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Showing papers on "Pichia pastoris published in 2002"


Journal ArticleDOI
TL;DR: The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels.

603 citations


Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

208 citations


Journal ArticleDOI
TL;DR: It is reported that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto β(1,4)-linked glucan chains, and it is concluded that this Arabidopsis gene encodes an β-glucan synthase activity involved in xyloglucan biosynthesis.
Abstract: Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of α-xylosyltransferase and β-glucan synthase activities. Here we report that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto β(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 α-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 14C-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 α(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an α-xylosyltransferase activity involved in xyloglucan biosynthesis.

205 citations


Journal ArticleDOI
TL;DR: Optimize G+C content, regardless of corresponding codon optimization, appears to be the major contributor to increased translational efficiency in this heterologous expression host.

178 citations


Journal ArticleDOI
TL;DR: A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air and describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.
Abstract: A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L–1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L–1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.

149 citations


Journal ArticleDOI
TL;DR: 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, and the recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1.
Abstract: 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.

149 citations


Journal ArticleDOI
TL;DR: This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant, and the cloning of a honeybee CSP gene called ASP3c is reported.
Abstract: 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

139 citations


Journal ArticleDOI
TL;DR: This work has developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression and is able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher.

126 citations


Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

118 citations


Journal ArticleDOI
TL;DR: The results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.

115 citations


Journal ArticleDOI
TL;DR: To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, gene variants with yeast-preferred codon usage and lower repetitive AT and GC content are prepared and its apparent ability to act as negative expression regulator is discussed.

Journal ArticleDOI
TL;DR: The rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1, suggesting some control of expression at the post-transcriptional level.

Journal ArticleDOI
TL;DR: This is the first successful expression of a full-length malarial parasite integral membrane protein in yeast and expands on the role of Pfcrt in conferring CQR and defines a productive route for analysis of important P. falciparum transport proteins and membrane associated vaccine candidates.

Journal ArticleDOI
TL;DR: 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 and in a higher level of homogeneity of the isolated enzyme.

Journal ArticleDOI
TL;DR: LTPs, the allergens from peach and apple, have a main clinical relevance in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities.
Abstract: 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. Objetive To isolate and characterize cDNAs for Pru p 3 and Mal d 3,and to produce recombinant Pru p 3 in the yeast Pichia pastoris. Methods cDNAs for both allergens were isolated by polymerase chain reaction using non-degenerated 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.

Journal ArticleDOI
TL;DR: The 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 and transglycosylation activity could be demonstrated by MALDI-TOF MS analysis of products formed during degradation of cellopentaose.

Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

Journal ArticleDOI
TL;DR: 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.

Journal ArticleDOI
TL;DR: It is shown 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.

Journal ArticleDOI
TL;DR: The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg, and it is concluded that the recombinant enzyme is properly folded.

Journal ArticleDOI
TL;DR: The first heterologous expression of human CBG is described, showing 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 and specificities for dietary Xenobiotics than for other aryl-glycosides.
Abstract: Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic β-glucosidase (CBG) that hydrolyses various β-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 ≈ 10 mg·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-β-d-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (β-d-fucosides, α-l-arabinosides, β-d-glucosides, β-d-galactosides, β-l-xylosides, β-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 (Km) and specificities (kcat/Km) 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.

Journal ArticleDOI
TL;DR: 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
Abstract: 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.

Journal ArticleDOI
TL;DR: 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, which are incorporated into a turnover mechanism for oxalates oxidase.
Abstract: 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.

Journal ArticleDOI
TL;DR: The development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation is described, which produced higher levels of laccase activity than those grown in the absence of alanine.
Abstract: 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 μM 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 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. Journal of Industrial Microbiology & Biotechnology (2002) 29, 55–59 doi:10.1038/sj.jim.7000268

Journal ArticleDOI
TL;DR: 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.

Journal ArticleDOI
TL;DR: 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, and analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of theprotein upon treatment with DTT.
Abstract: 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 α-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...

Journal ArticleDOI
TL;DR: By this strategy, the production of intact recombinant hirudin Hir65 reached 0.7 g l−1 in fed-batch fermentation, and its proportion was 35% to all forms of hirUDin.
Abstract: The effects of the specific growth rate and methanol concentration on the degradation of hirudin produced by recombinant Pichia pastoris were investigated. When a methanol-limited state and the specific growth rate of 0.02 h−1 were maintained during the fermentation, a minimum degradation of hirudin and a maximum specific hirudin production rate were achieved. By this strategy, the production of intact recombinant hirudin Hir65 reached 0.7 g l−1 in fed-batch fermentation. Its proportion was 35% to all forms of hirudin.

Journal ArticleDOI
TL;DR: Results suggest that the extracellular domain of the human AChR α subunit expressed in P. pastoris has an apparently near native conformation, which makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies.

Journal ArticleDOI
TL;DR: 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 are employed and can serve as a framework for the design of other PID feedback control systems in biological processes.
Abstract: 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

Book ChapterDOI
TL;DR: Although the P. pastoris expression system is not suitable for the production of all foreign proteins, many are compatible with the system and should increase the range of proteins that can be produced by this methylotrophic host.
Abstract: Although the P. pastoris expression system is not suitable for the production of all foreign proteins, many are compatible with the system. For these proteins, there are major advantages to production in this yeast. Relative to higher eukaryotic expression systems, culturing costs are lower, yields (foreign protein/L of culture medium) are usually higher, and for secreted proteins, the initial purity of the product in the culture medium is higher. Relative to bacterial expression systems, the chances of recovering a eukaryotic foreign protein in its biologically active form are significantly higher with P. pastoris. Although few major P. pastoris- produced human Pharmaceuticals are on the market, several are in the clinical trial pipeline, including the anti-tumor drugs angiostatin and endostatin (44). In addition, P. pastoris is one of the only lower eukaryotic expression systems that is available to academic researchers as a complete kit (see Invitrogen catalog). Recent improvements in the system, including new marker host combinations and alternative promoters, should increase the range of proteins that can be produced by this methylotrophic host. As more proteins are successfully (or unsuccessfully) synthesized in P. pastoris, parameters involