, 298
{Advances in eukaryotic expression systems}; Gao Y et al.; The increasing popularity of eukaryotic expression systems can be attributed to their capability of performing many post-translational modifications . At present, There mainly are three expression systems including yeast expression system, insect cell expression system and mammalian cell expression system . The methylotropic yeast Pichia Pastoris usually utilizes alcohol oxidase promoter to drive the expression of foreign gene . Recently, a continuous fermentation has been developed in Pichia Pastoris with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter . The baculovirus-mediated insect cell expression system is considered to be safe, powerful, but cell-lytic . Baculovirus-S2 system uses the popular and genetically well understood Drosphila S2 cells which do not appear to be lysed after infection . In mammalian cell expression system, recombinant adenovirus are attracting a great deal of attention as a highly efficient gene transfer vehicle . The frequency of Ad vector rescue by homologous recombination in E . coli and Cre-mediated site-specific recombination is significantly higher than by homologous recombination in vivo . Tetracycline-regulatable system is a widely used mammalian cell inducible expression system due to its high efficiency and stringency.

Yeast, 2003 Jan 15, 20(1), 39 - 51
Analysis of the hypoxia-induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast; Passoth V et al.; We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP . This system was used to analyse hypoxia-dependent PsADH2 regulation . Reporter gene activity was only found under oxygen limitation on a fermentable carbon source . The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae . Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction . This motif was different from the known hypoxia response elements of S . cerevisiae but showed some similarity to the mammalian HIF-1 binding site . Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation . Similar to the S . cerevisiae heme sensor system, the promoter was induced by Co(2+) . Cyanide was not able to mimic the effect of oxygen limitation . The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co(2+) but not by cyanide . Thus, the very first promoter analysis in P . stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast .

Biochemistry, 2002 Dec 24, 41(51), 15195 - 202
Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography; Kawano S et al.; Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid . We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography . The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model . The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds . The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89 . In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79 . Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.

Clin Chim Acta, 2003 Jan, 327(1-2), 171 - 9
Expression of TIMP-1 in Pichia pastoris . Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library; Wozniak G et al.; To quantitate tissue inhibitor of metalloproteinase (TIMP)-1 in biological samples, a strategy for isolation of monoclonal antibodies was applied that employs a phage-displayed single-chain Fv (scFv) . In order to obtain sufficient amounts of TIMP-1 to use as an antigen, high-level expression in Pichia pastoris was achieved under the control of the AOX-1 promotor . Purified protein antigen was then used for panning to achieve enrichment of specific phage from naive scFv library . In five subsequent panning rounds, antibody fragments that display specificity to TIMP-1 were selected . Regions encoding scFv were subcloned into a vector allowing production of scFv-alkaline phosphatase (AP) fusion proteins . Two such conjugates displaying dose-dependent reactivity with TIMP-1 were isolated and characterised, providing the basis for the construction of a TIMP-1 quantitation assay based entirely on recombinant proteins.

Microbiology, 2002 Dec, 148(Pt 12), 4003 - 14
Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host; Soden DM et al.; The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter . The native Ple . sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic . pastoris . The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization . The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1 . The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38 . The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively . With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5 . The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 115 - 24
Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization; Ogawa M et al.; We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B . stearothermophilys neutral protease for improving thermostability {J . Agric . Food Chem., 46 (1998) 1655} . The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity . The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor . Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris . Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin . Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity . Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity . The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.

Curr Genet, 2002 Nov, 42(2), 114 - 22 Epub 2002 Nov 21.
Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T . harzianum T-203); Viterbo A et al.; The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups . CHIT37 from T . harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level . Northern analysis showed that chit36Y from T . asperellum is regulated both by glucose and nitrogen levels . Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene . The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants . Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression . The R . solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (<12 kDa) . CHIT36 recombinant protein from the yeast Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 291 - 8
Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica; Mayson BE et al.; The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter . Methanol concentrations during the induction phase directly affect cellular growth and protein yield . Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P . methanolica expressing the human transferrin N-lobe protein . The PMAD18 P . methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype . Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration . Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time .

J Gen Appl Microbiol, 2002 Feb, 48(1), 55 - 65
A phylogenetic study of ubiquinone-7 species of the genus Candida based on 18S ribosomal DNA sequence divergence; Suzuki M et al.; To clarify phylogenetic relationships among ubiquinone 7 (Q7)-forming species of the genus Candida, we analyzed the nearly complete sequences of 18S ribosomal RNA genes (18S rDNAs) from fifty strains (including 46 type strains) of Candida species, and from 8 type strains of species/varieties of the genera Issatchenkia, Pichia and Saturnispora . Q7-forming Candida species were divided into three major groups (Group I, II, and III) and were phylogenetically distant from a group that includes the type species of the genus Candida . Group I included four clusters with basal branches that were weakly supported . The first cluster comprised C . vartiovaarae, C . maritima, C . utilis, C . freyschussii, C . odintsovae, C . melinii, C . quercuum, Williopsis saturnus var . saturnus, and W . mucosa . The second cluster comprised C . norvegica, C . montana, C . stellimalicola, C . solani, C . berthetii, and C . dendrica . Williopsis pratensis, W . californica, Pichia opuntiae and 2 related species, P . amethionina (two varieties), and P . caribaea were also included in this cluster . The third cluster comprised C . pelliculosa (anamorph of P . anomala), C . nitrativorans, and C . silvicultrix . The fourth cluster comprised C . wickerhamii and C . peltata, which were placed in the P . holstii - C . ernobii clade with Q8-containing species . Group II comprised C . pignaliae, C . nemodendra, C . methanolovescens, C . maris, C . sonorensis, C . pini, C . llanquihuensis, C . cariosilignicola, C . ovalis, C . succiphila (including its two synonyms), C . methanosorbosa, C . nitratophila, C . nanaspora, C . boidinii (including its two synonyms), W . salicorniae, and P . methanolica . Group III was composed of four clusters with strong bootstrap support . The first cluster comprised C . valida (anamorph of P . membranifaciens), C . ethanolica, C . pseudolambica, C . citrea, C . inconspicua, C . norvegensis, C . rugopelliculosa, and C . lambica . Three species and two varieties of the genus Issatchenkia were also included in this cluster . The second cluster comprised C . diversa, C . silvae, 4 Saturnispora species, and P . besseyi . The third comprised C . sorboxylosa, and the fourth comprised C . vini . Based on this 18S rDNA sequence analysis, it is evident that Q7-forming Candida species and the genera Pichia and Williopsis are polyphyletic . The genus Issatchenkia is suggested to be congeneric with the genus Pichia . The genus Saturnispora is phylogenetically definable.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1392 - 9
Design of methanol Feed control in Pichia pastoris fermentations based upon a growth model; Zhang W et al.; The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut(+) (methanol utilization plus) strain . Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity . An accurate methanol control is required in the methanol fed-batch phase (induction phase) in the fermentation . A simple "on-off" control strategy is inadequate for precise control of methanol concentrations in the fermentor . In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model . The closed-loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor . First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters K(c) (controller gain), tau(I) (controller integral time constant), and tau(D) (controller derivative time constant) . Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed-batch phase . Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings . The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved . The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.

Reprod Fertil Dev, 2002, 14(5-6), 327 - 32
Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris; Fidler AE et al.; The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians . To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris . The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCE The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro . Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF . This source of biologically active marsupial SCF may prove useful in future studies of marsupial development.

Protein Expr Purif, 2002 Dec, 26(3), 496 - 505
Expression of CB2 cannabinoid receptor in Pichia pastoris; Feng W et al.; To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris . The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P . pastoris alcohol oxidase 1 gene . A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification . In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins . Radioligand binding assays demonstrated that the CB2 receptors expressed in P . pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems . Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin . MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor . ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor . In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P . pastoris for purification.

Protein Expr Purif, 2002 Dec, 26(3), 406 - 15
Expression and characterization of a synthetic protein C activator in Pichia pastoris; Kunes YZ et al.; Protein C activators are proteases that activate protein C in the mammalian coagulation system . A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway . We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence . This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence . A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth . A two-step purification scheme was devised to purify ILPCA to approximately 80% purity . The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa . Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1) . In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.

Protein Expr Purif, 2002 Dec, 26(3), 394 - 405
Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris; Baumgartner P et al.; The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris . To optimise yields of PHA-E, transformants of P . pastoris were selected for high-level production of the recombinant protein . A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported . PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography . The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa . Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein . The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E . The data presented here suggest that, using P . pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.

Protein Expr Purif, 2002 Dec, 26(3), 386 - 93
Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent; Brucato CL et al.; Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains . It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation . The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies . To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris . Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity . A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.

Protein Expr Purif, 2002 Dec, 26(3), 349 - 56
Purification of full-length recombinant human and rat type 1 11beta-hydroxysteroid dehydrogenases with retained oxidoreductase activities; Nobel CS et al.; 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum . This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase . Previous attempts to purify the native enzyme have yielded a protein without reductase activity . To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11beta-HSD1 with retention of their natural oxidoreductase activities . This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography . Both enzymes were 90-95% pure and exhibited dehydrogenase and reductase activities with K(M) values in agreement with those reported in the literature.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 25, 782(1-2), 307 - 16
Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions; Weinberger SR et al.; Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed . When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species . The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations . In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution . Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS) . The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces . Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions . The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures . Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode . Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2177 - 9 Epub 2002 Nov 23.
Crystallization of Pichia pastoris lysyl oxidase; Lee M et al.; A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms . PPLO is a glycoprotein . The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa) . Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution . Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution . Native data have been recorded from each crystal form at 100 K using synchrotron radiation . A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit.

Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 241 - 6
Heterologous expression of cry2 gene from a local strain of Bacillus thuringiensis isolated in Nigeria; Ogunjimi AA et al.; A cry2 gene encoding a larvicidal crystal protein was isolated from a strain of Bacillus thuringiensis found in soil samples in Nigeria . This gene was cloned into plasmid pUC19 and subcloned into both pBluescript (sk(+/-)) and pPICZ alpha B placed under a T7/AOXI (alcohol oxidase I) promoter respectively and transformed into Escherichia coli and Pichia pastoris . Clones were induced for expression, and the cellular proteins extracted and analysed by SDS/PAGE . Integration of an insert into the yeast chromosome was confirmed by PCR amplification using AOXI primers designed to monitor the intactness of the insertion into the chromosome . The expression cassettes constructed were both expressed in E . coli strain (XL1-blue) and P . pastoris (SMD1168) respectively . An approximately 70 kDa recombinant toxin was obtained both in P . pastoris and E . coli in different quantities . Expression was confirmed by Northern-blot analysis of 2.0 kb transcripts, obtained from clones induced for RNA transcripts, which hybridized with a {(32)P}dCTP-labelled probe prepared from a 641 bp fragment of restriction-endonuclease- Hae II-digested PCR product of the cry2 gene.

Int Arch Allergy Immunol, 2002 Nov, 129(3), 212 - 8
Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis; Labrada M et al.; BACKGROUND: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients . This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated . Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins . METHODS: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris . Spleen cells were fused with myeloma cells using polyethylene glycol . Hybridoma screening was performed using a direct ELISA with recombinant allergen . MAb specificity to rBlo t 13 was tested by immunoblotting . Topography of binding sites and binding of MAb to native allergen was studied by ELISA . Reactivity of MAb against allergenic extract of B . tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition . In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P . pastoris expression was compared . RESULTS: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated . These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE . In ELISA, the binding of 5G3 MAb to B . tropicalis and D . siboney extracts was inhibited by rBlo t 13 . Both MAbs showed the highest reactivity when the allergen was expressed in P . pastoris . CONCLUSION: Two MAbs specific for Blo t 13 were obtained . These MAbs recognized the same or close epitopes on the rBlo t 13 molecule . The occurrence of homologous allergens to Blo t 13 in D . siboney is suggested by the ELISA inhibition assay .

Infect Immun, 2002 Dec, 70(12), 6961 - 7
Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria; Stowers AW et al.; A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens . To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate . Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy . We evaluated the efficacy of a form of P . falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P . falciparum parasites . Significant protection from this otherwise lethal challenge with P . falciparum was observed . Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized . The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial . The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.

Infect Immun, 2002 Dec, 70(12), 6948 - 60
In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response; Kennedy MC et al.; Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate . While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P . falciparum have been described . The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P . falciparum strains is unknown . We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies . Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P . falciparum clones . Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity . Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections . However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure . Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.

Biol Chem, 2002 Jul-Aug, 383(7-8), 1125 - 31
Cold-adapted and mesophilic brachyurins; Gudmundsdottir A; Two different types of brachyurins, termed I and II, have been described in the literature . Within type I there are two subtypes, Ia and Ib . The prototype for the type I brachyurins is Fiddler crab collagenase I . Its cold-adapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics . Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen . The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin . A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical . As a cold-adapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I . It is also more sensitive to thermal inactivation and autolysis . Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I . Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes . Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin . Substitution of a highly exposed residue in the 'autolysis loop' of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.

Biochem J, 2003 Mar 1, 370(Pt 2), 417 - 27
A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition; Crepin VF et al.; Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall . The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties . We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates . A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris . The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no . AJ293029) . The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate) . The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action.

Biotechnol Bioeng, 2003 Jan 5, 81(1), 27 - 32
Biooxidation of n-hexanol by alcohol oxidase and catalase in biphasic and micellar systems without solvent; Karra-Chaabouni M et al.; Alcohol oxidase from Pichia pastoris together with catalase from bovine liver was used to oxidize n-hexanol to hexanal . For this purpose, an aqueous buffer solution was mixed with large amounts of hexanol by simple agitation, yielding a biphasic system, or by adding the nonionic surfactant Brij 35 . Initial velocities and reaction yields after 24 h were measured as a function of various parameters such as the amounts of enzymes, hexanol, or surfactant . High enzymatic activity was determined for hexanol concentrations of between 20 mass% and 80 mass% without using any additional organic solvent . The homogenization of the biphasic systems with the help of Brij 35 did not yield a significant improvement of the bioconversion, which would justify the use of surfactants .

J Mol Biol, 2002 Nov 15, 324(1), 165 - 75
The disulphide mapping, folding and characterisation of recombinant Ber e 1, an allergenic protein, and SFA8, two sulphur-rich 2S plant albumins; Alcocer MJ et al.; We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris . We show that both proteins were secreted at high levels and that the purified proteins were properly folded . We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity . The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family . A model three-dimensional structure of the allergen was generated . During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation . The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 725 - 30
{High expression of a heat-stable phytase in Pichia pastoris}; Peng RH et al.; It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding . The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris . Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical . After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell . The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis . After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid . The activity of phytase was 130 000 u per microlitre fluid . The thermostable phytase remained 40% active after exposure at 90 degrees for 80 min.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 697 - 702
Increasing bioactivity of Flt3 ligand by fusing two identical soluble domains; Lu CM et al.; Flt3 ligand (FL) is a hematopoietic growth factor, initiating its in tracellular signaling cascade by binding to counterpart receptor and driving receptor dimerization . The native form of soluble FL in vivo is mainly monomeric . In this study, we constructed a rFL-FL fusion protein cDNA by linking two copies of cDNA encoding the soluble domain of FL in tandem and expressed it in Pichia pastoris . On SDS-polyacrylamide gel electrophoresis, the rFL-FL fusion protein showed a molecular weight of 43 kD, agreeing well with the predicted value . The 43 kD protein was further confirmed by Western blot using polyclonal rabbit anti-human FL antibody . The rFL-FL fusion protein exhibited about 10-fold increment in its activity on colony formation of bone marrow progenitor cells . RFL-FL fusion protein also exerted more potent effect than monomeric FL on extending the survival of starving Raji cells.

FEBS Lett, 2002 Nov 6, 531(2), 265 - 72
Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity; Takai T et al.; Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1 . The active recombinant variants will be the basis for various future studies . The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity . Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested.

Chin Med J (Engl), 2002 Sep, 115(9), 1352 - 7
Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth; Wang Y et al.; OBJECTIVES: Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro . We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro . METHODS: cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method . After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system . The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening . Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation . His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free . His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418 . PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome . Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol . The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth . RESULTS: In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data . The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K . SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature . The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells . CONCLUSION: Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.

Protein Expr Purif, 2002 Nov, 26(2), 290 - 300
High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris; Reddy ST et al.; Mannose 6-phosphate receptors (MPRs) form essential components of the lysosomal enzyme targeting system by binding newly synthesized acid hydrolases with high (nM) affinity . We report the use of Pichia pastoris as a host to efficiently express the extracytoplasmic ligand-binding domain of the cation-dependent mannose 6-phosphate receptor . A truncated and glycosylation-deficient form of the receptor AF-Asn(81)/Stop(155) was secreted into the culture medium, yielding approximately 28mg/L after purification, which is an improvement of 10-100-fold compared to expression in baculovirus-infected insect cells and mammalian cells, respectively . Enzymatic deglycosylation indicated high-mannose sugars at the single potential glycosylation site of Asn 81 . The extent and heterogeneity of N-glycans were revealed by applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . In the case of AF-Asn(81)/Stop(155), the majority (75%) of the oligosaccharides contained chain lengths of Man(8-10)GlcNAc(2) while Man(11-12)GlcNAc(2) comprised the remaining (25%) N-linked sugars . A comparative MALDI-TOF spectra of Asn(81)/Stop(155) purified from insect cells indicated that Man(2-3)GlcNAc(2) and GlcNAcMan(2-3)GlcNAc(2) share the oligosaccharide pool . The receptor isolated from yeast was functional with respect to ligand binding and acid-dependent dissociation properties, as determined by pentamannosyl phosphate-agarose affinity chromatography . In addition, the protein was biochemically and functionally similar to Asn(81)/Stop(155) expressed in insect cells concerning its oligomeric state and binding affinity to the lysosomal enzyme, beta-glucuronidase (K(d)=1.4nM) . These results demonstrate that P . pastoris is a convenient system for the production of large quantities of functional recombinant MPRs suitable for structure-function studies.

Protein Expr Purif, 2002 Nov, 26(2), 202 - 10
Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification; Paus EJ et al.; Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies . The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter . Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp . These were then integrated at the HIS locus of P . pastoris GS115 (his4) . Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s) . Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP . Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW . Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86% . Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized . The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

Photochem Photobiol, 2002 Oct, 76(4), 457 - 61
Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris; Jorissen HJ et al.; The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris . Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively) . Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore . The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min . Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved . The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.

Yeast, 2002 Nov, 19(15), 1365 - 72
Cation/H+ antiporters mediate potassium and sodium fluxes in Pichia sorbitophila . Cloning of the PsNHA1 and PsNHA2 genes and expression in Saccharomyces cerevisiae; Banuelos MA et al.; Pichia sorbitophila grows rapidly in the presence of very high NaCl concentrations . Under these conditions, even when the K(+) concentration is low, P . sorbitophila cells can maintain low Na(+) and high K(+) contents . This remarkable capacity of P . sorbitophila fails when the external pH is not acidic . This indicates that Na(+) efflux is mediated by an electroneutral Na(+)/H(+) antiporter . We have cloned and sequenced two genes designated as PsNHA1 and PsNHA2, which probably encode two antiporters of this type . The genes present high similarity with the corresponding genes from other yeasts . The heterologous expression of PsNHA1 or PsNHA2 in a Saccharomyces cerevisiae mutant lacking the Na(+) efflux systems and sensitive to high concentrations of Na(+) and K(+) rescued the tolerance and the ability to extrude both cations . The Accession Nos of the sequenced DNA fragments are: PsNHA1, AJ496431; PsNHA2, AJ496432 . (TC 2.A.36)

Appl Biochem Biotechnol, 2002 Jul-Dec, 102-103(1-6), 367 - 79
Biosynthetic activity of recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II; Bhatia Y et al.; The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii beta-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described . With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively . In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively . The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides . In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained . The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10% . Detailed kinetic analysis indicated a significant (about twofold) increase in Vmax/KM of synthetic reactions in the presence of DMSO . A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose.

Biosens Bioelectron, 2002 Dec, 17(11-12), 983 - 91
Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor; von Tiedemann B et al.; Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis . It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins . The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family . Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding . The ligand-binding site of the receptor is localized on extracellular Ig-like domains . The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore) . Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods . Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6 . Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e . sample volume . This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain.

Eur J Biochem, 2002 Nov, 269(21), 5295 - 302
Brassica napus soluble epoxide hydrolase (BNSEH1); Bellevik S et al.; Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses . We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique . The cDNA encodes a soluble protein containing 318 amino acid residues . The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants . A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris . The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana . BNSEH1 was shown to be a monomer by gel filtration analysis . The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1 . The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis . The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop.

Biochem J, 2003 Feb 1, 369(Pt 3), 593 - 601
The C-terminal segment of the 1,3-beta-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization; Palomares O et al.; Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses . Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries . Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris . The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells . The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC . Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure . Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48 . The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen . These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy . Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.

Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 393 - 6
Expression and purification of human endostatin in Pichia pastoris and its inhibition on the growth of mouse pulmonary adenocarcinoma cell line LA795; Xia H et al.; OBJECTIVE: To obtain yeast strain Pichia pastoris that highly expresses human endostatin by gene recombination technology, and to evaluate the inhibitory effect of recombinant human endostatin (rhES) on the growth of mouse pulmonary adenocarcinoma cell line LA795 . METHODS: The gene coding for human endostatin was cloned into the genome of Pichia pastoris through LiCl transformation method, and the clones with high soluble rhES expression were selected . Purification of rhES was performed with heparin affinity chromatography . Effects of rhES on bFGF-induced proliferation of human endothelial cell line ECV-304 cells were observed . T739 mice with subcutaneous inoculation of LA795 cells were treated with injections of either rhES or PBS for 14 consecutive days, and the volume of the tumors were measured . RESULTS: Clones with high rhES expression were obtained and purified rhES potently inhibited the proliferation of ECV-304 cells and the growth of LA795 cells in T739 mice . CONCLUSION: rhES produced by Pichia pastoris possesses good biological activities and conspicuously inhibits the growth of LA795 cells.

Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 60 - 6 Epub 2002 Aug 29.
Study of two-stage processes for the microbial production of 1,3-propanediol from glucose; Hartlep M et al.; The microbial production of 1,3-propanediol (1,3-PD) from glucose was studied in a two-stage fermentation process on a laboratory scale . In the first stage, glucose was converted to glycerol either by the osmotolerant yeast Pichia farinosa or by a recombinant Escherichia coli strain . In the second stage, glycerol in the broth from the first stage was converted to 1,3-PD by Klebsiella pneumoniae . The culture broth from P . farinosa was shown to contain toxic metabolites that strongly impair the growth of K . pneumoniae and the formation of 1,3-PD . Recombinant E . coli is more suitable than P . farinosa for producing glycerol in the first stage . The fermentation pattern from glycerol can be significantly altered by the presence of acetate, leading to a significant reduction of PD yield in the second stage . However, in the recombinant E . coli culture acetate formation can be prevented by fed-batch cultivation under limiting glucose supply, resulting in an effective production of 1,3-PD in the second stage with a productivity of 2.0 g l(-1) h(-1) and a high yield (0.53 g/g) close to that of glycerol fermentation in a synthetic medium . The overall 1,3-PD yield from glucose in the two stage-process with E . coli and K . pneumoniae reached 0.17 g/g.

Heart Vessels, 2002 Sep, 16(6), 260 - 3
Native valve endocarditis due to Pichia ohmeri; Joao I et al.; Candida species can cause clinical manifestations in various organs of the cardiovascular system, i.e., the pericardium, myocardium, and endocardium, with endocarditis being the best-known clinical entity . Endocarditis is seen primarily in intravenous drug users and in individuals with damaged native valves, especially in congenital heart disease or rheumatic valvular diseases, and in prosthetic heart valves . The authors present a case of Pichia ohmeri endocarditis in an intravenous drug user, with an unusual presentation form . This is a case of a 42-year-old man, an intravenous heroin user, who was admitted to our Vascular Surgery Department because of fever and acute serious ischemia of the left inferior limb . He presented with fever (39 degrees C), a pale and cold left limb, absence of the left popliteal pulse, and a pansystolic murmur at the cardiac apex . The transthoracic echocardiogram showed a large vegetation on the anterior leaflet of the mitral valve and severe mitral regurgitation with good left ventricular systolic function . Empirical antibiotic therapy was started . Six days after admission, embolectomy was performed with partial clinical recovery . Three blood cultures and the embolus showed a teleomorphic form of Candida guilliermondii - Pichia ohmeri . Therapy with intravenous liposomal amphotericin B, fluocitosin, imipenem, and aztreonam was started . Two weeks later, his clinical condition deteriorated with acute heart failure refractory to medical therapy, mandating mechanical ventilation and high-dose vasopressor and inotropic amine support . He underwent urgent mitral valve replacement with a biologic prosthetic valve . Rapid stabilization of the cardiac status occurred, but ischemic limb lesions required further vascular interventions.

J Biol Chem, 2002 Dec 6, 277(49), 47205 - 12 Epub 2002 Oct 08.
Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region; Hancock MK et al.; The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands . Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region . Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes . Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.

Nat Cell Biol, 2002 Oct, 4(10), 750 - 6
De novo formation of transitional ER sites and Golgi structures in Pichia pastoris; Bevis BJ et al.; Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER . Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris . The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo . tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation . Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth . Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

J Biochem Mol Biol, 2002 Sep 30, 35(5), 476 - 81
Secretion of Pem-CMG, a peptide in the CHH/MIH/GIH family of Penaeus monodon, in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae; Treerattrakool S et al.; The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement . In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P . monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter . In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed . The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted . The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively . The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed . This produced the mature Pem-CMG peptide.

Protein Expr Purif, 2002 Oct, 26(1), 96 - 105
Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris; Sinclair G et al.; The lysosomal hydrolase glucocerebrosidase catalyzes the penultimate step in the breakdown of membrane glycosphingolipids . An inherited deficiency in this enzyme leads to the onset of Gaucher disease, the most common lysosomal storage disorder . Exogenous sources of this protein are required for biochemical and biophysical investigations and enzyme replacement therapy of Gaucher disease . Heterologous expression of glucocerebrosidase has been successful in mammalian and insect cell lines and although its use in enzyme replacement therapy of Gaucher disease has proven efficacious, current production levels limit the availability of the enzyme . Initial attempts to express human glucocerebrosidase using the methylotrophic yeast Pichia pastoris had limited success, despite significant levels of transcription . Using fragments of the glucocerebrosidase cDNA fused to the luciferase cDNA as a translational read-through reporter, the impact of synonymous codon usage bias on protein expression in P . pastoris was examined . A table of preferred codons was determined for P . pastoris and the codon usage of a 186-bp fragment of the glucocerebrosidase gene was optimized to that of the P . pastoris preferred set . A second construct with altered G+C content but no codon optimization was created for comparison . While the native glucocerebrosidase coding region limited luciferase activity to baseline levels, the codon optimized and G+C altered constructs increased luciferase activity 10.6- and 7.5-fold, respectively . Optimized G+C content, regardless of corresponding codon optimization, appears to be the major contributor to increased translational efficiency in this heterologous expression host.

Protein Expr Purif, 2002 Oct, 26(1), 65 - 70
Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme; Bellevik S et al.; Epoxide hydrolases are enzymes involved in metabolism and defense of plants . Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana . To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed . We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis . We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A . thaliana . A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose . The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis . The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively . The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C . Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively . The activity was 50-fold lower towards cis-stilbene oxide . The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.

Protein Expr Purif, 2002 Oct, 26(1), 28 - 34
Expression, purification, and characterization of equine lactoferrin in Pichia pastoris; Paramasivam M et al.; Lactoferrin is an 80kDa iron-binding glycoprotein . It is secreted by exocrine glands . Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it . In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector . The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column . The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin . Its N-terminal sequence was identical to that of the native ELF . The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly . The recombinant protein appears to be hyperglycosylated by the host strain, GS115 . This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P . pastoris system . An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.

Biotechnol Bioeng, 2002 Dec 5, 80(5), 490 - 7
Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChE; Weill CO et al.; In a previous report, Morel and Massoulie showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulie, 2000) . They reported that a 5' Bungarus fragment could partially transfer this property to a CAT expression vector . This appeared to offer the possibility of increasing the production of recombinant proteins . In the present paper, we show that insertion of this fragment in the transcribed region, before the polyadenylation site, may have either stimulatory or inhibitory effects, depending on the vector and on the reporter gene . Since the stimulatory effect of Bungarus coding region could not be attached to a small number of discrete motifs, we reasoned that it might result from a general feature of the sequence . Therefore it might be possible to partially transfer this property to the very homologous human AChE (hHAChE) coding sequence by modifications based on synonymous codons, which increased nucleotide identity between the 5' fragment (721 nucleotides) of bBAChE and hHAChE from 71% to 85% . The production of human AChE in transfected COS cells was increased nearly 2-fold with this modified construct, but still remained about 4-fold smaller than that of Bungarus AChE . There was no change in expression level in transformed Pichia pastoris . We thus confirm that coding sequences can strongly influence gene expression, but in a manner that depends on the context and cannot yet be predicted .

J Biol Chem, 2002 Dec 20, 277(51), 49767 - 75 Epub 2002 Sep 25.
Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast; Zhang H et al.; Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D . A., Nomura, T., Talley, A . K., Cooper, R . A., Dzekunov, S . M., Ferdig, M . T., Ursos, L . M., Sidhu, A . B., Naude, B., Deitsch, K . W., Su, X . Z., Wootton, J . C., Roepe, P . D., and Wellems, T . E . (2000) Mol . Cell 6, 861-871) . Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter . Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles . Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression . Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure . We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast . To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast . Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild type versus CQR-conferring mutant Pfcrt function, which may include effects on H(+) transport (Dzekunov, S., Ursos, L . M . B., and Roepe, P . D . (2000) Mol . Biochem . Parasitol . 110, 107-124), and to perfect a rapid purification of biotinylated Pfcrt . These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of important P . falciparum transport proteins and membrane associated vaccine candidates.

Curr Opin Biotechnol, 2002 Aug, 13(4), 329 - 32
Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris; Cereghino GP et al.; The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels . Using this system, recent advances have been made in the quality of recombinant proteins in fermenter culture and in the quality of the protein product, namely improved secretion signals and glycosylation patterns.

Yeast, 2002 Oct, 19(14), 1203 - 20
SHAM-sensitive alternative respiration in the xylose-metabolizing yeast Pichia stipitis; Shi NQ et al.; SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood . We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption . The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum . Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool . Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose . Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis . We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism . Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source . We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P . stipitis.

Yeast, 2002 Oct, 19(14), 1191 - 202
Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris; Hirose M et al.; We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris . rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial . The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT) . In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns . The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated . The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components . The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column . Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components . The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components . Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels .

Mol Biochem Parasitol, 2002 Aug 28, 123(2), 125 - 34
A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis; Hussein AS et al.; A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species . Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M) . All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs . Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans . Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine . A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine . This model is consistent with mutational analysis of the nematode enzymes . Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms . These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity .

J Biotechnol, 2002 Oct 23, 99(2), 97 - 110
Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen; Bisht H et al.; A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris . Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates . Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs) . Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein . Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data .

J Biol Chem, 2002 Nov 15, 277(46), 44035 - 43 Epub 2002 Aug 30.
Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger; Tahir TA et al.; The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis . Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined . The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced . The low specific activities of Y6A and Y89A were entirely accounted for by a change in k(cat) and K(m), respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K(m) and decreased k(cat) . Tyr(6), Tyr(10), Tyr(89), Tyr(164), and Trp(172) are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A . niger xylanase in complex with the modeled xylobiose . All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity . Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism . The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase . The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration . A close inspection of the three-dimensional structure of A . niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.

Eur J Biochem, 2002 Sep, 269(18), 4586 - 96
Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier; Briand L et al.; Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication . We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein . The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide . ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing . The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs . CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs . Gel filtration analysis showed that ASP3c is monomeric at neutral pH . Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range . It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual) . This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Infect Immun, 2002 Oct, 70(10), 5676 - 83
Secreted metalloprotease gene family of Microsporum canis; Brouta F et al.; Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors . Microsporum canis, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported . Using Aspergillus fumigatus metalloprotease genomic sequence (MEP) as a probe, three genes (MEP1, MEP2, and MEP3) were isolated from an M . canis genomic library . They presented a quite-high percentage of identity with both A . fumigatus MEP and Aspergillus oryzae neutral protease I genes . At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these M . canis genes (MEP genes) encode a zinc-containing metalloprotease gene family . Furthermore, MEP3 was found to be the gene encoding a previously isolated M . canis 43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in Pichia pastoris . Reverse transcriptase nested PCR performed on total RNA extracted from the hair of M . canis-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process . This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

J Clin Oncol, 2002 Sep 15, 20(18), 3792 - 803
Phase I study of recombinant human endostatin in patients with advanced solid tumors; Herbst RS et al.; PURPOSE: Endostatin, a 20-kd fragment of collagen XVIII, is a potent inhibitor of angiogenesis . We evaluated recombinant human endostatin (rh-Endo) in a phase I trial designed to assess safety, pharmacokinetics, and serum markers of angiogenesis in patients with solid tumors . PATIENTS AND METHODS: Twenty-six patients were enrolled onto a dose-finding trial of rh-Endo administered as an intravenous bolus over a 20-minute period once daily . Three patients each were treated at dose levels of 15, 30, 60, 120, 180, and 600 mg/m(2)/d, and seven patients were treated at 300 mg/m(2)/d . Treatment consisted of a minimum of two 28-day cycles . Evaluations included noninvasive imaging, pharmacokinetics, and serum biomarkers . RESULTS: Twenty-five patients were treated with rh-Endo . Treatment was well tolerated; there were no dose-limiting toxic effects . Bacteremia from frequent central line access was the most common problem . The pharmacokinetic disposition of rh-Endo was linear and best described using a two-compartmental open model . The overall mean half-life was 10.7 +/- 4.1 hours . A dose of 300 mg/m(2) achieved an area under the concentration-time curve associated with activity in preclinical models . In two patients, there was evidence of antitumor activity, but no responses were seen . Serum markers of angiogenic activity did not provide insight into rh-Endo's activity . Serum antibodies were observed against both rh-Endo and the Pichia pastoris vector, but no allergic reactions were observed . CONCLUSION: rh-Endo was safe and well tolerated . rh-Endo pharmacokinetic profiles achieved area under the concentration-time curves associated with activity in preclinical models . Evidence of minor antitumor activity was observed and further studies are indicated.

Am J Physiol Regul Integr Comp Physiol, 2002 Oct, 283(4), R885 - 96
Crocodile transthyretin: structure, function, and evolution; Prapunpoj P et al.; Structure and function were studied for Crocodylus porosus transthyretin (crocTTR), an important intermediate in TTR evolution . The cDNA for crocTTR mRNA was cloned and sequenced and the amino acid sequence of crocTTR was deduced . In contrast to mammalian TTRs, but similar to avian and lizard TTRs, the subunit of crocTTR had a long and hydrophobic NH(2)-terminal region . Different from the situation in mammals, triiodothyronine (T(3)) was bound by crocTTR with higher affinity than thyroxine (T(4)) . Recombinant crocTTR and a chimeric construct, with the NH(2)-terminal region of crocTTR being replaced by that of Xenopus laevis TTR, were synthesized in the yeast Pichia pastoris . Analysis of the affinity of the chimeric TTRs showed that the NH(2)-terminal region modulates T(4) and T(3) binding characteristics of TTR . The structural differences of the NH(2)-terminal regions of reptilian and amphibian TTRs were caused by a shift in splice sites at the 5' end of exon 2 . The comparison of crocodile and other vertebrate TTRs shows that TTR evolution is an example for positive Darwinian evolution and identifies its molecular mechanism.

Int Arch Allergy Immunol, 2002 Aug, 128(4), 264 - 70
Expression systems for production of recombinant allergens; Schmidt M et al.; Recombinant allergenic proteins have been produced in a variety of different expression systems . This review gives examples of and compares prokaryotic expression systems, such as Escherichia coli, and eukaryotic systems including the yeasts, Saccharomyces cerevisiae and Pichia pastoris, baculovirus-insect cell systems, mammalian cell systems and plant systems . Important factors to consider in choosing an expression system are discussed and examples of applications are given .

Mol Immunol, 2002 Sep, 39(1-2), 93 - 101
Influence of the 3D-conformation, glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen . Use of recombinant isoforms and specific monoclonal antibodies as immunological tools; Gonzalez EM et al.; Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area . Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia pastoris by recombinant methods, have been used as tools to determine the role of the three-dimensional (3D)-folding, the glycan component and several point changes of the amino acid sequence in the binding of murine IgG mAbs and human IgE to the olive allergen . Seven mAb families (F1-F7) were established, two of which (F1 and F2) recognize continuous epitopes . The carbohydrate moiety of Ole e 1 was involved in the binding to F2 and F4, whereas F3 and F7 were able to bind to all Ole e 1 variants . The remaining families of IgG murine antibodies exhibited different affinities for the antigens assayed in a native or denatured conformation . Although the binding of human IgE to Ole e 1 was not affected by heat treatment, it was shown to be strongly dependent on the integrity of the disulfide bridges and was partially inhibited by F3-F7 IgG antibodies, their individual values ranging from 12 to 31% and reaching 53% with their mixture . The IgE from sera of olive-allergic patients showed a significant diversity of binding capacity to the members of the Ole e 1-like family due to the microheterogeneity of their polypeptide sequences, in spite of their highly conserved primary structures . Whereas one of the isoforms of Ole e 1 exhibits a highly similar behavior to the natural form, being a putative molecule for diagnostic purposes, other ones can be considered as hypoallergenic variants of this allergen and, thus, potential candidates to be used in immunotherapy.

Yeast, 2002 Sep 15, 19(12), 1067 - 73
Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica; Nakagawa T et al.; The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2 . The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2 . A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%) . The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1 . The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction . The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range . Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level . These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p .

Vet Immunol Immunopathol, 2002 Oct 8, 89(1-2), 13 - 27
Cloning, expression, purification, and biological activity of five feline type I interferons; Wonderling R et al.; Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals . In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity . Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses . We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity . FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes . Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60% . Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site . Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated . The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro . In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris . The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.

J Agric Food Chem, 2002 Sep 11, 50(19), 5313 - 7
Effect of glycosylation modification (N-Q-(108)I --> N-Q-(108)T) on the freezing stability of recombinant chicken Cystatin overexpressed in Pichia pastoris X-33; Jiang ST et al.; The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host . The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated . A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence . The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM) . They acted as a competitive inhibition reaction against papain . According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one . However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P . pastoris.

J Biotechnol, 2002 Oct 9, 99(1), 51 - 62
High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris; Boettner M et al.; The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins . In order to provide proteins for the 'protein structure factory', a structural genomics initiative, we are working on the high-throughput expression of human proteins . Therefore, cDNAs are cloned for intracellular expression . The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins . Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter . We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression . The screening procedure is based on a culture volume of 2 ml in a 24-well format . Lysis of the cells occurs via a chemical lysis without mechanical disruption . Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher .

J Biotechnol, 2002 Oct 9, 99(1), 23 - 39
Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris; Sarramegna V et al.; The human mu-opioid receptor (HuMOR) was fused in its N-terminus end to the green fluorescent protein (GFP) or/and to the c-myc and six histidines tags in its C-terminus end, and expressed in the methylotrophic yeast Pichia pastoris . Neither the C- nor the N-terminal tagging of the receptor does modify its pharmacological properties as compared to the untagged receptor . Expression levels of fusion receptors determined by GFP fluorescence measurements strongly correlates with the number of sites expressed per cell detected through saturation studies (Bmax value), thus showing that GFP is an efficient and reliable reporter of the HuMOR functional expression . The N- and C-terminus tags have allowed to show that the entire molecule is overexpressed . They have permitted in-situ localization experiments using fluorescence and electron microscopy techniques and have shown a dense intracellular labelling . Above all, the quantification of expression levels made possible through fluorescence intensity analysis, have revealed that huge amounts of receptor are produced that could not be detected through classical binding experiments: for a Bmax value of 1 pmol mg(-1) of receptor determined through binding studies, 16 pmol were found in membrane preparations using fluorescence and 100 pmol in whole cells . These results should be very useful for large-scale production and structural biology of HuMOR, and other G-protein coupled receptors (GPCRs) .

Anal Biochem, 2002 Aug 15, 307(2), 287 - 96
Quantitative analysis of c-myc-tagged protein in crude cell extracts using fluorescence polarization; Sun S et al.; A fluorescence polarization (FP) assay was developed to determine the concentration of a c-myc-tagged recombinant protein in a crude cell extract . The basis of the assay was a competition between a c-myc-tagged protein and a fluorescein-labeled c-myc peptide for a c-myc antibody Fab . Fluorescein-labeled c-myc peptide produced a high-fluorescence polarization signal upon binding to the c-myc antibody, which can be inhibited in the presence of a c-myc-tagged protein . Quantitation of a c-myc-tagged protein was realized by measuring the decrease in fluorescence polarization . The observed IC(50) values in the competition FP assay were similar among all monomeric c-myc-tagged proteins tested, indicating that the interaction of the c-myc tag with the antibody was independent of the fusion protein sequence . The c-myc-tagged protein concentrations measured by FP were found to correlate well with values derived from a spike experiment and with values obtained by quantitative immunoblot . This assay was not perturbed by the presence of crude cell lysate, dithiothreitol or detergents, and worked with both native and denatured samples from several expression systems, including Escherichia coli, Pichia, insect cells, and mammalian cells . The assay under the current condition can detect as low as 0.05% expression level of c-myc-tagged protein with regards to total proteins, depending on the expression system . This assay is both quantitative and rapid (less than 15min) and is therefore suitable for the optimization of recombinant protein expression conditions as well as for the monitoring of protein purification procedures.

Protein Eng, 2002 Jul, 15(7), 595 - 601
Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity; Schmitt J et al.; The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis . The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil . Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity . Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed . Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14 . The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site . In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates . This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.

Biochemistry, 2002 Sep 3, 41(35), 10849 - 57
Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity; Schilling S et al.; Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins . In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation . Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions . CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya . The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC . However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E . coli . Expression of QC in E . coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P . pastoris . Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity . Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT . In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 348 - 51
{Effects of different methanol feeding strategy on hirudin production in high-density fermentation by recombinant Pichia pastoris}; Zhou XS et al.; Four different methanol feeding modes were evaluated in the hirudin production in high-density fermentation by Pichia pastoris . It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level . On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography . However, the stable methanol concentration was perfectly achieved by the on-line monitoring with methanol sensor . The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase . Therefore, the high cell dry weight (162 g/L) and high hirudin activity (2.4 x 10(4) ATU/mL or 1.7 g/L) was obtained in the fed-batch fermentation of recombinant Pichia pastoris by methanol-glycerol mixed feeding.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 295 - 9
{Production of SAM by recombinant Pichia pastoris}; Li DY et al.; To utilize Pichia pastoris to produce S-adenosyl-L-methionine (SAM), an intracellular expression vector harboring S . cerevisiae SAM2 was transformed into GS115 . A recombinant strain having 2 copies of expression cassette was obtained through G418 resistance screening . This strain had higher SAM synthetase activity and higher SAM production capacity than the original strain, when cultured in medium containing methanol and methionine . The carbon source and nitrogen source of medium was optimized . The results showed SAM production by this strain was closely related to carbon metabolism . With supplementation of 0.2% glycerol every day from the beginning of 3rd day, this strain produced 1.58 g/L SAM when cultured in a medium containing 0.75% L-methionine and optimized carbon and nitrogen source after 6 days.

Mikrobiol Z, 2002 May-Jun, 64(3), 13 - 9
{Search for lectin producers among the yeast of Pichia Hansen emend . Kurtzman genus}; Podgorskii VS et al.; Strains (103) of yeast from Pichia genus: P . anomala, P . guilliermondii and P . membranaefaciens species have been investigated . The researchers have found 87 strains (84.5%) capable to form extracellular and surface lectins with activity to 512 hemagglutinating units . The overwhelming number of strains (75.7-92.6%) synthesized lectins of the both forms . Considerably less quantity of lectin producers of some form--extracellular or surface ones--have been found . Availability of producers of extracellular lectins was characteristic to greater extent of the yeast of P . anomala and P . guilliermondii, and producers of surface lectins--mainly of P . guilliermondii . The most active producers of lectins have been isolated from human and animal organisms, soils and wine.

Med Dosw Mikrobiol, 2002, 54(2), 167 - 71
{Yeast-like fungi as etiologic agents of blood infections in patients hospitalized in 1998-1999}; Swoboda-Kopec E et al.; The aim of the study was the analysis of frequency of yeast-like fungi as etiological agents of fungemias in patients hospitalized in operative and conservative wards of Medical Academy Central Clinical Hospital in Warsaw in 1998-1999 . Peripheral blood samples and collected from vascular catheters were incubated in BacT/Alert system(Organon Teknika, USA) . Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France) (the time of cultivation from 48 h to 7 days at 30 C) and on chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA) . Fungal strains were identified by standard mycological procedures using ID 32 C strips (ATB system, bioMerieux, France) and tests of Sanofi Diagnostics Pasteur (France) . The total number of positive blood cultures was 1724 . Fifty eight fungal strains were isolated from blood samples (3.36%) . Strains belonged to 4 genera: Candida (55), Trichosporon (1), Saccharomyces (1) and Pichia (1) . Thirty eight fungal strains were isolated from peripheral blood samples . Forty seven fungal strains were cultured from patients hospitalized in operative wards . Among fungi isolated from peripheral blood samples C . albicans (10), C . glabrata (9) and C . parapsilosis (5) strains dominated . From blood samples collected from vascular catheters most often C . albicans (7), C . glabrata (4) and C . parapsilosis (3) were isolated.

Protein Expr Purif, 2002 Aug, 25(3), 416 - 25
Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris; Koganesawa N et al.; A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris . An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter . Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein . To overcome this problem, we utilized a high-density fermentation method . The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases . Recombinant GBP was purified through three reverse-phase HPLC columns . We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing . Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP . Up to 50mg GBP was recovered per 1L of yeast culture supernatant.

Protein Expr Purif, 2002 Aug, 25(3), 400 - 8
High level expression and secretion of truncated forms of herpes simplex virus type 1 and type 2 glycoprotein D by the methylotrophic yeast Pichia pastoris; van Kooij A et al.; Herpes simplex virus type 1 and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell . Availability of substantial amounts of these proteins, or large fragments thereof, will be needed to allow studies at the molecular level . We studied the potency of the Pichia pastoris yeast expression system to produce soluble forms of gD . The DNA sequences encoding the extracellular domains of gD {amino acids 1-314 (gD-1(1-314)) and amino acids 1-254 (gD-1(1-254)) of gD-1 and amino acids 1-314 of gD-2 (gD-2(1-314))} were cloned into the P . pastoris yeast expression vector pPIC9 . Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1(1-314His) and gD-1(1-254His)) to facilitate their purification . Large amounts of gD-1(1-314) and gD-1(1-314His) (280-300mg/L induction medium) were produced . The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L . SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa . The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa . All recombinant proteins produced by P . pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, and AP7) . In addition, we showed that gD-1(1-314), gD-2(1-314), and gD-1(1-254His) were able to interfere with binding of HSV to susceptible cells . These results indicate that the conformations of the recombinant proteins closely resemble those of native gD.

Anticancer Res, 2002 Jul-Aug, 22(4), 2001 - 7
Targeting of interleukin-2 to human MK-1-expressing carcinoma by fusion with a single-chain Fv of anti-MK-1 antibody; Matsumoto H et al.; BACKGROUND: Targeting of cytokines into the tumor sites using antibody-cytokine fission proteins represents a novel approach in cancer immunotherapy . We previously reported a novel monoclonal antibody, FU-MK-1, which recognizes a glycoprotein antigen (termed MK-1 antigen) that is overexpressed on the surface of a majority of carcinomas . MATERIALS AND METHODS: To target IL-2 and cytotoxicity of effector cells to MK-1-expressing tumor cells, we genetically fused recombinant human interleukin-2 (rhIL-2) to a single chain variable fragment (scFv) antibody derived from FU-MK-1 . The resulting fission protein, designated FUscFv/IL-2 was expressed in Pichia pastoris, purified by Ni-affinity chromatography, and characterized for the MK-1-binding specificity and the IL-2 biological activity . RESULTS: The FUscFv/IL-2 fusion protein effectively introduced a specific cytotoxicity of lymphokine-activated killer cells to the tumor cells and consequently suppressed the tumor growth in a SCID mouse xenograft model . CONCLUSION: This approach may be used for in vivo administration to localize IL-2 to tumor tissues, enhancing the immune response to human MK-1-expressing tumors while reducing systemic side-effects.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1328 - 36
Cloning, characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp . I-11; Kakugawa K et al.; A cDNA clone of the lipase secreted by Kurtzmanomyces sp . I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase . The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively . The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases . The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved . A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1295 - 300
Characterization of partially truncated human midkine expressed in Pichia pastoris; Murasugi A et al.; Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichiapastoris . Approximately 640 mg of rh-midkine was secreted into one liter of medium of the high cell-density fermentation . The protein processing of the rh-midkine was done efficiently and correctly in P . pastoris, and O-mannosylation was not detected in the purified rh-midkine . However, only about 30% of the purified rh-midkine was intact . The other ones lost 5-12 amino acid residues from the amino-termini, provably by proteolysis . Even the mixture of these truncated midkines could promote CHO cell proliferation as well as the authentic rh-midkine.

J Ind Microbiol Biotechnol, 2002 Aug, 29(2), 55 - 9
Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris; O'Callaghan J et al.; A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library . The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P . pastoris strains KM71 and GS115 . Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS . No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains . The presence of at least 200 microM copper was necessary for optimal laccase activity in the culture supernatants . During growth of P . pastoris on minimal medium the pH of the medium was reduced to <3.0 . If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days . Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine . This study describes the development of a medium that allows convenient pH control of P . pastoris without the need for continuous neutralisation.

Eur J Endocrinol, 2002 Aug, 147(2), 227 - 33
The (ProB27, ThrB28) human insulin analogue is more potent and more rapidly absorbed from subcutis than human insulin; Clausen R et al.; OBJECTIVE: To test the physiological properties of human insulin in which the amino acids Thr (B27) and Pro (B28) are interchanged (PT insulin) . This was hypothesised to prevent dimerisation and accelerate the absorption from s.c . tissue without altering the affinity for the insulin receptor . DESIGN: PT insulin was expressed in Pichia pastoris and processed in vitro . The purified compound was used for physiological investigations . METHODS: Receptor binding activity to insulin and IGF receptors was evaluated in a competition assay using iodinated PT insulin and recombinant receptors while growth induction properties were evaluated by thymidine incorporation . Absorption kinetics from pig subcutis was investigated by measuring the disappearance of iodinated PT insulin . The potency was evaluated by measuring the blood glucose lowering activity in mice . RESULTS: The absorption of PT insulin was accelerated compared with human insulin, although still slower than Asp (B28) insulin . Human and PT insulin had similar affinities for the human insulin receptor (K(d)=3.6 x 10(-12) vs 5.2 x 10(-12) mol/l) while the affinity for the IGF receptor was four times higher for PT insulin than for human insulin (K(d)=3.4 x 10(-8) vs 1.3 x 10(-7) mol/l) . This resulted in a slightly higher DNA synthesis when assayed in intermediary insulin concentrations . The blood glucose lowering effect in mice exceeded the effect of human insulin (integral 0-60 min: 61.4+/-7 vs 30+/-4, n=6, P=0.046) . CONCLUSIONS: PT insulin is absorbed faster and is more potent than human insulin . Although PT insulin stimulates growth more than human insulin, this will not prevent its use in the clinic, but the main interest will probably focus on investigations to clarify the paradox of full biological activity in connection with the recently described lack of structure in the B-chain.

Eur J Biochem, 2002 Aug, 269(15), 3645 - 58
Towards the development of selective amine oxidase inhibitors . Mechanism-based inhibition of six copper containing amine oxidases; Shepard EM et al.; Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO) . Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined . Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation . Striking differences in selectivity and rates of inactivation were observed . For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4 . In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site . Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations . Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities . Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 208 - 11
{Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product}; Han XQ et al.; E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector . After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal . The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P . pastoris culture contained protein E2 . The results of the study on the immunological activity indicated that the protein E2 expressed in P . pastoris can elicit animal bodies to produce antibodies against protein E2.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 187 - 92
{Expression and characterization of envelope protein 2 gene of hepatitis G virus in Pichia pastoris}; Wang ZH et al.; A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV-C/HGV fused with Schistosoma japonicum, glutathione S-transferase(GST) was amplified with PCR from plasmid pGEX-E2 . The amplified DNA fragment was inserted into plasmid pGEX-5X-1, at the downstream of the coding sequences of GST, in the same reading frame with the gene of GST . The fusion gene fragment of GST-E2 was amplified with PCR, using the recombinant plasmid pGEX-5X-1-E2 as the template . The amplified 1324 bp DNA fragment of GST-E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with alpha-factor secreting signal peptide . The plasmid pPIC9K-GST-E2 was transformed into Pichia pastoris GS115 with electroporation . The transformants (His+ Muts) were selected and induced to express the 54kD GST-E2 fusion protein, which could be specially recognized by both the antisera directed against E2 and against GST . The GST-E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95% . The expression was optimized to achieve the highest expression level of GST-E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant . The GST-E2 protein derived from the recombinant Pichia pastoris was proved possessing antigenicity and high specificity by ELISA, probed with sera from the patients infected by GBV-C/HGV.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 172 - 7
{Expression and purification of recombinant huwentoxin-I in Pichia pastoris}; Nie DS et al.; HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity . rHWTX-I expressed by P . pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity . The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.

J Exp Bot, 2002 Aug, 53(375), 1723 - 34
Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv . Chandler); Blanco-Portales R et al.; Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis . By a differential screening of a strawberry (Fragariax ananassa cv . Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1) . Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots . In addition, the gene presented a differential expression in fruits along the ripening process . Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2) . Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns . Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry . RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed . The Fxacad1 cDNA was expressed in E . coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide . The antibodies obtained were used for immunolocalization studies . The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers) . Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein . The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.

Clin Chem, 2002 Aug, 48(8), 1232 - 40
Detection of human kallikrein 4 in healthy and cancerous prostatic tissues by immunofluorometry and immunohistochemistry; Obiezu CV et al.; BACKGROUND: Human kallikrein 4 (gene, KLK4; protein, hK4), a recently discovered member of the kallikrein gene family, shares many characteristics with prostate-specific antigen, the best available marker for prostate cancer . Because the protein has not been detected in any human tissue, we attempted to develop immunologic methods for hK4 analysis and use them to detect hK4 in healthy and cancerous tissue extracts and biological fluids . METHODS: We extracted total RNA from 20 pairs of matched (healthy-cancer) prostate tissue samples . KLK4 cDNA was amplified by reverse transcription-PCR (RT-PCR) and cloned in a pPICZalphaA expression vector . We then transformed the construct product into Pichia pastoris yeast strains and induced secreted recombinant protein production by addition of methanol . We purified the recombinant protein by nickel ion-affinity chromatography and used it as an immunogen in rabbits and mice to generate polyclonal anti-hK4 antibodies . These antibodies were used to develop a sandwich-type immunoassay suitable for hK4 quantification in biological fluids and tissue extracts . RESULTS: The immunoassay had a detection limit of 0.1 microg/L . We detected hK4 in 10 of 21 matched (healthy-cancer) prostate tissues, and hK4 was frequently higher in healthy tissues . In one matched-sample pair, the hK4 content was relatively high in both the healthy {4.62 microg/g of total protein (TP)} and the cancerous (1.22 microg/g of TP) prostate tissue . Among tissue extracts, we found the highest concentrations of hK4 in healthy (0.0-4.62 microg/g of TP) and cancerous (0.0-1.72 microg/g of TP) prostatic extracts and in placental extracts (0.0-0.05 microg/g of TP) . We also detected traces of hK4 protein immunoreactivity in amniotic fluid (<0.1-0.6 microg/L), human breast milk (<0.1-0.75 microg/L), and seminal plasma (0.2-0.9 microg/L) . Immunohistochemical studies showed cytoplasmic staining for hK4 protein in both malignant and benign epithelial cells of the prostate . However, we did not detect hK4 in cerebrospinal fluid, healthy and cancerous ovarian tissue extracts, and many other human tissue extracts . CONCLUSIONS: hK4 protein is present in some prostatic tissue extracts but at relatively low concentrations, although KLK4 mRNA is readily detectable by RT-PCR . We propose that the protein either is not synthesized efficiently or is degraded very quickly.

Mikrobiologiia, 2002 May-Jun, 71(3), 368 - 72
{The reversion of Pichia guilliermondii transformants to the wild-type phenotype}; Piniaga IuV et al.; Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB7 of this enzyme . Experiments showed that the presence of an ARS element and the promoter region of the gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype . Different types of recombination in the yeast P . guilliermondii are discussed.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1314 - 21 Epub 2002 Jul 20.
Structure of beta-cinnamomin, a protein toxic to some plant species; Rodrigues ML et al.; Phytophthora and Pythium species are among the most aggressive plant pathogens, as they invade many economically important crops and forest trees . They secrete large amounts of 10 kDa proteins called elicitins that can act as elicitors of plant defence mechanisms . These proteins may also induce a hypersensitive response (HR) including plant cell necrosis, with different levels of toxicity depending on their pI . Recent studies showed that elicitins function as sterol carrier proteins . The crystallographic structure of the highly necrotic recombinant beta-cinnamomin (beta-CIN) from Phytophthora cinnamomi has been determined at 1.8 A resolution using the molecular-replacement method . beta-CIN has the same overall structure as beta-cryptogein (beta-CRY), an elicitin secreted by Phytophthora cryptogea, although it shows a different surface electrostatic potential distribution . The protein was expressed in Pichia pastoris and crystallized in the triclinic space group with two monomers in the asymmetric unit . The interface formed by these two monomers resembles that from beta-CRY dimer, although with fewer interactions.

Protein Expr Purif, 2002 Jul, 25(2), 291 - 9
A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris; Piefer AJ et al.; The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome . HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli . Maximal IN expression was observed at 48h post-induction for the yeast system and 2h post-induction for E . coli . Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification . IN from both sources was routinely greater than 90% pure with yields exceeding 1.5mg of purified IN per liter of culture for P . pastoris . The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis . Specific activities for IN purified from E . coli and P . pastoris were calculated from in vitro 3(') processing assays and were comparable . In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3(') processing and strand transfer assays . Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3(') processing pH of 8.3 . IN from both sources showed no enhancement in activity with MnCl(2) concentrations greater than 5mM . The specific activity of P . pastoris purified IN was 0.35 product (pmol)/h/microg IN, and E . coli produced IN was 0.48 product (pmol)/h/microg IN.

Protein Expr Purif, 2002 Jul, 25(2), 270 - 82
Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris; Woo JH et al.; The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation . The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin . However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae . This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P . pastoris under the control of AOX1 (alcohol oxidase 1) promoter . The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P . pastoris because of several AT-rich regions, which induce an early termination of transcription . After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture . No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes . Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation . The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography . The immunotoxin purified from P . pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line . Our results demonstrate that P . pastoris is an ideal system for expression of toxin-based fusion proteins.

Protein Expr Purif, 2002 Jul, 25(2), 256 - 62
Purification and characterization of recombinant rat mast cell protease 7 expressed in Pichia pastoris; Lawson C et al.; Rat mast cell protease 7 (rMCP7) is a neutral serine protease and a component of mast cells, where it is stored in secretory granules . Mast cells express numerous proteases so in order to characterize rMCP7, it was cloned and expressed as a recombinant protein in Pichia pastoris . During expression, rMCP7 protein was cleaved from the alpha-mating factor signal at the engineered KEX2 cleavage site to produce active rMCP7 . The protein produced was stable at pH 5.5 and active in the absence of heparin . The rMCP7 was glycosylated and treatment with N-glycosidase F resulted in a protein of the predicted molecular mass of 30 kDa . The rMCP7 was purified via an ammonium sulfate precipitation, using casein as a carrier protein, followed by cation exchange chromatography . The purified protein was assayed using a range of substrates and where possible, k(m) and k(cat) values were determined . The substrate profile displayed by the recombinant rMCP7 was consistent with that of tryptase isolated from rat skin . The expression and purification of recombinant rMCP7 offer an efficient, low-cost method of producing large amounts of protein . It also offers the opportunity of easy manipulation and mutagenesis of rMCP7 for further biochemical, structural, and physiological studies.

Protein Expr Purif, 2002 Jul, 25(2), 229 - 36
Soluble expression and purification of porcine pepsinogen from Pichia pastoris; Yoshimasu MA et al.; This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris . The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter . After P . pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin . The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography . Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing . When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation . A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable . The above results indicate that the P . pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).

Eur J Biochem, 2002 Jul, 269(14), 3372 - 82
The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p is biochemically different from mammalian PP2A; Zabrocki P et al.; The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids . We have overexpressed S . cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity . Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained . The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose . Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes . Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate . Furthermore, selected inhibitors and metal ions affected their activities to the same extend . In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine . Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p . The N-terminal extension appears also responsible for interactions with phospholipids . Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents . These properties make the S . cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.

Yeast, 2002 Aug, 19(11), 949 - 56
Molecular genetics of 6-phosphofructokinase in Pichia pastoris; Edelmann A et al.; Previously, studies on glucose-induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose-induced selective autophagy-1-protein is the alpha-subunit of 6-phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway . In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P . pastoris . Investigating the yeast cell-free extract by Western blot analysis, two distinct signals of Pfk were obtained . In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the beta-subunit of Pfk from P . pastoris with a deduced molecular mass of 103.7 kDa . On the basis of these results, a hetero-oligomeric structure of Pfk in P . pastoris became obvious . Because the molecular and kinetic properties of a homo-oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P . pastoris as a heterologous production system . Furthermore, the 3'- and 5'-non-coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region .

Traffic, 2002 Aug, 3(8), 560 - 74
Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes; Hazra PP et al.; During peroxisomal matrix protein import, the peroxisomal targeting signal receptors recognize cargo in the cytosol and interact with docking and translocation subcomplexes on the peroxisomal membrane . Using immunoprecipitations of multiple protein components, we show that in Pichia pastoris the docking subcomplex consists of the unique peroxins Pex13p, Pex14p and Pex17p, whereas the putative translocation subcomplex has all three RING-finger peroxins, Pex2p, Pex10p and Pex12p, as unique constituents . We identify Pex3p as a shared component of both subcomplexes . In pex3delta cells, the unique constituents of the docking subcomplex interact as they do in wild-type cells, but the assembly of the translocation subcomplex is impaired and its components are present at reduced levels . Furthermore, several interactions detected in wild-type cells between translocation and docking subcomplex components are undetectable in pex3delta cells . Contrary to previous reports, pex3delta cells have peroxisome remnants that pellet during high-speed centrifugation, associate with membranes on floatation gradients and can be visualized by deconvolution microscopy using antibodies to several peroxins which were not available earlier . We discuss roles for Pex3p in the assembly of specific peroxisomal membrane protein subcomplexes whose formation is necessary for matrix protein import.

Infect Immun, 2002 Aug, 70(8), 4471 - 6
High-level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion; Kocken CH et al.; Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria . Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses . Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris . In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures . Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein . High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P . falciparum by immunofluorescence . In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P . falciparum.

Biotechnol Bioeng, 2002 Aug 20, 79(4), 438 - 49
Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris; Hong F et al.; Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system . The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures . Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol) . The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature . The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature . The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase . A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0% . The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(5), 587 - 589
Secretory Expression of Human Insulin in Methylotrophic Yeast Pichia pastoris; Wang Y et al.; The porcine insulin precursor (PIP) gene and its derivative form sp-PIP gene, which had a nona-peptide (called spacer peptide, sp) added at the 5' terminus of PIP gene, were inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP, respectively . P.pastoris GS115 was transformed by pPIC9/PIP or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were selected by dot-blotting . The expression levels of PIP and sp-PIP were 10 mg/L and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer peptide could increase the expression . The expression level of PIP (sp-PIP) in P.pastoris was higher than that of PIP in S.cerevisiae and K.lactis reported in this laboratory . The expression level of sp-PIP was 250 mg/L in 10 L fermentor . Recombinant human insulin was obtained by means of transpeptidation of PIP or sp-PIP . The receptor binding capacity is identical with that of porcine insulin . In vivo biological activity of the recombinant human insulin is 27 IU/mg.

Plant Physiol, 2002 Jul, 129(3), 1252 - 64
Investigation of the microheterogeneity and aglycone specificity-conferring residues of black cherry prunasin hydrolases; Zhou J et al.; In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase . Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase . Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members . Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs . Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites . Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns . Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates . Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris . Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis . The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.

Yeast, 2002 Jul, 19(10), 849 - 62
Post-translational modifications of the AFET3 gene product: a component of the iron transport system in budding cells and mycelia of the yeast Arxula adeninivorans; Wartmann T et al.; The yeast Arxula adeninivorans is characterized by a temperature-dependent dimorphism . A . adeninivorans grows as budding cells at temperatures up to 42 degrees C, but forms mycelia at higher temperatures . A strong correlation exists between morphological status and iron uptake, achieved by two transport systems that differ in iron affinity . In the presence of high Fe(II) concentrations (>2 microm), budding cells accumulate iron concentrations up to seven-fold higher than those observed in mycelia, while at low Fe(II) concentrations (<2 microm), both cell types accumulate similar amounts of iron . The copper-dependent Fe(II) oxidase Afet3p, composed of 615 amino acids, is a component of the high-affinity iron transport system . This protein shares a high degree of homology with other yeast iron transport proteins, namely Fet3p of Saccharomyces cerevisiae, Cafet3p of Candida albicans and Pfet3p of Pichia pastoris . Expression of the AFET3 gene is found to be strongly dependent on iron concentration but independent of the morphological stage; however, cell morphology was found to influence post-translational modifications of the gene product . O-glycosylation was observed in budding cells only, whereas N-glycosylation occurred in both cell types . The N-glycosylated 103 kDa glycoprotein matures into the 108.5 kDa form, further characterized by serine phosphorylation . Both N-glycosylation and phosphorylation occur at low iron concentrations (< or =5 microm) . The mature Afet3p of 108.5 kDa is uniformly distributed within the plasma membrane in cells of both morphological stages .

Anal Bioanal Chem, 2002 Jul, 373(4-5), 211 - 4 Epub 2002 Jun 06.
Development of a flow-injection analysis (FIA) enzyme sensor for fructosyl amine monitoring; Ogawa K et al.; An enzyme-sensor system with flow-injection analysis (FIA) has been developed for the detection of fructosyl amine compounds; the sensor utilizes fructosyl amine oxidase isolated from the marine yeast Pichia sp . N1-1 strain . With this FIA system 0.2 to 10 mmol L(-1) fructosyl valine can be determined . The sensor is approximately five times more sensitive to fructosyl valine, a model compound for glycated hemoglobin HbA1c, than to N(epsilon)-fructosyl lysine, a model compound for glycated albumin . This FIA system can also be used to detect fructosyl dipeptides . The operational stability of the sensor enabled more than 120 consecutive sample injections over a period of approximately 20 h.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(6), 680 - 684
Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene; He ZY et al.; Total RNA was isolated from normal Chinese human liver cell line L02 . A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid . Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394) . The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported . The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned . Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA . The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115 . SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days . The longer the transformant was induced, the higher the ratio of glycosylated protein was.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(1), 59 - 62
High Expression and Purification of Recombinant Human Serum Albumin from Pichia pastoris; Qiu RD et al.; Recombinant human serum albumin (rHSA) was produced in methylotrophic yeast Pichia pastoris . By optimization of expression, about 150 mg/L of rHSA was obtained from broth of Pichia pastoris GS115/HAS (his+Mut(S)) supernatant . The rHSA was isolated and purified by hollow-fiber ultrafiltration, Phenyl-Sepharose hydrophobic chromatography and antibody-immunoadsorbent chromatography . Finally, rHSA was purified to electrophoretic purity.

Can J Microbiol, 2002 May, 48(5), 418 - 26
Characterization of two novel yeast strains used in mediated biosensors for wastewater; Trosok SP et al.; After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater . 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii . While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses . Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch . Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains . Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater . The lower detection limits of SPT1- and SPT2-based K3Fe(CN)6-mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively . Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min . Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.

Antiviral Res, 2002 Aug, 55(2), 279 - 90
Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica; Choi J et al.; We expressed the Hepatitis B virus polymerase (HBV P protein) using a recently introduced yeast system, Pichia methanolica . HBV (1-680 amino acids) and Duck Hepatitis B virus (DHBV, 1-780 amino acids) polymerase were expressed and showed DNA dependent DNA polymerase (DDDP) . The DHBV polymerase had RNA dependent DNA polymerase (RDDP) and RNase H activities . We present a new simplified way of obtaining active viral P protein using the yeast expression system . The viral P proteins proved to be stable and were not aggregated in the yeast system .

Clin Exp Allergy, 2002 Jul, 32(7), 1048 - 53
High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris; Jacquet A et al.; BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease . Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success . OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1 . METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence . The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity . RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa . Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1 . During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1 . Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities . However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1 . CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(2), 139 - 144
Expression of the Recombinant Hepatitis B Virus Surface Antigen Carrying PreS Epitopes in Pichia pastoris; Yang JY et al.; Many studies have suggested that hepatitis B surface antigen(HBsAg) including PreS sequences could be an ideal candidate for highly effective hepatitis B virus vaccine . Modified surface antigens S1S, SS1 and S2S which carried PreS epitopes, were expressed in Pichia pastoris . The characterization of antigenicity and particle assembly demonstrated that the expression products could be assembled into particles which presented S, PreS1 or PreS2 antigenicity, respectively . The expression was more efficient than that in Saccharomyces cerevisiae.

J Biochem (Tokyo), 2002 Jul, 132(1), 77 - 82
Secretion of glycosylated alpha-lactalbumin in yeast Pichia pastoris; Saito A et al.; The secretion of N-linked glycosylated alpha-lactalbumin was much higher in the expression system of yeast Pichia pastoris carrying goat alpha-lactalbumin cDNA than in mammalian milk . This is possibly because of the presence of N-linked glycosylation signal sequences, Asn(45)-Asp(46)-Ser(47) and Asn(74)-Ile(75)-Ser(76), in wild-type alpha-lactalbumin . Attempts to elucidate the mechanism of the higher secretion of glycosylated alpha-lactalbumin in P . pastoris were made . Mutant N45D that deleted the N-linked glycosylation signal sequence at position 45 predominantly secreted nonglycosylated protein . On the other hand, mutant D46N with another N-glycosylation signal site at position 46 only secreted N-linked glycosylated alpha-lactalbumin, i.e . not the nonglycosylated protein . The total secreted amount of mutant N45D was greatly enhanced, while the secreted amounts of the wild-type and mutant D46N were very low, suggesting that the increase in the number of glycosylation sites greatly reduced the secretion of alpha-lactalbumin . It seems likely that the glycosylated alpha-lactalbumin may be degraded by the quality control system.

J Virol Methods, 2002 Jul, 104(2), 167 - 72
Purification and reactiongenicity of human immunodeficiency virus type 2 total external glycoprotein expressed in Pichia Pastoris; Zhang YJ et al.; The purification of human immunodeficiency virus type 2 total external glycoprotein gp105 expressed in Pichia pastoris was investigated . Expression conditions were optimized by an orthogonal test . The results from tests of variance analyses showed that the most important parameter for efficient expression of total gp105 in P . pastoris is adequate aeration during methanol induction . The optimum induction conditions for gp105 expression were: more than 85% aeration, induction for 3 days, the initial pH 6.0-7.0 and a final methanol concentration of 1.0% . Under these conditions, the expressed total gp105 was secreted into fermentation broth and reached a yield of 23%, approximately 141 mg/l . Expressed gp105 was isolated and purified by salting out and Sephadex G-100 chromatography and the yield of gp105 was 40% . gp105 was purified to electrophoretic purity and its isoelectric point (pI) was about 5.2 by SDS-PAGE and isoelectrofocusing . The purified gp105 contained approximately 35% carbohydrate, which proved that the expressed gp105 was a glycoprotein . Its N-terminal amino acid was arginine by Dansyl-Cl and the result indicated that expressed gp105 was secreted and cleaved correctly . The results from gp105 ELISA demonstrated that the purified total gp105 showed good reactiongenicity and antigenic specificity.

Glycoconj J, 2001 Jun, 18(6), 439 - 47
Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase; Wilson IB; Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Galbeta1,3(Fucalpha1,4)GlcNAc, on some N-glycans . By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian alpha1,3/4-fucosyltransferases . The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (K(m) 16 microM) to lacto-N-tetraose (Galbeta1,3GlcNAcbeta1,3Galbeta1,4Glc; K(m) 80 microM) as well as to beta1,3- and beta1,4-galactosylated N-glycans . It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato.

Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 81 - 4
Processing of the Hepatitis C virus precursor protein expressed in the methylotrophic yeast Pichia pastoris; Acosta-Rivero N et al.; The expression and processing of the Hepatitis C virus core protein (HCcAg) were analyzed in the methylotrophic yeast Pichia pastoris . Two proteins with 21 (p21) and 23 kDa (p23) were detected in immunoblot with a serum from a chronic carrier patient, as the major products for HCcAg . Both proteins, p21 and p23, produced by proteolytic processing in P . partoris, share the same N-terminal region and reacted with a monoclonal antibody towards the first 35 amino acids of HCcAg . The proteolytic processing of the recombinant polypeptide, having the HCcAg and the first 148 aa of E1 protein, was also confirmed by immunoblot analysis using mAbs with HCcAg and E1 specificities, respectively . The 32 kDa glycosilated E1 protein was then immuno-identified, as well as the processed HCcAg . These data demonstrated the usefulness of P . pastoris, as expression system, to study the processing of HCV structural proteins . (c) 2002 Elsevier Science (USA).

Protein Eng, 2002 Jun, 15(6), 529 - 36
High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein; Riley ML et al.; There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrP(Sc), from its normal (cellular) form, PrP(c) . Recently, the crystal structure of the human prion protein in a dimeric form was reported . Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris . FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system . Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms . Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrP(Sc) structure and are not infectious . Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell . The glycosylated proteins might act as powerful tools for crystallization trials, PrP(c)/PrP(Sc) conversion studies and other applications in the life cycle of prions.

Microb Pathog, 2002 Apr, 32(4), 165 - 72
Cloning, expression and characterization of the CHO cell elongating factor (Cef) from Vibrio cholerae O1; McCardell BA et al.; CHO cell-elongating factor (Cef) is a recently identified putative virulence factor of Vibrio cholerae . Our previous studies show that this 85 kDa protein elongates CHO cells, causes fluid accumulation in suckling mice and has esterase activity . In this study, the cef gene was cloned in Escherichia coli using a yeast vector and subsequently expressed in the yeast Pichia pastoris . The cef genes from V . cholerae candidate vaccine strains JBK 70 and CVD 103-HgR were sequenced and found to be nearly identical (100 and 99.9% respectively) with an open reading frame (ORF) from the published sequence of V . cholerae N16961 . Cloned toxin was purified to homogeneity in 3 steps using anion exchange, hydrophobic interaction and gel filtration chromatography . The size of cloned Cef on SDS-PAGE gels was 114 kDa . The increased size was probably due to glycosylation by the yeast since cloned protein reacted strongly with a glycoprotein stain . The cloned protein could not be directly sequenced, but when treated with trypsin, yielded a protein fragment with an amino acid sequence that matched the sequence predicted for the Cef protein . The purified cloned protein had esterase and CHO cell activity, but no suckling mouse activity .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 239 - 242
Expression of Smad4 Gene in Pichia pastoris and Identification of the Protein; Liu YP et al.; Smad4 is a novel tumor suppressor gene which is mutated or deleted in about 50% of pancreatic carcinoma and 30% of colon cancer . SMAD4 was expressed in Pichia pastoris and was characterized . The molecular weight of the expression product was shown to be about 67 kD, and 13 amino acids in N terminal were determined and were identical with the putative sequence from Smad4 cDNA, and it was able to specifically react with the antibody against SMAD4.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 217 - 222
Expression of Human Flt3 Ligand in Pichia pastoris and Its Biological Characteristics; Xu ZX et al.; Flt3 ligand(FL) is a cytokine that stimulates the proliferation and differentiation of hematopoietic stem cells/progenitors . In order to obtain high level of recombinant human soluble FL(rhFL) production, an artificial gene for rhFL was synthesized by using favored genetic codons of the yeast Pichia pastoris . Then the gene was cloned into the vector pPICzalphaA and the resulting construct was introduced into Pichia pastoris for expression . It was found that it was possible to obtain biologically active rhFL with a yield of over 30 mg/L of yeast culture . The rhFL stimulated colony formation from cord blood . rhFL, SCF, GM-CSF and IL-3 are the most promising combination for the in vitro expansion of stem/progenitor cells . It was also showed that rhFL increased the induction of dendritic cells from cord blood in combination with GM-CSF, TNFalpha and IL-4 . Interestingly, rhFL stimulated the growth of an endothelial cell line this effect has not been reported before.

Eur J Biochem, 2002 Jun, 269(12), 3086 - 92
IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1; Pomes A et al.; Sensitization to cockroach allergens is associated with the development of asthma . Bla g 1 is a German cockroach allergen that shows allergenic cross-reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino-acid repeats . Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1 . By molecular mass, purified natural Bla g 1 would contain approximately two duplexes . We investigated the pattern of IgE antibody binding to this repeated structure, and whether one or two duplexes are sufficient for IgE binding . Recombinant (r)Bla g 1 duplexes were expressed in Escherichia coli and in Pichia pastoris, and analyzed for monoclonal antibody and IgE antibody binding by ELISA and/or immunoblotting . Optimal rBla g 1 expression was obtained using methanol-inducible P . pastoris (> 95% pure protein, yield approximately 48 mg x L(-1)), and rBla g 1 was produced as multiple molecular forms of molecular mass 43, 32, 21 and 6 kDa, that were the result of proteolytic cleavage . There was an excellent correlation between IgE antibody binding to natural and recombinant Bla g 1 (r = 0.91, n = 29, P < 0.001), and immunoblot analysis showed that a single Bla g 1 duplex was sufficient for IgE antibody binding . The rBla g 1 is suitable for structural studies and a candidate for clinical use in diagnosis of cockroach allergy and development of new forms of immunotherapy.

Protein Expr Purif, 2002 Jun, 25(1), 41 - 9
Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris; Wang SH et al.; Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene . Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs . In contrast, P . pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form . Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 microM) resulted in significant improvements in expression of rPLF from P . pastoris . Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L) . P . pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column . The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF .

Biochemistry, 2002 Jun 25, 41(25), 8058 - 67
Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6); Cai J et al.; Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues . The normal function of MRP6, including its physiological substrate(s), remains unknown . To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris . The protein was expressed in the membrane fraction as a stable 170 kDa protein . Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-{alpha-(32)P}ATP and compared to those of the drug efflux pump MRP1 . rMrp6 can bind 8-azido-{alpha-(32)P}ATP in a Mg(2+)-dependent and EDTA-sensitive fashion . Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-{alpha-(32)P}ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot . Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-{alpha-(32)P}adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion . This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-{alpha-(32)P}ADP to the protein . By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins . The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, {(125)I}-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds . The P . pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6 . In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in {(125)I}-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.

Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 268 - 72
{Riboflavin overproduction in 4-aminopyrazole{3,4-d}pyrimidine-treated yeast Pichia guilliermondii}; Kutsiaba VI et al.; More than 90 mutants resistant to the adenine analogue 4-aminopyrazole{3,4-d}pyrimidine (4-APP), were isolated from a wild-type strain of yeast Pichia guilliermondii . Some of Appr mutants accumulated noticeable amounts of products absorbing at 260 nm in the culture medium, probably nucleotides and their derivatives . In comparison to the parent strain, the mutant Appr-27 synthesized greater amounts of xanthine and uracil suggesting the presence of defects in the regulation of de novo biosynthesis of purines and pyrimidines . The regulatory mutations rib80 and rib81 are known to cause riboflavin (RF) overproduction and derepression of RF-producing enzyme synthesis in P . guilliermondii . The mutant Appr-27 was crossed to the rib81 strain . The yield of RF biosynthesis in some meiotic segregants was significantly higher than that in segregants from the diploid rib81/RIB81 . Apparently, rib81 and appr mutations were combined in single genome on the favorable genetic background . An increase in RF production was also found in strains with appr mutations induced directly in the genome of the RF oversynthesizing strain rib80 rib81 . These results indicate that introduction of appr mutations into genome of P . guilliermondii can intensify their RF overproduction.

J Biotechnol, 2002 Aug 7, 97(2), 107 - 16
Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast; Nigam JN; Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate has been utilized as a substrate for ethanol production using Pichia stipitis NRRL Y-7124 . Hydrolysate fermentability was considerable improved by boiling, and overliming up to pH 10.0 with solid Ca(OH)(2) in combination with sodium sulfite . The percent total sugar utilized and ethanol yield (Y(p/s)) for the untreated hydrolysate were 20.15+/-0.17% and 0.19+/-0.003 g(p) g(s)(-1), respectively, compared with 76.0+/-0.32% and 0.35 g(p) g(s)(-1), respectively for the treated material . The fermentation was very effective at an aeration rate of 0.02 v/v/m, temperature 30+/-0.2 degrees C and pH 6.0+/-0.2 . However, the volumetric productivity (Q(p)) was still considerably less than observed in a simulated synthetic hydrolysate medium with a sugar composition similar to the hemicellulose acid hydrolysate . L-Arabinose was not fermented but assimilated . The presence of acetic acid in the hydrolysate decreased the ethanol yield and productivity considerably.

Biophys Chem, 2002 Mar 28, 95(3), 211 - 25
Roles of hydration water molecules in molecular packing of the killer toxin from Pichia farinosa in its crystalline state investigated by cryogenic X-ray crystallography; Nakasako M et al.; The hydration structures around the killer toxin from Pichia farinosa were investigated by cryogenic X-ray crystallography . In particular, those contributing to the molecular association and the crystal contacts were analyzed with respect to the geometry and the networks of hydrogen bonds . The hydration water molecules attached on the surface so as to make up the surface shape in the contact complementary and mediated the intermolecular interactions through the networks of hydrogen bonds . Careful inspection of the contact area led to a proposal as to the molecular association mode of the toxin to determine the biological function in cells . In addition, the water-associated protein-protein interactions were approximated well by a simple theoretical equation on the solvation force expected in confined geometry . The present analysis may provide a way to analyze the crystal contact and molecular recognition in macromolecules in aqueous solution.

Mol Plant Microbe Interact, 2002 Jun, 15(6), 549 - 56
Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica; Doyle EA et al.; Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle . Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases . Using differential screening, a class III pectate lyase, Mj-pel-1 (M . javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes . DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family . In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted . When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active . The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin . Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+ . The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 503 - 508
Purification and Characterization of Recombinant Hepatitis B Virus Surface Antigen SS1 Expressed in Pichia pastoris; Yang JY et al.; The purification of recombinant hepatitis B surface antigen, SS1 protein, expressed in Pichia pastoris, and the investigation of its physiochemical characters and immunogenicity were described here . Employing McAb immunoaffinity chromatography, this protein was purified to purity of 95% . The results of ELISA and Western blotting showed good antigenicity of this purified SS1 protein . CsCl gradient centrifugation and electron microscopy assay proved that this purified protein could be assembled into particles similar to the HBV subviral particles . Strong antibody responses against both the HBs and PreS1 epitopes were induced in BALB/c mice immunized with this purified protein . The simultaneous injection of a CpG adjuvant induced a Th1-like immune response against both the HBs and PreS1 epitopes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 463 - 468
Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor; Ma L et al.; Four cDNA clones, encoding truncated Flt-1 mutants consisting of loop 2, 1-2, 2-3 and 1-3, were amplified by PCR from human placenta cDNA library and the corresponding gene fragments were named Flt-1(2) Flt-1(1-2) Flt-1(2-3) and Flt-1(1-3) . In order to detect which part of the extracellular domain was involved in ligand binding, the interaction between Flt-1 mutants and hVEGF(165) was studied with yeast two-hybrid system . The data presented here suggest that both Flt-1(2-3) and Flt-1(1-3) were able to bind hVEGF(165) . Two recombinant expression plasmids, pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3), were constructed and transformed into Pichia pastoris strain GS115, respectively . After 4 days of methanol induction, the amount of the expressed Flt-1s reached 60% of total proteins in supernatant by SDS-PAGE . Recombinant proteins were purified with CM-Sepharose Fast Flow and Sephacryl S-100 chromatography . Biological activities analysis proved that 1-3 loop had slightly better biological activity than 2-3 loop in VEGF(165) binding and in inhibition of HUVEC proliferation stimulated by hVEGF(165).

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(6), 562 - 568
Expression of Human Soluble CD40 Ligand in Pichia pastoris and Its Effects on Dendritic Cells and Malignant B Cells; Shi HZ et al.; CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) superfamily and is expressed primarily on the activated CD4( )T lymphocytes . The CD40 molecule, the cognate receptor of CD40L presents on many immunocytes such as B lymphocytes, dendritic cells (DCs) as well as on some neoplastic cells . Triggering of CD40 through CD40L plays a central role in the initiation and regulation of the human immune response . In order to further investigate the possible biological roles of CD40 signaling triggered by CD40L, we subcloned the DNA fragment encoding the extracellular region of human CD40L into the pSK plasmid . After being sequenced, the target fragment was introduced into the pPICZalphaA plasmid to construct the pPICZalphaA-sCD40L expressing vector which was then transduced into Pichia pastoris GS115 cells by electroporation . The tansformant expressed sCD40L in culture supernatants with a maximum yield of about 35 mg/L . Furthermore, we found that the recombinant human soluble CD40 ligand (rhsCD40L) could effectively induced human peripheral blood monocytes(PBMCs) in vitro in the absence of TNFalpha into dendritic cells (DCs) with the typical morphology and special surface markers of dendritic cells including CD1a, CD80, CD83, and HLA-DR etc . To our surprise, the rhsCD40L also could inhibit directly in vitro proliferation of the CD40-positive multiple myeloma cell line XG-2 and the B lymphoma cell line Daudi significantly at an optimal concentration from 2.5 to 15.0 mg/L, while CD40 negative ovarian carcinoma cell lines, SKB and SKR, were not effected by either high or low concentration of rhsCD40L . Moreover, rhsCD40L had the same effects as CD40L-transfected cell in inducing XG2 cell apoptosis . Our results demonstrated that functional human soluble CD40L could be successfully expressed in the Pichia pastoris system and that the recombinant human soluble CD40L might be a potential immune adjuvant and a new powerful molecule for tumor bio-therapy.

Med Mycol, 2001 Oct, 39(5), 395 - 400
Immuno-crossreactivity of an anti-Pichia anomala killer toxin monoclonal antibody with a Williopsis saturnus var . mrakii killer toxin; Guyard C et al.; A monoclonal antibody (mAbKT4), produced against the Pichia anomala ATCC 96603 killer toxin (PaKT) was used to detect the toxin (WmKT) produced by Williopsis saturnus var . mrakii MUCL 41968 which inhibits the growth of a PaKT-sensitive P . anomala strain MUCL 41969 . Immunofluorescence studies revealed that mAbKT4 specifically labels the surface of P . anomala and W . saturnus var . mrakii, suggesting that both taxa secrete a killer toxin bearing a common epitope . Immunoblot analyses of concentrated supernatants from P . anomala and W . saturnus var . mrakii cultures showed that in both taxa mAbKT4 reacts with high molecular weight secreted proteins ranging 85-200 kDa . However, immunoblot experiments showed that the molecular weights of PaKT and WmKT are quite different, indicating that the two toxins are related but not identical molecules.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 325 - 330
Expression, Purification and Biological Activity Analysis of Human Vascular Endothelial Growth Factor (VEGF(165)) in Pichia pastoris; Ma L et al.; The human VEGF(165) cDNA was amplified by PCR, and was inserted, after confirming by DNA sequence analysis, into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of alpha factor gene to form a recombinant expression plasmids pPIC9K/hVEGF(165) . This recombinant plasmid was transformed into KM71 . Transformants were screened, cultured inflasks and induced by the addition of 1% methanol . After 4 days of methanol induction, the expressed hVEGF(165) came up to 60% of total proteins in medium supernatant as shown by SDS-PAGE . Western blot assay proved that the expressed hVEGF(165) had good antigenicity and high specificity . The recombinant protein was further purified by using Heparin-Sepharose CL6B affinity chromatography, and was proved that it had good biological activity to stimulate HUVEC proliferation and to promote collateral blood vessel formation in an acquired limb artery occlusion animal model.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 291 - 295
Expression of Human Angiostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity; Xin L et al.; Human angiostatin cDNA was amplified from human hepatoma cell line HepG2 using RT-PCR and was cloned into pPIC9K vector . Recombinant Pichia pastoris strain with 4 copies of angiostatin gene was obtained . Recombinant protein was purified by lysine-affinity Sepharose column and the finally purified angiostatin was 25 mg/L, higher than previously reported 17 mg/L . Amino acid sequence analysis revealed the identity of our protein the same with that previously reported . Recombinant angiostatin inhibited specifically the proliferation of bovine aortic endothelial cell stimulated by bFGF, with ED(50) being about 3 mg/L.

FEMS Microbiol Lett, 2002 May 7, 210(2), 187 - 91
Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris; Blanco P et al.; A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris . PG secretion was efficiently directed by the S . cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P . pastoris . The level of PGU1 activity achieved in P . pastoris was significantly enhanced when compared to activity using the same gene in S . cerevisiae . Expression of PG proteins, engineered by site-directed mutagenesis, in P . pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity . Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.

Biochemistry, 2002 Jun 11, 41(23), 7241 - 52
Evidence of an odorant-binding protein in the human olfactory mucus: location, structural characterization, and odorant-binding properties; Briand L et al.; Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily . They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules . Putative human OBP genes (hOBP) have recently been described {Lacazette et al . (2000) Hum . Mol . Genet . 9, 289-301}, but the presence of the corresponding proteins remained to be established in the human olfactory mucus . This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located . On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate . To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized . It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs . By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids . A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes . The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(4), 405 - 408
Expression of HIV-2 gp 105 Gene and Characterization of gp105 in Methyltrophic Pichia pastoris; Zhang YJ et al.; To explore a new approach to express HIV-2 external glycoprotein gp105 in methyltrophic yeast, Pichia pastoris, the gp 105 gene was obtained by PCR amplification and was cloned into the secreted expression vector pHILS1 . The positive recombinant Pichia pastoris strains were screened and identified by PCR after electroporation and induction by methanol of the gp105 expression . SDS-PAGE and Western blot analyses showed that gp105, unlike gp120 from HIV-1, could be secreted into medium by recombinant Pichia pastoris strain, and the expressed product of a 90 kD glycoprotein showed satisfactory antigenicity with specific antibody.

Microb Ecol, 2002 Apr, 43(3), 388 - 96 Epub 2002 Mar 05.
The identification and characterization of osmotolerant yeast isolates from chemical wastewater evaporation ponds; Lahav R et al.; Ramat Hovav is a major chemical industrial park manufacturing pharmaceuticals, pesticides, and various aliphatic and aromatic halogens . All wastewater streams are collected in large evaporation ponds . Salinity in the evaporation ponds fluctuates between 3% (w/v) and saturation and pH values range between 2.0 and 10.0 . We looked for microorganisms surviving in these extreme environmental conditions and found that 2 yeast strains dominate this biotope . 18S rDNA sequence analysis identified the isolates as Pichia guilliermondii and Rhodotorula mucilaginosa . Both isolates grew in NaCl concentrations ranging up to 3.5 M and 2.5 M, respectively, and at a pH range of 2-10 . There was a distinct difference between the Rhodotorula and Pichia strains and S . cerevisiae RS16 that served as a control strain with respect to accumulation of osmoregulators and internal ion concentrations when exposed to osmotic stress . The Pichia and Rhodotorula strains maintained high glycerol concentration also in media low in NaCl . Utilization of various carbon sources was examined . Using a tetrazolium-based assay we show that the Rhodotorula and Pichia strains are capable of utilizing a wide range of different carbon sources including anthracene, phenanthrene, and other cyclic aromatic hydrocarbons.

Blood, 2002 Jun 15, 99(12), 4400 - 5
Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis; Burns S et al.; A drug that specifically inhibits erythropoiesis would be clinically useful . The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose . Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103 . Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity . To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris . Using this method milligram quantities of highly purified Epo (R103A) are obtained . The yeast-expressed Epo (R103A) is properly processed and glycosylated and specifically inhibits Epo-dependent cell growth and (125)I-Epo binding . Epo (R103A) does not, however, directly induce apoptosis in 32D cells expressing EpoR . Epo (R103A) inhibits erythropoiesis of human CD34(+) hematopoietic cells and completely blocks erythroid burst-forming unit formation in normal human bone marrow colony assays . Yeast-expressed Epo (R103A) is a specific inhibitor of primary erythropoiesis suitable for testing in animal models.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 659 - 664
Expression of the Recombinant Human Interleukin-11 in Pichia pastoris; Wang TY et al.; Full length cDNA of human IL-11 was synthesized by DNA synthesizer . An expression plasmid, pGENYk, containing the recombinant DNA fragment, was linearized and transformed into Pichia pastoris . This recombinant gene was highly expressed in this yeast, and the expression product was purified by a three-step chromatography method . Analysis of the purified recombination protein with SDS-PAGE, Western blot and biological activities showed that the activity of the protein was the same as the Neumega expressed in E.coli.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 653 - 658
Expression of Human Trefoil Factor 3 in Pichia pastoris and Its Biological Activity Analysis; Wang YR et al.; The human TFF3 (trefoil factor 3) DNA fragment was amplified by polymerase chain reaction (PCR) from human fetal placenta cDNA . The gene was cloned into the Pichia pastoris expression vector pPIC9K containing AOX 1 promoter and alpha-factor leader sequence . Multi-copies insertion transformants were screened on G418 plates . After the induction by 2% methanol for 48 hours, the expression of dimeric hTFF3 came up to 45% of total proteins in medium, as identified by SDS-PAGE and Western blot assay . The recombinant protein was further purified by S-Sepharose, Q-Sepharose ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography to the 95% purity, as shown by densitometric scanning . The N-terminus and molecular weight of the recombinant hTFF3 was in good agreement with the native hTFF3 . The recombinant protein was proved to have good biological activity of preventing rats from the gastric ulcer induced by hydrochloric acid.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 600 - 606
PCR Based Cloning and Sequence Analysis of the Pichia pastoris Cystathionine beta-Synthase Gene; Li DY et al.; The cystathionine beta-Synthase (CBS) gene of Pichia pastoris has been cloned by homology to the CBS gene of Saccharomyces cerevisiae . First, based on the homology alignment of CBS genes from different sources, a pair of degenerate PCR primers were designed to amplify a conservative fragment of Pichia pastoris CBS gene . After the sequence was revealed, 5' RACE and 3' RACE were performed separately . The whole sequence of Pichia pasotris CBS gene was assembled . This gene encodes a polypeptide of 501 residues . Comparison of deduced amino acid sequence of this new gene with that of S . cerevisiae CBS showed 54% identity . Disruption of CBS gene resulted in a Cys(-) auxotrophy in Pichia pastoris . Thesequence was submitted to GenBank/EMBL/DDBJ under Accession No.AF367364.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7797 - 802
An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis; Faik A et al.; Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of alpha-xylosyltransferase and beta-glucan synthase activities . Here we report that etiolated pea microsomes contain an alpha-xylosyltransferase that catalyzes the transfer of xylose from UDP-{(14)C}xylose onto beta(1,4)-linked glucan chains . The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues . Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode alpha-xylosyltransferases involved in xyloglucan biosynthesis . To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above . One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity . Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the (14)C-labeled xylose mainly to the second glucose residue from the nonreducing end . Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an alpha(1,6)-linkage to the glucan chain . We conclude that this Arabidopsis gene encodes an alpha-xylosyltransferase activity involved in xyloglucan biosynthesis.

Eur J Biochem, 2002 May, 269(10), 2538 - 45
Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed; Palomares O et al.; 2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor . The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced . The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn . Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris . The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC . Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein . As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity . The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed . In conclusion, the recombinant expression of napin precursors in P . pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.

Microb Ecol, 2001 Aug, 42(2), 186 - 192
Yeast-Yeast Interactions in Guava and Tomato Fruits; Abranches J et al.; The host specificity of yeast-yeast interactions was investigated for two yeast types, represented by six pairs of Pichia membranifaciens clade yeasts (Pichia membranifaciens or Issatchenkia occidentalis) with apiculate yeasts (Kloeckera apis, Kloeckera africana, or Saccharomycodes ludwigii), commonly found in fruits . Competitive interactions between the two types were detected in both ripe tomato and guava fruit pulp . The differences in growth rates and carrying capacities depended on fruit type (host) and culture conditions (monocultures versus bicultures) . These differences were probably due to nutrient composition of each fruit . Pichia membranifaciens did not show host dependent responses, but the apiculate yeasts and Issatchenkia occidentalis did . Depending on yeast strain and culture conditions (i.e., monoculture or biculture), carbon, nitrogen, and vitamins were investigated as potential limiting growth factors in guava fruit . Both singular and multiple limiting nutrients were implicated.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 318 - 22
{Specific antibodies against recombinant MSP1 of Plasmodium falciparum strongly inhibit the parasite growth in vitro}; Zhang DM et al.; In order to produce large amounts of protein for vaccine trials, a synthetic msp1-42 gene was inserted into Pichia pastoris expression vector and the plasmid was introduced into Pichia pastoris SMD1168 by electroporation . The expressed MSP1-42 was secreted into the protein-free medium . To measure the conformational properties of MSP1-42,16 monoclonal antibodies (11 recognizing conformational epitopes) were allowed to interact with the Pichia-derived MSP1-42, and all antibodies specific for conserved and K1 protype interacted with the protein . Interestingly, three monoclonal antibodies (e.g . 9.8, 13.1 and 7.3), that were shown not to interact with CHO-derived MSP1, could interact with the Pichia-derived MSP1-42 . Rabbits were immunized with recombinant MSP1-42 formulated with CFA adjuvant four times . The rabbits were bled on the day 3 after last immunization, and total IgG isolated by protein A column from the immunized rabbits was shown to strongly inhibit the parasite growth in vitro dose-dependently, whereas IgG from rabbit with adjuvant had no inhibition.

Biochem J, 2002 Sep 1, 366(Pt 2), 603 - 11
Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris; Lee GC et al.; The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis . An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium . The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C . rugosa lipase . The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C . The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min . Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters . Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis . Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate . The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate . In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate . From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.

J Biol Chem, 2002 Jul 26, 277(30), 26994 - 7005 Epub 2002 May 15.
Structural and functional genomics of the CPT1B gene for muscle-type carnitine palmitoyltransferase I in mammals; van der Leij FR et al.; Muscle-type carnitine palmitoyltransferase I (M-CPT I) is a key enzyme in the control of beta-oxidation of long-chain fatty acids in the heart and skeletal muscle . Because knowledge of the mammalian genes encoding M-CPT I may aid in studies of disturbed energy metabolism, we obtained new genomic and cDNA data for M-CPT I for the human, mouse, rat, and sheep . The introns of these compact genes are 80% (mouse versus rat) and 60% (mouse versus human) identical . Sheep and goat, but not cow, pig, rodent, or human promoter sequences contain a short interspersed repeated sequence (SINE) upstream of highly conserved regulatory elements . These elements constitute two promoters in humans, sheep, and mice, and, contrary to previous reports, there is a second promoter in rats as well . Thus, the transcriptional organization of these genes is more uniform than previously supposed, with interspecies differences in the 5'-ends of the mRNAs reflecting differences in splicing; only in humans extensive splicing and splice variation is found in the 5'- and 3'-untranslated regions . In the mouse, intron retention was detected in heart, muscle, and testes and may indicate an additional mechanism of regulation of M-CPT I expression . Splice variation in the coding region was previously proposed to lead to expression of CPT I enzymes with altered malonyl-CoA sensitivity (Yu, G . S., Lu, Y . C., and Gulick, T . (1998) Biochem . J . 334, 225-231) . However, when expressed in the yeast Pichia pastoris, none of three earlier described splice variants had CPT I activity . Therefore, the involvement of splice variation of M-CPT I in the modulation of malonyl-CoA inhibition of fatty acid oxidation may be less relevant than hitherto assumed.

J Biol Chem, 2002 Jul 26, 277(30), 26980 - 6 Epub 2002 May 15.
Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris . Role of glycosylation in alpha-bungarotoxin binding; Psaridi-Linardaki L et al.; The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris . The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer . halpha-(1-210) bound (125)I-alpha-bungarotoxin with a high affinity (K(d) = 5.1 +/- 2.4 nm), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K(i) approximately 7.5 mm) . Interestingly, (125)I-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210) . Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to halpha-(1-210), as did antibodies from a large proportion of myasthenic patients . These results suggest that the extracellular domain of the human AChR alpha subunit expressed in P . pastoris has an apparently near native conformation . The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies.

FEMS Microbiol Lett, 2002 Mar 19, 209(1), 57 - 62
Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid, a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid; Iacazio G et al.; During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth . The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments . The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.

J Biol Chem, 2002 Jul 12, 277(28), 25735 - 40 Epub 2002 May 02.
Manipulating monomer-dimer equilibrium of bovine Beta -lactoglobulin by amino acid substitution; Sakurai K et al.; Bovine beta-lactoglobulin, a major protein in cow's milk composed of nine beta-strands (betaA-betaI) and one alpha-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0 . It is assumed that the intermolecular beta-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation . Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation . Various I-strand mutants showed decreases in K(a), suggesting that the intermolecular beta-sheet is essential for dimer formation . By substituting either Asp(33) or Arg(40) on the AB-loop to oppositely charged residues (i.e . R40D, R40E, and D33R), a large decrease in K(a) was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg(40) on one monomer and Asp(33) on the other monomer in the wild-type dimer . However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg(33) and Asp(40) of different molecules . These results suggested that protein-protein interactions of bovine beta-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.

Clin Exp Allergy, 2002 Jan, 32(1), 87 - 92
cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family; Diaz-Perales A et al.; BACKGROUND: Lipid-transfer proteins (LTPs) have been identified as major allergens of Rosaceae fruits in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities . Pru p 3 and Mal d 3, the allergens from peach and apple, respectively, have a main clinical relevance in these areas . OBJECTIVE: To isolate and characterize cDNAs for Pru p 3 and Mal d 3, and to produce recombinant Pru p 3 in the yeast Pichiapastoris . METHODS: cDNAs for both allergens were isolated by polymerase chain reaction using nondegenerated primers . Expression of Pru p 3 was performed in P . pastoris using the pPIC 9 vector . The recombinant product was purified by gel-filtration chromatography followed by RP-HPLC . Immunodetection and immunoblot inhibition assays were carried out with sera from peach-allergic patients . RESULTS: The cDNAs for both Pru p 3 and Mal d 3 showed a 273 open reading frame coding for the 91 amino acid mature polypeptides . The deduced amino acid sequences exhibited N-terminal regions fully identical to those previously determined for the natural peach and apple allergens . Pru p 3 was expressed in P . pastoris at 20 mg/L of culture medium . The recombinant allergen showed the same N-terminal sequence (plus a glutamic acid added for proper extracellular expression) and apparent molecular size as natural Pru p 3 . Both the recombinant and natural forms of Pru p 3 displayed similar immunoglobulin (Ig)E-binding capacity in immunodetection and immunoblot inhibition assays . CONCLUSIONS: Comparison of the complete primary structures of mature Pru p 3 and Mal d 3 deduced from their corresponding cDNA clones supports the close relationship between both allergens . Recombinant Pru p 3 binds IgE in vitro like its natural counterpart . Therefore, it can be a useful tool for specific diagnosis and structural studies.

Res Microbiol, 2002 Apr, 153(3), 173 - 80
Feasibility of copper uptake by the yeast Pichia guilliermondii isolated from sewage sludge; de Siloniz MI et al.; Copper is one of the most abundant toxic heavy metals in municipal wastewaters and, in consequence, in sewage sludge and compost . The ability of a strain of the yeast Pichia guilliermondii, which was isolated from sewage sludge, to eliminate copper has been evaluated, using both viable and nonviable biomass . It has been found that raising concentrations of copper affected both morphology and physiological parameters of the viable yeast, and it is thought that a process of bioaccumulation may be involved in its copper uptake . The growth rate of nonadapted cells decreased with increasing concentrations of copper, mainly due to a decrease in the biomass yield . The cells could be adapted by training with increasing copper concentrations up to 317.7 mg/l . This adaptation was an all-or-nothing process: once cells had adapted, the biomass yield, metabolic flux and consequent growth rate were constant and independent of the external copper concentration . Also, it was determined that up to 20 mg of copper per gram of viable adapted biomass could accumulate from the medium (i.e., double the amount when using nonadapted viable biomass) . Finally, adsorption data on nonviable cells were found to be well modeled by the Langmuir isotherm (qmax = 9.09 mg/g of biomass).

Curr Microbiol, 2002 Jun, 44(6), 391 - 5
Secretion of beta-1,3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine; Masih EI et al.; Pichia membranifaciens strain FY-101, isolated from grape skin, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mold disease of the grapevine . When grown together on solid as well as in liquid media, the yeast brings about the inhibition of Botrytis cinerea, which in turn loses its ability to produce the grey mold symptoms on the grapevine plantlets . The secretion of beta-1,3-glucanases by P . membranifaciens is one of the possible mechanisms related to this antagonism . In vitro experiments confirm that this yeast can be used as a biological control organism against B . cinerea . An account of this antagonism and the production of beta-1,3-glucanases by P . membranifaciens is given here.

Insect Mol Biol, 2002 Jun, 11(3), 197 - 205
Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans; Hamilton JV et al.; Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen . We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal . The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion . Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen . This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

J Exp Bot, 2002 May, 53(372), 1367 - 76
The apoplastic oxidative burst in response to biotic stress in plants: a three-component system; Bolwell GP et al.; The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells . It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating . However, one mechanism may be dominant in any given species . NADPH oxidases have been implicated in a number of systems and have been cloned and characterized . However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase . The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses . The third component, the actual reductant/substrate, has remained elusive . The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation . The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates . The mechanism has proved to be complex and may involve a number of low molecular weight components . Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production . This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase . The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme . Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens . Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.

Biochim Biophys Acta, 2002 Apr 12, 1574(3), 293 - 303
A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp . E2; Harhangi HR et al.; Anaerobic fungi have very high cellulolytic activities and thus degrade cellulose very efficiently . In cellulose hydrolysis, beta-glucosidases play an important role in prevention of product inhibition because they convert oligosaccharides to glucose . A beta-glucosidase gene (cel1A) was isolated from a cDNA library of the anaerobic fungus Piromyces sp . E2 . Sequence analysis revealed that the gene encodes a modular protein with a calculated mass of 75800 Da and a pI of 5.05 . A secretion signal was followed by a negatively charged domain with unknown function . This domain was coupled with a short linker to a catalytic domain that showed high homology with glycosyl hydrolases belonging to family 1 . Southern blot analysis revealed the multiplicity of the gene in the genome . Northern analysis showed that growth on fructose resulted in a high expression of cel1A . The cel1A gene was successfully expressed in Pichia pastoris . The purified heterologously expressed protein was shown to be encoded by the cel1A gene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of a tryptic digest . Purified heterologous Cel1A was active towards several artificial and natural substrates with beta-1-4 linked glucose molecules with a remarkably high activity on cellodextrins . The enzyme was strongly inhibited by D-glucono-1,5-delta-lactone (K(i)=22 microM), but inhibition by glucose was much less (K(i)=9.5 mM) . pH and temperature optimum were 6 and 39 degrees C, respectively . The enzyme was fairly stable, retaining more than 75% of its activity when incubated at 37 degrees C for 5 weeks . Transglycosylation activity could be demonstrated by MALDI-TOF MS analysis of products formed during degradation of cellopentaose.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 23 - 6
{Immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris}; Tong Y et al.; BACKGROUND: To study to immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris . METHODS: BALB/c mice were immunized with the recombinant HEV ORF2 protein . The ability of antiserum to bind HEV was tested using affinity-capture reverse transcription polymerase chain reaction (RT-PCR) . Moreover, the recombinant protein was used to immunize BALB/c mice by different routes with different adjuvants . Serum conversion rate of anti-HEV antibody and the ELISA titer were detected . RESULTS: The antiserum could capture native HEV for RT-PCR . As to the immunization effect, the immune response by intramuscular route was better than that of the intraperitoneal route . The protein with alum and CpG adjuvant could elicits more significant immune responses than using the alum adjuvant alone . The best way was to immunize with the protein with alum and CpG adjuvant by intramuscular route with a boosted injection on the 4th week after the first immunization . The ED50 was 0.023 microgram . This is the first report that the antibody elicited by recombinant HEV ORF2 protein expressed in pichia pastoris recognizes native HEV . High immunogenicity of this kind of ORF2 was also demonstrated by inducing strong immune response in mice with good ED50 result . CONCLUSIONS: The high immunogenicity of this kind of HEV ORF2 may make a foundation for the development of new type of hepatitis E vaccine.

Mol Biochem Parasitol, 2002 Apr 30, 121(1), 99 - 105
Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries; Mathieu MA et al.; Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals . The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues . Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL) . We first screened Sm32 using a P1-diverse library . This library demonstrated the absolute specificity of Sm32 for asparagine at P1 . To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized . The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi . The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2 . Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3 . However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn . Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.

Mol Biochem Parasitol, 2002 Apr 30, 121(1), 49 - 61
SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni; Caffrey CR et al.; Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma . A novel cathepsin B endopeptidase from adult S . mansoni has been isolated and characterized . The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31 . A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material . SmCB2 has been functionally expressed in and purified from Pichia pastoris . Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE . Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates . We conclude that the recombinant enzyme is properly folded . The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg . By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes . By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females . The enzyme may be lysosomal and function at the host parasite-interface.

Eur J Biochem, 2002 Apr, 269(8), 2083 - 92
Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases; Grobe K et al.; Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls . They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs . Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall . Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension . Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases . Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein . We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable . Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules . Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins . These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.

Biochim Biophys Acta, 2002 Apr 1, 1596(1), 76 - 82
Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D; Cater SA et al.; The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated . Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited . The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D . Re-evaluation of the potato inhibitor revealed that it too was more potent (>20-fold) towards yeast proteinase A than cathepsin D and so might be renamed the potato inhibitor of proteinase A . The potency towards yeast proteinase A may reflect a similarity between this fungal enzyme and aspartic proteinases produced by fungal pathogens which attack tomato and/or potatoes.

Electrophoresis, 2002 Apr, 23(7-8), 993 - 7
Combination of zymography and immunodetection to analyze proteins in complex culture supernatants; Poppelmann M et al.; The physiological function of an allergen might be an important factor for the allergenicity . The major grass pollen allergen Phl p 1 shows sequence similarities to the consensus sequences of cysteine proteases . However, up to now, the proteolytic activity of Phl p 1 is controversial . The culture supernatant of Phl p 1-transfected clones from Pichia pastoris showed a proteolytic activity but this might be due to Phl p 1 or irrelevant yeast contaminants . To solve this question, we made use of the zymogram technique and improved it . Substrate as well as substrate concentration was changed from 1% casein to 0.25% skimmed milk powder . For staining, we used a colloidal Coomassie stain (RotiBlue) with a higher sensitivity and better practicability than the conventional Coomassie staining . The proteins in the zymogels and in the SDS-PAGE gels showed similar electrophoretic mobility . Furthermore, the zymogels could be blotted and immunostained . Thus, the molecular mass of the proteolytic bands could be determined and directly compared with immunoblotting results . To clearly assign the protease, we separated the culture supernatant of the Phl p 1-transfected P . pastoris clone by affinity chromatography with monoclonal antibody . Our studies demonstrate that the proteolytic activity did not belong to the recombinant allergen but to the yeast proteins . The enzyme was classified by zymogram inhibition tests as a strong serine protease.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 40 - 4
{Expression of the human soluble low density lipoprotein receptor in methylotropic yeast}; Hu J et al.; To obtain the expression of human low density lipoprotein receptor ligand binding domain in methylotropic yeast, firstly the DNA fragment encoding for human low density lipoprotein receptor ligand binding domain was amplified by RT-PCR with human hepatoma Bel-7402 total RNA as template . The nucleotide sequencing analysis indicated that the sequence of the cloned DNA fragment was as same as the reported human LDLR cDNA sequence . Then the expression vector pPIC9K-sLDLr was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation . The recombinant sLDLR was identified by SDS-PAGE, Western blot and Ligand binding blot in supernatant of GS115/pPIC9K-sLDLr . The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sLDLR was about 36 kD . And the ligand binding blot analysis indicated the expressed sLDLR has the biological activity . The sLDLR, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS115).

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 6 - 10
Cloning, expression and purification of gussurobin, a thrombin-like enzyme from the snake venom of Gloydius ussuriensis; Yang Q et al.; Total RNAs were extracted from the venom gland of the snake Gloydius ussuriensis . The cDNA of gussurobin, a thrombin-like enzyme from Gloydius ussuriensis, was cloned and amplified by RT-PCR . Assay of the nucleotide sequence of the cDNA allowed postulation of the complete amino acid sequence for Gloydius ussuriensis, Chinese Viperdae . Its amino acid sequence exhibits significant homology with that of other snake thrombin-like enzymes . The cDNA of gussurobin was inserted into the vector pPIC 9K and expressed successfully in Pichia pastoris, strain GS115 . The recombinant gussurobin was separated and purified from 500 ml culture and showed one band on SDS-PAGE.

Zhonghua Yi Xue Za Zhi, 2002 Feb 10, 82(3), 198 - 202
{Synthesis and expression of 42 kD C-terminal region of the major merozoite surface protein (MSP1 - 42) of P . falciparum 3D7 strain in pichia pastoris}; Zhang D et al.; OBJECTIVE: Production of 3D7/MSP1 - 42 recombinant protein with correct conformation in Pichia pastoris for vaccine efficiency assay . METHODS: Asymmetric PCR-based method was utilized to synthesize the 1 202 bp 3D7/msp1 - 42 gene . The expressing plasmid containing the synthetic gene was introduced into Pichia pastoris by electroporation . The secreted product was detected by Western Blot . RESULTS: The redesigned entire 3D7/msp1 - 42 gene was generated with error-free, and expressed to produce 42 kD recombinant protein in secreted form . Conformational monoclonal antibody specific for MSP1 C-terminal can interact with the recombinant protein . CONCLUSION: The redesigned 3D7/msp1 - 42 gene was expressed in P . pastoris with full length of recombinant protein which resembled most likely to the native protein.

Mol Plant Microbe Interact, 2002 Mar, 15(3), 243 - 50
A tobacco S-like RNase inhibits hyphal elongation of plant pathogens; Hugot K et al.; Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica . Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment . After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P . parasitica zoospores and from Fusarium oxysporum conidia in vitro . Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs . Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P . parasitica . Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space . The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Mikrobiol Z, 2002 Jan-Feb, 64(1), 20 - 6
{On the identity of Candida utilis and Pichia jadinii yeast species}; Ignatova EA et al.; Life cycles of Candida utilis (Henneberg) Lodder et Kreger-van Rij imperfect yeast (13 strains) used in industry were the study subject . When the strains were mated, we detected one of the stages of sexual process--conjugation of cells of the opposite mating type . Most of the studied cultures conjugated on the 2d-3d day . No ascospores were formed . Haploidy and heterothallism of the studied C . utilis strains were confirmed by hybridization of auxotrophic mutants . Based on PCR assay results, the yeasts are demonstrated to belong to the ascospore perfect yeast species of Pichia jadinii (A . et R . Sartory . We ill et Meyer, Wickerham) Kurtzman.

Clin Exp Allergy, 2002 Mar, 32(3), 448 - 54
What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?
Diaz-Perales A, Sanchez-Monge R, Blanco C, Lombardero M, Carillo T, Salcedo G.
BACKGROUND: Class I chitinases are the major panallergens in fruits associated with the latex-fruit syndrome . These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain . The role of these domains in their allergenic capacity is still controversial . OBJECTIVE: We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model . METHODS: Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector . Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography . The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection . Immunoblot, RAST and CAP inhibition assays were also performed . RESULTS: Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies . In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex-fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays . Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5 . RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70-90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results . CONCLUSIONS: Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro . The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments . RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens.

Curr Genet, 2002 Feb, 40(5), 339 - 44 Epub 2001 Dec 08.
A Penicillium chrysogenum gene ( aox) identified by specific induction upon shifting pH encodes for a protein which shows high homology to fungal alcohol oxidases; Holzmann K et al.; Parallel cultures of Penicillium chrysogenum were grown in controlled bioreactors under conditions of penicillin production and one was shifted from the initial pH 6.0 to pH 8.0 . RNA was isolated from both cultures and used for a differential hybridization experiment to identify genes specifically induced upon this pH shift . About 2,000 plaques of a cDNA library constructed from pH 8.0 material were screened with a pH 8.0 to pH 6.0 subtracted probe . Two independent clones of which the RNA was highly abundant in pH 8-shifted material and essentially not present in pH 6 material were isolated . Both clones were found to belong to one specific gene, which could be identified by sequence homology as an alcohol oxidase . The identified aox gene encoded for a peptide of 666 amino acids, interrupted by nine introns; and it showed high homology (>65% identity) to alcohol oxidases from Cladosporium fulvum and the methanol-utilizing yeasts Candida boidinii, Hansenula polymorpha (now Pichia angusta) and P . pastoris . The transcription start was identified by primer extension analysis . Southern analysis revealed that related genes are widely distributed among fungal species.

Protein Eng, 2002 Mar, 15(3), 251 - 5
Alternative CUG codon usage (Ser for Leu) in Pichia farinosa and the effect of a mutated killer gene in Saccharomyces cerevisiae; Suzuki C et al.; The halotolerant yeast Pichia farinosa KK1 strain produces a killer toxin termed SMKT (salt-mediated killer toxin) . Mass spectrometry and Edman sequencing of peptides from the mature SMKT and secreted protoxin demonstrate that positions specified by the CUG codon contain unmodified serine (Ser) in P.farinosa . In order to express the authentic SMK1 product in Saccharomyces cerevisiae, which uses the universal genetic code, the three CUG codons corresponding to Ser87, Ser137 and Ser206 in the SMK1 gene were changed to universal Ser codons by site-directed mutagenesis . The expression of the modified SMK1 gene with universal Ser codons was lethal in S.cerevisiae, as well as that of the unmodified SMK1 gene with the CUG codons . The secretion of protoxin with the authentic amino acid sequence from the modified SMK1 was significantly increased, whereas the transcription level of SMK1 was not affected in the presence or absence of CUG codon . Our results provide the first in vivo evidence that non-universal decoding of CUG is used in a hemiascomycetous yeast, P.farinosa.

Acta Microbiol Pol, 2001, 50(3-4), 291 - 9
Application of Saccharomyces cerevisiae and Pichia stipitis karyoductants to the production of ethanol from xylose; Kordowska-Wiater M et al.; Karyoductants of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 with the ability to ferment D-xylose to ethanol were isolated . The ability of these isolates to assimilate different sugars, ethanol tolerance and ethanol production from D-xylose was investigated . Karyoductants didn't grow on starch, lactose and cellobiose, like S . cerevisiae, but showed good growth on xylose and L-arabinose, like P . stipitis . All isolates fermented xylose to ethanol slower than P . stipitis and with lower yields, 0.09 - 0.16 g/g . They secreted also about 3.4 - 7.1 g/dm3 of xylitol to the culture medium (P . stipitis only 0.06 g/dm3) . The karyoductants showed an average tolerance to ethanol when compared with the parent strains and fermented glucose in the presence of 6% alcohol whereas parent strain S . cerevisiae and P . stipitis showed exogenic ethanol tolerance of 9% and 3%, respectively.

Fungal Genet Biol, 2002 Apr, 35(3), 261 - 76
Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey; Wang GY et al.; cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy . The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pI of 8.4 . The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites . Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif . The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M . fructicola . MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni(2+)-nitrilotriacetic acid affinity chromatography . Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.

Scand J Infect Dis, 2002, 34(2), 140 - 1
Pichia ohmeri prosthetic valve endocarditis and review of the literature; Reina JP et al.; Fungal prosthetic valve endocarditis (PVE) is a serious complication of valve replacement surgery . We report the first case of documented Pichia ohmeri PVE in an immunocompetent man who was successfully treated with valve replacement and antifungal therapy with amphotericin B.

Parasitology, 2002 Mar, 124(Pt 3), 237 - 46
Immunogenic properties of the Plasmodium vivax vaccine candidate MSP1(19) expressed as a secreted non-glycosylated polypeptide from Pichia pastoris; Soares IS et al.; The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) is one of the most promising vaccine candidates against the erythrocytic forms of malaria . In the present study, a gene encoding the Plasmodium vivax MSP1(19) epitope (PvMSP1(19)) and the Pan-Allelic DR epitope (PADRE) was expressed in the methylotrophic yeast Pichia pastoris . A non-glycosylated form of the recombinant protein rPvMSP1(19)-PADRE was purified from culture supernatants . This recombinant protein maintains its antigenicity, being recognized by a very high percentage (85.6%) of sera from Brazilian individuals naturally exposed to P . vivax . The antibody immune response elicited by rPvMSP1(19)-PADRE was compared in C57BL/6 mice immunized with different adjuvant formulations . After 3 immunizing doses, antibody titres induced in the presence of the adjuvants monophosphoryl lipid A, trehalose dicorynomycolate and cell wall skeleton or alum plus CpG ODN 1826 were as high as titres generated by Complete Freund's Adjuvant . Based on these immunological studies, we concluded that rPvMSP1(19)-PADRE deserves further evaluation in pre-clinical immunizations against P . vivax in non-human primates.

J Biol Chem, 2002 Jun 28, 277(26), 23702 - 8 Epub 2002 Mar 28.
Increased expression of Hsp40 chaperones, transcriptional factors, and ribosomal protein Rpp0 can cure yeast prions; Kryndushkin DS et al.; The Sup35 (eRF3) translation termination factor of Saccharomyces cerevisiae can undergo a prion-like conformational conversion, thus resulting in the {PSI(+)} nonsense-suppressor determinant . In vivo this process depends critically on the chaperone Hsp104, whose lack or overexpression can cure {PSI(+)} . The use of artificial prion {PSI(+)PS} based on a hybrid Sup35PS with prion domain from the yeast Pichia methanolica allowed us to uncover three more chaperones, Ssb1, Ssa1, and Ydj1, whose overexpression can cure prion determinants . Here, we used the {PSI(+)PS} to search a multicopy yeast genomic library for novel factors able to cure prions . It was found that overexpression of the Hsp40 family chaperones Sis1 and Ynl077w, chaperone Sti1, transcriptional factors Sfl1 and Ssn8, and acidic ribosomal protein Rpp0 can interfere with propagation and manifestation of {PSI(+)PS} in a prion strain-specific manner . Some of these factors also affected the manifestation and propagation of conventional {PSI(+)} . Excess of Sfl1, Ssn8, and Rpp0 influenced at least one of the tested chaperone-specific promoters, SSA4, HSP104, and model promoters, with either the heat shock or stress response elements . Thus, the induction of chaperone expression by these proteins could explain their prion-curing effects.

Receptors Channels, 2001, 7(6), 453 - 69
Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris; Schiller H et al.; In the present report, the successful solubilization and purification of the ETB receptor heterologously produced in the methylotrophic yeast P . pastoris is described for the first time . In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of recombinant P . pastoris cells was much easier and less time consuming . Recombinant P . pastoris clones producing two different ETB receptor constructs were grown in a fermenter to a density of about 360 g/l . After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells . A method for the large-scale preparation of membranes was established . Solubilization of the recombinant ETB receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside . The stability of the solubilized and ligand-bound receptor was examined in detail . Subsequently, two purification methods for two different receptor constructs were tested and a large-scale procedure for isolation of recombinant receptor was established . In general, the purification methods described herein will be adaptable to other G protein-coupled receptors heterologously produced in heterologous expression systems including P . pastoris.

Appl Environ Microbiol, 2002 Apr, 68(4), 1955 - 61
Yeast species associated with orange juice: evaluation of different identification methods; Arias CR et al.; Five different methods were used to identify yeast isolates from a variety of citrus juice sources . A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX . Twenty-three different species were identified representing 11 different genera . Distribution of the species was considerably different depending on the type of sample . Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ) . Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species . Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates . Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis . The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively . Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H . occidentalis, H . vineae, Pichia fermentans, and Saccharomycopsis crataegensis . With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

Acta Crystallogr D Biol Crystallogr, 2002 Apr, 58(Pt 4), 683 - 6 Epub 2002 Mar 22.
Expression, purification and preliminary crystallographic studies of alpha-amylase isozyme 1 from barley seeds; Robert X et al.; The germinating barley seed contains two major alpha-amylase isozyme families, AMY1 and AMY2, involved in starch degradation to provide energy used by the plant embryo for growth . Many years of difficulty in growing three-dimensional crystals of natural AMY1 have now been overcome by a nonapeptide truncation of the enzyme C-terminus . The truncated enzyme was overexpressed in Pichia pastoris, purified and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as precipitant and 2-propanol as an additive . Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 88.36, b = 72.82, c = 61.74 A and one molecule per asymmetric unit.

DNA Seq, 2001 Dec, 12(5-6), 367 - 80
Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins; Tan H et al.; Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized . The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP . The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2 . Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units . Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development . Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only . The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes . The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1) . Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers . However, the 50 kDa protein was absent in other cotton tissues.

Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 683 - 7
{Decolorization and isolation of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris}; Zhou WB et al.; Schemes for decolorization of broth and isolation of recombinant hirudin from the fermentation supernatants of recombinant yeast Pichia pastoris were developed to remove a great deal of non-desired proteins and coloring matters . Based on the stability of hirudin, heating is desirable to pre-treat the fermentation broth . Several different column chromatographies, including anion-exchange, cation-exchange, hydrokyapatite adsorption, hydrophobic interaction, gel filtration and their combination were investigated on the efficiencies of decolorization and isolation . Among these methods, the combination of cation-exchange and anion-exchange was found to be a success in both decolorization and isolation of hirudin.

Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 648 - 51
{Studies on the expression condition of human trefoil factor 3 in Pichia pastoris}; Wang YR et al.; In order to enhance the expression level of human trefoil factor 3 (hTFF3) in Pichia pastoris, we optimized the transformant growth conditions in shake flasks including carbon sources in growth medium, inoculation ratio, methanol concentration, pH rotation speed and inducing time . The transformant could grow on the glucose to OD600 5.0 after 14 hours inoculation . The best inoculation ration of 100 mL growth medium to the induction medium was 1:1 . The expression level of dimeric human trefoil factor 3 induction with 1% methanol for 48 hours at pH 6.0, agitation speed 240 r/min could reach 20 mg/L with OD600 15 . The protein was expressed in 5-liter fermentor with 2% methanol induction for 32 hours, finally the cell density reached OD600 120 . 100 mg/L of recombinant hTFF3 was obtained in the supernatant.

Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 643 - 7
{Expression, purification and identification of human matrix metalloproteinase-2}; Ding XY et al.; The expression sequence of matrix metalloproteinase-2 (MMP-2) has been obtained by PCR amplifying, restriction enzyme cut and sequencing analysis demonstrate that the sequence is correct . The recombinant expression plasmid pPIC9/MMP-2 containing MMP-2 is constructed and transformed the yeast Pichia pastoris . Recombinant matrix metalloproteinase-2 protein was expressed in Pichia pastoris in great deal after induction by methanol . The purity of the recombinant MMP-2 filtrated through Sephacryl S-200 reached to electrophoresis purity . With the ability to degrade gelatin and IV type collagen, recombinant MMP-2 has the similar substrate specificity with natural MMP-2 . The recombinant MMP-2 with 50 kD molecular weight is smaller than natural MMP-2, which suggested degradation occurred to it.

Allergy, 2002 Mar, 57(3), 215 - 20
Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens; Au LC et al.; BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms . The N-terminal amino acid sequences of some acidic Cyn d 1 isoforms were found to be different from those of Cyn d 1 cDNA clones identified previously . METHODS: A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library . The reactive clones were isolated and verified by sequencing . Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins . RESULTS: All four cDNA clones encode the full-length Cyn d 1 with mature proteins of 244 amino acid residues . A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified . The predicted isoelectric focusing (pI) values of the newly identified Cyn d 1s are acidic while pIs of the previously identified Cyn d 1s are basic . The two recombinant acidic Cyn d 1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cyn d 1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay . CONCLUSIONS: The present study identified full-length cDNAs encoding new isoallergens of Cyn d 1, and separated Cyn d 1 gene into an acidic group and a basic group.

Blood, 2002 Apr 1, 99(7), 2562 - 8
Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma; Zhu D et al.; Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site . Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional V(H) genes as a consequence of somatic mutation . FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation . Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P <.001) . Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 {41%} patients) . Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively . In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv in Pichia pastoris, and glycosylation was demonstrated . These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL . Most novel sites are located in the complementarity-determining regions . V(H) sequences of nonfunctional V(H) genes contained few sites, arguing for positive selection in FL . One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment . This common feature of FL may be critical for tumor behavior.

Eur J Biochem, 2002 Mar, 269(6), 1753 - 60
Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene; Xu B et al.; Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis . The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5 . The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns . The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence . The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1 . The expression level is strongly affected by the induction temperature . A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase . However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained . The specific activity of the purified recombinant M . edulis beta-mannanase was close to that of the wild-type enzyme . Also pH and temperature optima were the same as for the native protein . In conclusion, P . pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase . This is the first time a mollusc beta-mannanase has been characterized at the DNA level.

FEMS Microbiol Lett, 2002 Jan 22, 207(1), 97 - 102
Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris; Poirier Y et al.; Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers . A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris . The PHA synthase was expressed under the control of the promoter of the P . pastoris acyl-CoA oxidase gene . Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium . PHA accumulated as inclusions within the peroxisomes . P . pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plants.

Biochim Biophys Acta, 2002 Feb 28, 1580(2-3), 95 - 109
Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis; Nykiforuk CL et al.; cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L . cv Jet Neuf) . BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1 . Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells . Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose . TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT) . The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity . The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide . Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level . In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.

J Virol Methods, 2002 Apr, 102(1-2), 175 - 90
Erratum to "characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E . coli and P . pastoris"; Watelet B et al.; The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris . A hexa-histidine tag was introduced at the C terminus of the E . coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography . The P . pastoris expressed HBcAg was isolated following heat treatment . The two recombinant HBcAgs were purified further on a sucrose gradient . Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P . pastoris and reaction with Ellman's reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E . coli or P . pastoris . Electron microscopy indicated that the E . coli and the P . pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm . Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P . pastoris derived particles suggesting structural discrepancies between the two recombinant molecules . The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum . The preliminary results indicate that the P . pastoris HBcAg produced intracellularly is more suitable than the renatured E . coli HBcAg for detection of anti-HBc in this diagnostic assay.

Zhonghua Xue Ye Xue Za Zhi, 2001 Mar, 22(3), 138 - 40
{High efficient expression of recombinant human platelet factor IV in pichia pastoris}; Wang C et al.; OBJECTIVE: To express recombinant human platelet factor 4 (PF4) in methanol yeast . METHODS: PF4 cDNA amplified by PCR was cloned into the mating factor alpha (MFalpha) signal sequence, downstream the alcohol oxidase 1 (AOX 1) promoter of the pPIC9 vector . The reconstructed vector was subsequently integrated into the methanol yeast P . pastoris strain GS115 . The highly expressing strain was selected and the protein sequences and their biological activities were assayed . RESULTS: The amino acid sequence of rhPF4 was the same as that of natural one . The rhPF4 can neutralize the anticoagulation effect of heparin in a dose-dependent manner . CONCLUSION: The amount of rhPF4 expressed in methanol yeast meets well the scale for manufacturing . The biological activities of the rhPF4 were the same as that of natural PF4.

Appl Environ Microbiol, 2002 Mar, 68(3), 1232 - 9
Molecular cloning of XYL3 (D-xylulokinase) from Pichia stipitis and characterization of its physiological function; Jin YS et al.; XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs . Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present . Southern analysis of P . stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes . Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol . These results indicate that XYL3 is critical for ethanol production from xylose but that P . stipitis has another pathway for xylose assimilation . Expression of XYL3 using its P . stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol . In vitro, D-xylulokinase activity in recombinant S . cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number . These results confirmed the function of XYL3 in S . cerevisiae.

Biomed Environ Sci, 2001 Dec, 14(4), 302 - 11
Expression of human vascular endothelial growth factor (VEGF165) in Pichia pastoris and its biological activity; Ma L et al.; OBJECTIVE: To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it . METHODS: By inserting hVEGF165 cDNA coding 165 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 was expressed under the induction of 1% methanol . RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4 d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed . Western blot showed good antigenicity and specificity of expressed product . After being purified by Heparin-Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90% . Biological assays proved that the expressed product could stimulate the proliferation of HUVEC . CONCLUSION: hVEGF165 was successfully expressed . The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

Biomed Environ Sci, 2001 Dec, 14(4), 292 - 301
Expression of human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia pastoris, purification of the expressed product and detection of its biological activity; Ma L et al.; OBJECTIVE: To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia . pastroris, and to purify the expressed product and detect its biological activity . METHODS: By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol . RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed . Western blot showed good antigenicity and specificity of expressed product . After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90% . Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165 . CONCLUSION: Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed . The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

J Biol Inorg Chem, 2002 Jan, 7(1-2), 136 - 45 Epub 2001 Jul 24.
Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris; Whittaker MM et al.; Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids . An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium . The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley . No superoxide dismutase activity was detected in the recombinant oxalate oxidase . EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase . The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex . Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex . The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate . EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites . These features are incorporated into a turnover mechanism for oxalate oxidase.

Med Mycol, 2002 Feb, 40(1), 61 - 71
Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection; Schoen C et al.; An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var . rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA) . The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose . Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids . The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids . The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus . Low stringency Southern blot analysis of R . microsporus DNA suggested the presence of other homologous genes . Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant . Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region . Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro . Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.

J Biol Chem, 2002 May 10, 277(19), 16498 - 504 Epub 2002 Feb 21.
Peroxisomal membrane protein import does not require Pex17p; Harper CC et al.; Of the approximately 20 proteins required for peroxisome biogenesis, only four have been implicated in the process of peroxisomal membrane protein (PMP) import: Pex3p, Pex16p, Pex17p, and Pex19p . To improve our understanding of the role that Pex17p plays in PMP import, we examined the behavior of PMPs in a Pichia pastoris pex17 mutant . Relative to wild-type cells, pex17 cells appeared to have a mild reduction in PMP stability and slightly aberrant PMP behavior in subcellular fractionation experiments . However, we also found that the behavior of PMPs in the pex17 mutant was indistinguishable from PMP behavior in a pex5 mutant, which has no defect in PMP import, and was far different from PMP behavior in a pex3 mutant, which has a bona fide defect in PMP import . Furthermore, we found that a pex14 mutant, which has no defect in PMP import, lacks detectable levels of Pex17p . Based on these and other results, we propose that Pex17p acts primarily in the matrix protein import pathway and does not play an important role in PMP import.

Protein Expr Purif, 2002 Mar, 24(2), 221 - 6
Characterization of a Brassica napus myrosinase expressed and secreted by Pichia pastoris; Hartel FV et al.; In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1) . Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins . These large complexes impede purification and characterization of MB and MC myrosinases from the plant . We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B . napus to allow further characterization of myrosinase belonging to the MB gene family . The purified recombinant myrosinase hydrolyzes sinigrin with a K(m) of 1.0 mM; the specific activity and calculated k(cat)/K(m) were 175 U/mg and 1.9 x 10(5) s(-1) M(-1), respectively . A novel in-gel staining method for myrosinase activity is presented .

Protein Expr Purif, 2002 Mar, 24(2), 212 - 20
Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris; Sarramegna V et al.; The expression of the EGFP-human mu-opioid receptor fusion protein in the methylotrophic yeast Pichia pastoris was optimized and monitored using both fluorescence and ligand-binding experiments . A set of parameters, including gene copy number, strain type, temperature, pH, and methanol inducer levels, was studied for its effect on the production of the recombinant protein . We show here that the expression level is optimal after 10 h of promoter induction and that the maximum is reached at a lower temperature and a higher pH than normally used . The optimized conditions have allowed a fourfold increase of the ligand-binding active form of the receptor, whereas the total expression level determined by EGFP fluorescence measurements was not modified .

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 542 - 5 Epub 2002 Feb 21.
Crystallization and X-ray analysis of native and selenomethionyl beta-mannanase Man5A from blue mussel, Mytilus edulis, expressed in Pichia pastoris; Xu B et al.; The glycohydrolase family 5 beta-mannanase Man5A from Mytilus edulis has been expressed in Pichia pastoris and purified in a form suitable for X-ray crystallographic analysis . Crystals were grown by the hanging-drop technique at 293 K using polyethylene glycol 5000 monomethylether as precipitant and dioxane as additive . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.8, b = 64.8, c = 90.7 A . Diffraction to 1.4 A resolution has been obtained at 100 K . Expression was also performed in the presence of selenomethionine . The incorporation of SeMet was estimated at 40% by amino-acid analysis and its presence in crystals was confirmed from the X-ray absorption scanning spectrum.

Genes Cells, 2002 Jan, 7(1), 75 - 90
Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy; Mukaiyama H et al.; BACKGROUND: In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded through direct engulfment by the vacuole in a process known as micropexophagy, but the mechanism of micropexophagy is not known . RESULTS: To gain molecular insights into micropexophagy, we used fluorescence time-lapse microscopy, coupled with gene-tagging mutagenesis to isolate P . pastoris mutants defective in micropexophagy . The relevant genes have been designated PAZ genes . Morphological and genetic analyses enabled us to postulate a schematic model for micropexophagy . This new model invokes the generation of new vacuolar compartments as an intermediate structure during micropexophagy . Different classes of paz mutants arrest micropexophagy at distinct stages of the process . Most of APG-related paz mutants ceased micropexophagy at Stage 1c and that GCN-family paz mutants ceased micropexophagy at Stage 2 . The paz2Delta strain shows a unique phenotype . Paz2 is the homologue of Saccharomyces cerevisiae Apg8, which is necessary for macroautophagy in that yeast . Our analysis revealed that in P . pastoris, Paz2 plays a key role in repressing the engulfment of peroxisomes by the vacuole before the onset of micropexophagy . Paz2 is proteolytically processed by another autophagy-related Paz protein Paz8, but this processing is not required for the ability of Paz2 to suppress aberrant micropexophagy . CONCLUSION: Micropexophagy has been dissected into a multistep reaction that involves 14 identified Paz gene products . Our studies indicate that Paz2 controls the engulfment of peroxisomes by the vacuole, pointing to a novel early function of this protein.

Eur J Biochem, 2002 Jan, 269(2), 671 - 9
Maturation of Pichia pastoris-derived recombinant pro-Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite; van Oort E et al.; The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen . Its enzymatic activity has been shown to have pro-inflammatory effects that could also negatively influence efficacy of allergen-specific immunotherapy . The aim of this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeast Pichia pastoris and to study its maturation . Expression was achieved at a concentration ranging from 45 mg.L-1 (methanol-induced expression) to 168 mg.L-1 (constitutive expression) . No significant spontaneous maturation of the secreted proenzyme was observed . rpro-Der p 1 with a sequence-based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50-60 kDa on SDS/PAGE . Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30-fold lower capacity to induce histamine release from human basophils . Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1-specific antibody reagents . CD spectra of rpro-Der p 1 and nDer p 1 revealed significant structural differences . Deglycosylation of rpro-Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1 . On removal of N-glycans from rpro-Der p 1, which harbours two putative N-glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared . This suggests that hyperglycosylation hampers spontaneous maturation . Maturation of the recombinant pro-enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1 . In conclusion, high-level expression of rpro-Der p 1 in P . pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen-specific immunotherapy.

Trends Biotechnol, 2002 Mar, 20(3), 103 - 5
Optimizing scale-up fermentation processes; Thiry M et al.; There are many aims associated with the optimization of fermentation processes . Optimization is expected to increase the yield of the final product but the process must be compliant with good manufacturing practices, the available equipment and the expected final scale of operation . Dealing with genetically modified microorganisms that overproduce recombinant protein has the advantage that the vast majority of the processes use only three different species, namely Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris . Standard processes for each organism are described in textbooks and serve as a basis for the development of a tailored process . This article outlines the general philosophy that we have devised to ensure an efficient approach of scaling up fermentation processes for biopharmaceutical purposes, in a multidisciplinary environment.

J Biotechnol, 2002 Mar 28, 94(2), 213 - 6
A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart; Hermida L et al.; Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice . Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC) . The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation . IMAC could be used as an alternative for high scale purification.

Chembiochem, 2001 Aug 3, 2(7-8), 576 - 82
Cloning, functional expression, and characterization of recombinant pig liver esterase; Lange S et al.; The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver . Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA . Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed . Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P . pastoris . Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate . Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations . This was confirmed by native gels, which also show that rPLE is active as a trimer . Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide . This is a substrate specificity for the so-called gamma subunit of PLE . The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively . The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa . The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).

Antonie Van Leeuwenhoek, 2001 Dec, 80(3-4), 237 - 44
Three new species of Candida from apple cider: C . anglica, C . cidri and C . pomicola; Kurtzman CP et al.; Three new anamorphic ascomycetous yeasts are described: Candida anglica (type strain NRRL Y-27079, CBS 4262), Candida cidri (type strain NRRL Y-27078, CBS 4241), and Candida pomicola (type strain NRRL Y-27083, CBS 4242) . These three species were isolated from cider produced in the United Kingdom, and their identification was determined from unique nucleotide sequences in the species-specific D1/D2 domain of large subunit (26S) ribosomal DNA . Phylogenetic analysis of D1/D2 sequences placed C . anglica near Candida fragi, C . cidri near Pichia capsulata, and C . pomicola in the Pichia holstii clade.

Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 371 - 5
Reclassification of Pichia scaptomyzae and Pichia galeiformis; Ueda-Nishimura K et al.; Based on the base composition of nuclear DNA and DNA/DNA hybridization, Pichia galeiformis IFO 10718T was reclassified as a synonym of Pichia manshurica, and Pichia scaptomyzae IFO 1073 1T was confirmed to be a synonym of Pichia membranifaciens . Comparison of 18S rRNA gene sequences indicated that IFO 10731T (P . scaptomyzae) is identical to P . membranifaciens IFO 10215T and IFO 10725, and IFO 10718T (P . galeiformis) is identical to P . manshurica IFO 10726T . These data were consistent with the view that P scaptomyzae and P membranifaciens should be conspecific, as should P . galeiformis and P manshurica . Variation among 26S rRNA gene domain D1/D2 sequences from three P membranifaciens strains indicated that this species encompasses a genetically heterogeneous population.

Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 353 - 61
Four new Candida species from geographically diverse locations; Kurtzman CP; Four new species of Candida are described based on their unique nucleotide sequences in the D1/D2 domain of large subunit (26S) ribosomal DNA . Candida peoriaensis (type strain NRRL YB-1497, CBS 8800) and C . ponderosae (type strain NRRL YB-2307, CBS 8801) are members of the Pichia anomala clade and were isolated in the U.S . from, respectively, the stump of an elm tree (Ulmus sp.) and from insect frass of a Ponderosa pine (Pinusponderosa) . Candida ghanaensis (type strain NRRL YB-1486, CBS 8798) is a phylogenetically divergent species from soil in Ghana and appears related to the Dipodascus/Geotrichum clade . Candida litsaeae (type strain NRRL YB-3246, CBS 8799) was isolated from the frass of an insect-infested Litsaea polyantha tree from India, and is a divergent species that is most closely related to Candida ontarioensis.

Carbohydr Res, 2002 Feb 5, 337(2), 139 - 46
Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC; Ferro V et al.; The promising new anticancer agent, PI-88, is prepared by the sulfonation of the oligosaccharide phosphate fraction of the extracellular phosphomannan produced by the yeast Pichia (Hansenula) holstii NRRL Y-2448 . The composition of the oligosaccharide phosphate fraction was determined by capillary electrophoresis (CE) with indirect UV detection using 6 mM potassium sorbate at pH 10.3 as the background electrolyte . Further confirmation of the composition was obtained by HPLC analysis of a sample dephosphorylated by treatment with alkaline phosphatase . The structure of the hexasaccharide component has been determined by isolation and NMR spectroscopic analysis of its dephosphorylated derivative . Additionally, the structure of a second, previously undetected tetrasaccharide component (a hexosamine) has been determined by isolation and NMR spectroscopic analysis of the acetate of its dephosphorylated derivative . It is demonstrated that CE is an ideal method for the quality control of the oligosaccharide phosphate fraction for use in the production of PI-88.

J Biol Chem, 2002 Apr 12, 277(15), 12613 - 21 Epub 2002 Jan 25.
Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit; Yao Y et al.; The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis . To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris . By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast . The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter . It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel . The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml) . The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR . In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine . Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein . Furthermore, the receptor fragment was efficiently (15)N-labeled in P . pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy . We conclude that the soluble AChR protein is useful for high resolution structural studies.

Protein Expr Purif, 2002 Feb, 24(1), 152 - 62
High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae; Li M et al.; The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described . Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A . The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry . It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality . In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra . The amino termini of both P . pastoris- and S . cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine . The steady-state kinetic properties of P . pastoris-expressed MAO-A are similar to those of S . cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine . Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction . Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite . These data demonstrate significant advantages in the heterologous expression of human MAO-A in P . pastoris compared with the published S . cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme .

Protein Expr Purif, 2002 Feb, 24(1), 83 - 9
Purification of the Type II and Type III isozymes of rat hexokinase, expressed in yeast; Su D et al.; C-terminally His-tagged versions of the Type II and Type III isozymes of rat hexokinase were expressed in Pichia pastoris and Schizosaccharomyces pombe, respectively . Milligram amounts of the homogeneous isozymes were readily obtained in good yield by chromatography on Ni-NTA columns . The specific activities were 133 +/- 4 and 76 +/- 3 u/mg for the purified Type II and Type III isozymes, respectively . The K(m)'s for glucose and ATP were in good agreement with values in the literature for the isozymes isolated from mammalian tissues . The Type III isozyme exhibited substrate inhibition at elevated levels of glucose, as previously observed for this isozyme isolated from mammalian tissue sources .

Protein Expr Purif, 2002 Feb, 24(1), 18 - 24
Optimization of the expression of equistatin in Pichia pastoris; Outchkourov NS et al.; To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, we prepared gene variants with yeast-preferred codon usage and lower repetitive AT and GC content . The full gene optimization approximately doubled the level of steady-state mRNA and protein accumulated in the culture medium . The removal of a short stretch of 12 additional nucleotides from the multiple cloning site (MCS) sequence in the vector pPIC9 had an enhancement effect similar to full gene optimization (factor 1.5) at the mRNA level . However, at the protein level, this increase was 4- to 10-fold . The optimized gene without the MCS sequence yielded 1.66 g/L active protein in a bioreactor and was purified by a new two-step procedure with a recovery of activity that was >95% . This production level constitutes an overall improvement of about 20-fold relative to our previously published results . The characteristics of the MCS sequence element are discussed in the light of its apparent ability to act as negative expression regulator .

J Biomed Sci, 2002 Jan-Feb, 9(1), 47 - 58
Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation; Harohalli K et al.; The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared . All albumin species were nitrosated by incubation with acidified NO(2)(-) . The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA . In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34 . Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris . The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA . Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA . Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions .

Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 346 - 8 Epub 2002 Jan 24.
Preparation and crystallization of selenomethionyl dextranase from Penicillium minioluteum expressed in Pichia pastoris; Larsson AM et al.; Dextranase from the fungus Penicillium minioluteum hydrolyses alpha-1,6-glycosidic bonds in dextran polymers . The enzyme has been expressed in Pichia pastoris in the presence of selenomethionine (SeMet) . The level of SeMet incorporation was estimated by amino-acid analysis to be 50% . The protein has been crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 103.6, b = 115.3, c = 49.8 A and one molecule per asymmetric unit . The crystals diffract to 2.0 A and the presence of SeMet in the crystals has been confirmed by an X-ray absorption spectrum.

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 531 - 3
{Construction and characterization of the chimeric protein consisting RGD-containing peptide and proUK}; Jiao JW et al.; On the basis of structure analysis and computer modeling of proUK, two-chain DNA fragment encoding RGD peptide was inserted into the corresponding proUK cDNA site between Gly118-Leu119, using site-directed mutagenesis and DNA recombinant techniques . The chimeric gene was expressed in methylotrophic yeast Pichia pastoris expression system . The chimeric protein was purified after two step purification of Zn2+ chelating column and SP cation exchange column . The specific activity was 65,000 IU/mg protein . The chimeric protein had somewhat lower catalytic efficiency (kcat/Km) on the substrate S2444 as compared to Urokinase . But it had high anti-platelet aggregation activity, and the half inhibit constant was 2.1 mumol/L . The results showed that the chimeric protein not only had higher thrombolytic activity but also obtained anti-thrombus function . Further evaluation of the thrombolytic and antithrombolytic potential in appropriate animal models seemed to be investigated.

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 520 - 5
{High level secretion expression of porcine somatoropin gene in Pichia pastoris and N-glycosylation analysis of products}; Ouyang J et al.; The porcine somatoropin gene was inserted into the Pichia pastoris expression vector of pPICZ alpha A which contains AOX I promoter and alpha-factor signal sequence . The recombinant plasmid of pPICZ alpha A-pST was linearnized by Sac I and transformed into X-33 by electroporation . The multi-copy insert transformants were selected and cultivated in flasks . SDS-PAGE and Western blot analysis showed that PST gene products were observed in the supernants with a little larger molecular weight size than the natural PST's, however, the molecular weight size of the PST gene products in the soluble cellular proteins were identical to the natural PST's . Retransformation of the linearnized pPICZ alpha-pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one . The expressed rPST accumulated up to about 956 mg/L . The N-glycosylation analysis showed rPST had no N-glycosylation.

Arch Microbiol, 2001 Dec, 177(1), 29 - 35 Epub 2001 Oct 12.
Conservation and release of osmolytes by yeasts during hypo-osmotic stress; Kayingo G et al.; In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity . In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated . All these yeasts released glycerol upon hypo-osmotic shock . However, Z . rouxii also released arabitol, whereas P . sorbitophila released erythritol in addition to arabitol and glycerol . Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone . Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions . The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity . Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock . The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S . cerevisiae, remains to be established.

Biosci Biotechnol Biochem, 2001 Nov, 65(11), 2405 - 11
Immunochemical and mutational analyses of P-type ATPase Spf1p involved in the yeast secretory pathway; Suzuki C; The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified . It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa . To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn . Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT . Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p . Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p . The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin . The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.

Plant Physiol, 2002 Jan, 128(1), 247 - 55
AtFXG1, an Arabidopsis gene encoding alpha-L-fucosidase active against fucosylated xyloglucan oligosaccharides; de La Torre F et al.; An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants . Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE . Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1 . In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1 . Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium . The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside . However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol . Thus, the former must be related to xyloglucan metabolism.

Bioseparation, 2001, 10(1-3), 51 - 6
Routine manufacture of recombinant proteins using expanded bed adsorption chromatography; Shepard SR et al.; Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30-44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography . Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-1 nominal fermentation volume) . The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins . The separations were operated using a 60-cm (d) column run at 14 l/min . For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10) . For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10) . Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.

Pharm Res, 2001 Dec, 18(12), 1775 - 81
In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time; Watanabe H et al.; PURPOSE: Recombinant human serum albumin (rHSA), secreted by a Pichia pastoris expression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose . The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed . METHODS: Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography . Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration . Rat experiments were performed with 125I-labeled albumin . RESULTS: Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA) . Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column . 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA . Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA . Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA . The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA . In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations . CONCLUSION: The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Mikrobiologiia, 2001 Nov-Dec, 70(6), 753 - 8
{Effect of rib83 mutation on riboflavin biosynthesis and iron assimilation in Pichia guilliermondii}; Stenchuk NN et al.; The monogenic rib83 mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondii and lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media . Mutants with the combined mutations of rib83 with rib80 and rib81 (the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) and to overproduce riboflavin in both iron-deficient and iron-sufficient media . This suggests that the rib83 mutation is epistatic with respect to the rib80 and rib81 mutations . The RIB83 gene may positively control both riboflavin synthesis and iron acquisition in the yeast P . guilliermondii.

Eur J Biochem, 2002 Jan, 269(1), 249 - 58
Functional expression of human liver cytosolic beta-glucosidase in Pichia pastoris . Insights into its role in the metabolism of dietary glucosides; Berrin JG et al.; Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic beta-glucosidase (CBG) that hydrolyses various beta-d-glycosides, but whose physiological function is not known . Here, we describe the first heterologous expression of human CBG, a system that facilitated a detailed assessment of the enzyme specificity towards dietary glycosides . A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of approximately 10 mg x L-1 . The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-beta-D-glucoside . Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (beta-D-fucosides, alpha-L-arabinosides, beta-D-glucosides, beta-D-galactosides, beta-L-xylosides, beta-D-arabinosides), similar to the native enzyme . For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (K(m)) and specificities (k(cat)/K(m)) for dietary xenobiotics than for other aryl-glycosides . These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.

J Parasitol, 2001 Dec, 87(6), 1454 - 8
Characterization and large-scale expression of the recombinant cysteine proteinase from adult Clonorchis sinensis; Park SY et al.; Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design . As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms . First, the partial cysteine proteinase gene from C . sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences . The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) . The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L-like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively . The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L) . The active recombinant enzyme was purified from culture media using a Ni2+-NTA-agarose affinity column and gel filtration chromatography . This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (L-trans-epoxysuccinylleucylamido(4-quanidino)butane).

Biochem Biophys Res Commun, 2002 Jan 11, 290(1), 300 - 4
In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep; Acosta-Rivero N et al.; The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied . HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties . After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm . HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient . The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein . Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV . (c)2002 Elsevier Science.

Biochemistry, 2002 Jan 8, 41(1), 282 - 91
Evidence that clusterin has discrete chaperone and ligand binding sites; Lakins JN et al.; Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins.Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2) . It is unknown whether clusterin binds to all of these many ligands via one or more binding sites . Furthermore, the region(s) of clusterin involved in these many binding interactions remain(s) to be identified . As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris . The resultant protein had variable proteolytic truncations of the C-terminal region of the alpha-chain and the N-terminal region of the beta-chain . We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum . We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-clusterin monoclonal antibodies . Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins, and unstressed ligands, respectively, and (ii) the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain . It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation . This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules.

Int J Food Microbiol, 2001 Dec 4, 71(1), 71 - 3
Identification of Pichia anomala isolated from yoghurt by RFLP of the ITS region; Caggia C et al.; Several packs of swollen retailed plain and flavoured yoghurt were examined . The most commonly found species was Pichia anomala, identified both by physiological tests and RFLP analysis of the rDNA internal transcribed spacer (ITS) region . The isolated strains did not ferment lactose and were positive for galactose fermentation, confirming the hypothesis that galactose-fermenting yeast could be the cause of spoilage in yoghurt.

Int J Food Microbiol, 2001 Nov 8, 70(3), 283 - 9
Studies on acetate ester production by non-saccharomyces wine yeasts; Rojas V et al.; A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters . Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains . The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H . uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate . The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S . cerevisiae was examined by growing the yeasts on synthetic microbiological medium . S . cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H . guilliermondii 11104 and P . anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

J Biotechnol, 2000 Aug, 74(2), 105 - 19
Microbial production of spider silk proteins; Fahnestock SR et al.; The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically . We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris . In E . coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors . No such phenomenon was observed in the yeast P . pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis . Spider dragline silk analog proteins could be secreted by P . pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 521 - 7
Differential expression of the Trichoderma reesei beta-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis; Den Haan R et al.; The transcriptional control of two native promoters and one heterologous promoter and the production of a heterologous protein from these promoters were evaluated in the xylose-fermenting yeast Pichia stipitis cultivated on xylose and glucose as carbon sources, using the beta-xylanase II xyn2 gene of Trichoderma reesei . The xyn2 gene open reading frame was fused to the P . stipitis xylose reductase gene (XYL1) promoter, the P . stipitis transketolase gene (TKL) promoter and the Saccharomyces cerevisiae phosphoglycerate kinase gene (PGKI) promoter DNA sequences on episomal plasmids . The plasmids were transformed into Pichia stipitis and gene expression and beta-xylanase production monitored . The XYL1 promoter was shown to be inducible in the presence of xylose, as xyn2 transcription and beta-xylanase activity could be measured when the recombinant strain was cultivated on xylose but not when it was cultivated on glucose . TKL promoter expression was found to be constitutive when either glucose or xylose was used as sole carbon source . The PGK1 promoter did not promote xyn2 transcription in P . stipitis . The molecular size of the recombinant Xyn2 protein produced by P . stipitis was 20.7 kDa, which is similar to that of the native T . reesei Xyn2 protein . This indicates no or minimal glycosylation of the recombinant protein . The recombinant xyn2-expressing strain also yielded twice the amount of biomass yielded by the control strain when cultivated in medium containing 1% birchwood xylan as sole carbon source.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 77 - 83
Two new anamorphic yeasts: Candida germanica and Candida neerlandica; Kurtzman CP et al.; Descriptions are given for the two new anamorphic ascomycetous yeasts Candida germanica (type strain NRRL Y-27064, CBS 4105) and Candida neerlandica (type strain NRRL Y-27057, CBS 434) . The species were isolated, respectively, from the atmosphere over Germany and from pressed yeast cake in The Netherlands . Phylogenetic analysis of 26S domain D1/D2 ribosomal DNA sequences places C . germanica near Pichia philogaea, whereas C . neerlandica is a member of the Lodderomyces elongisporus/Candida albicans clade.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2291 - 3
Comparison of three signals for secretory expression of recombinant human midkine in Pichia pastoris; Murasugi A et al.; The secretion signals of Saccharomyces cerevisiae alpha mating factor, human midkine itself, and Pichia pastoris acid phosphatase, were tried for the expression of human midkine under the control of the AOX1 gene promoter in P . pastoris . Approximately 28 mg/l, 1.5 mg/l, and 0.2 mg/l of midkine were secreted by using the a mating factor pre-pro-sequence, the midkine signal sequence, and the phosphatase signal sequence in flask cultures, respectively.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2265 - 70
Glycolic acid production using ethylene glycol-oxidizing microorganisms; Kataoka M et al.; Screening for microorganisms oxidizing ethylene glycol to glycolic acid was carried out . Among stock cultures, several yeasts and acetic acid bacteria showed high glycolic acid producing activity . Pichia naganishii AKU 4267 formed the highest concentration of glycolic acid, 35.3 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 26.0%) . Among soil isolates, Rhodotorula sp . 3Pr-126, isolated using propylene glycol as a sole carbon source, formed the highest concentration of glycolic acid, 25.1 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 18.5%) . Rhodotorula sp . 3Pr-126 showed higher activity toward 20% (v/v) ethylene glycol than P . naganishii AKU 4267 . Optimization of the conditions for glycolic acid production was investigated using P . naganishii AKU 4267 and Rhodotorula sp . 3Pr-126 . Under the optimized conditions, P . naganishii AKU 4267 and Rhodotorula sp . 3Pr-126 formed 105 and 110 g/l of glycolic acid (corrected molar conversion yields, 88.0 and 92.2%) during 120 h of reaction, respectively.

Yeast, 2002 Jan 15, 19(1), 37 - 42
Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase; Baerends RJ et al.; Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts . Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha . Unlike the recently described Pichia pastoris homologue, the H . polymorpha gene does not contain an intron . The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca . 41 kDa) with 82% identity to P . pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes . The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions . The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g . methylamine, is used as nitrogen source . These properties render the FLD1 promoter of high interest for heterologous gene expression . The availability of the H . polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter .

Int Arch Allergy Immunol, 2001 Nov, 126(3), 196 - 205
Immunochemical characterization of two Pichia pastoris-derived recombinant group 5 Dactylis glomerata isoallergens; van Oort E et al.; BACKGROUND: Grass pollen of the Poaceae grasses are known to be highly allergenic . Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available . METHODS: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris . Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA) . In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed . RESULTS: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids . Both mature isoforms were expressed in Pichia at a concentration of approximately 15 mg/l . SDS-PAGE analysis showed that rDac g 5 had an apparent M(r) approximately 10 kD above nDac g 5 . By mass spectrometry this difference was shown to be around 2.5 kD . Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M(r) . Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5 . RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5 . A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02 . Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA . CONCLUSIONS: Dac g 5 was successfully cloned and expressed in P . pastoris . Minor differences in primary structure between isoforms influence their immune reactivity .

Mol Pharmacol, 2002 Jan, 61(1), 214 - 21
Defining minimal structural features in substrates of the H(+)/peptide cotransporter PEPT2 using novel amino acid and dipeptide derivatives; Theis S et al.; The peptide transporter PEPT2, expressed in a variety of tissues, including kidney, lung, and the central nervous system, mediates the uphill transport of di- and tripeptides, as well as a variety of peptidomimetic drugs . To identify the essential molecular features of substrates that determine affinity and transport by PEPT2, we synthesized a series of amino acid derivatives as well as modified dipeptides . Kinetic constants for the interaction of test compounds with PEPT2 were obtained in a competition assay using Pichia pastoris yeast cells expressing mammalian PEPT2 . The two-electrode voltage-clamp technique in Xenopus laevis oocytes was used to assess the substrate's electrogenic transport properties . Whereas omega bar-amino fatty acids showed no affinity for PEPT2, the introduction of a single carbonyl group into the backbone increased both affinity and transport currents more than 30-fold . omega bar-amino fatty acids, at their amino or carboxyl group coupled to an alanine residue, allowed us to determine the importance of the spatial position of functional groups within the molecule . Affinity and transport function declined by elongating the omega bar-amino acid chain when located in the N-terminal position, whereas the elongation in the carboxyl terminal with an N-terminal alanine caused less pronounced effects . The results clearly establish that a free N terminus, a correctly positioned backbone carbonyl group, and a carboxylic group that is in a suitable distance from the intramolecular carbonyl function and the amino terminal head group are the main features for substrate recognition and transport by PEPT2 . This information provides the framework for a rational design of peptidomimetic drugs for delivery via PEPT2.

J Biol Chem, 2002 Mar 1, 277(9), 7287 - 92 Epub 2001 Dec 21.
Synthesis and characterization of high affinity inhibitors of the H+/peptide transporter PEPT2; Theis S et al.; In this study, we describe the rational synthesis and functional analysis of novel high affinity inhibitors for the mammalian peptide transporter PEPT2 . Moreover, we demonstrate which structural properties convert a transported compound into a non-translocated inhibitor . Starting from Lys{Z(NO(2))}-Pro (where Z is benzyloxycarbonyl), which we recently identified as the first competitive high affinity inhibitor of the intestinal peptide transporter PEPT1, a series of different lysine-containing dipeptide derivatives was synthesized and studied for interaction with PEPT2 based on transport competition assays in Pichia pastoris yeast cells expressing PEPT2 heterologously and in renal SKPT cells expressing PEPT2 . In addition, the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides . Synthesis and functional analysis of Lys-Lys derivatives containing benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by PEPT2 with K(i) values as low as 10 +/- 1 nm . This is the highest affinity of a ligand of PEPT2 ever reported . Moreover, based on the structure-function relationship, we conclude that the spatial location of the side chain amino protecting group in a dipeptide containing a diaminocarbonic acid and its intramolecular distance from the Calpha atom are key factors for the transformation of a substrate into an inhibitor of PEPT2.

Yeast, 2001 Dec, 18(16), 1471 - 8
Interaction of SMKT, a killer toxin produced by Pichia farinosa, with the yeast cell membranes; Suzuki C et al.; SMKT (salt-mediated killer toxin), a killer toxin produced by the halotolerant yeast, Pichia farinosa, kills yeasts of several genera, including Saccharomyces cerevisiae . To elucidate the killing mechanism of SMKT, we examined the interaction of SMKT with membranes using liposomes . Leakage of calcein from calcein-entrapped liposomes was observed in the presence of SMKT . Destruction of liposomes was observed by dark-field microscopy . Comparison of intact S . cerevisiae cells with SMKT-treated cells by dark-field microscopy indicated that the spherical cell membrane is disrupted by SMKT . Using sodium carbonate extraction, we obtained direct evidence for the first time that SMKT is associated with the membrane of sensitive cells . Our results indicate that SMKT kills sensitive S . cerevisiae by interacting with the yeast cell membrane .

Arch Biochem Biophys, 2001 Dec 15, 396(2), 213 - 8
Biochemical characterization of recombinant Drosophila type 1 serine/threonine protein phosphatase (PP1c) produced in Pichia pastoris; Szoor B et al.; The methylotrophic yeast Pichia pastoris was used to express Drosophila melanogaster type 1beta serine/threonine phosphoprotein phosphatase catalytic subunit (PP1beta9C) . A construct encoding PP1beta9C with a short NH(2)-terminal fusion including six histidine residues was introduced into the X-33 and KM71H strains of P . pastoris by homologous recombination . Recombinant protein was purified from cell free extracts 24 h after methanol induction . PP1beta9C was purified to a specific activity of 12,077 mU/mg by a three-step purification method comprising (NH(4))(2)SO(4)-ethanol precipitation followed by Ni(2+)-agarose affinity chromatography and Mono Q anion-exchange chromatography . This purification scheme yielded approximately 80 microg of active, soluble PP1beta9C per 1 L of culture . In contrast to recombinant PP1beta9C overexpressed in bacteria, which differs from native PP1c in several biochemical criteria including the requirement for divalent cations, sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity, recombinant PP1beta9C produced in P . pastoris has native-like properties . P . pastoris thus provides a reliable and convenient system for the production of active, native-like recombinant PP1beta9C . (c)2001 Elsevier Science.

Yeast, 2001 Nov, 18(15), 1441 - 8
Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala; De la Rosa JM et al.; The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant . The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the beta-isopropylmalate dehydrogenase deficiency of the mutants . The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S . cerevisiae (84% identity) and other yeast species . A sequence resembling the GC-rich palindrome motif identified in the 5' region of S . cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region . In addition, upstream of the PaLEU2 the 3'-terminal half of a gene of the same orientation, encoding a homologue of the S . cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found . The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S . cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation .

Mol Biol Cell, 2001 Dec, 12(12), 3821 - 38
Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris; Guan J et al.; Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy . In addition, there exist both constitutive and regulated forms of autophagy . For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy . Our studies have shown that the differing pathways of autophagy have many molecular events in common . In this article, we have identified a new member in the family of autophagy genes . GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains . We have shown that these proteins are required for pexophagy and autophagy in P . pastoris and the Cvt pathway, autophagy, and pexophagy in S . cerevisiae . In P . pastoris, Gsa12 appears to be required for an early event in pexophagy . That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant . Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface . Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

Pediatr Infect Dis J, 2001 Sep, 20(9), 843 - 8
Pichia anomala outbreak in a nursery: exogenous source?
Aragao PA, Oshiro IC, Manrique EI, Gomes CC, Matsuo LL, Leone C, Moretti-Branchini ML, Levin AS; IRIS Study Group.
BACKGROUND: Pichia anomala is a rare cause of fungemia . From February to April, 1998, eight cases of fungemia occurred in the intensive care and high risk units of the Nursery . There were four infants with P . anomala infection, one of whom also had Candida parapsilosis infection, two cases with C . parapsilosis infection and two with Candida albicans infection . OBJECTIVE: To determine factors associated with fungemia in the intensive care and high risk units of the Nursery, especially P . anomala . METHODS: A cohort study with 59 newborns . RESULTS: Factors associated with fungemia were: central venous catheter (CVC) (P = 0.0006); total parenteral nutrition (TPN) (P = 0.0005); lipid emulsion (P = 0.002); previous antimicrobial use (P = 0.002); and other invasive procedures (P = 0.002) . Factors associated with P . anomala fungemia were: CVC (P = 0.004); TPN (P = 0.018); previous antibiotic use (P = 0.037); and other invasive procedures (P = 0.037) . Evaluation of the units demonstrated that there were several technical problems involving administration of TPN that was manipulated in the Nursery without precautions . Changes in TPN formulation and education as to adequate technique were implemented . During follow-up (1998 to 1999) only two fungemias occurred that were caused by C . albicans . Cultures of hands of personnel were negative for P . anomala . Electrophoretic karyotyping of P . anomala showed three profiles . CONCLUSIONS: Factors associated with fungemia were catheter use, invasive procedures and total parenteral nutrition, suggesting that the acquisition of P . anomala was exogenous.

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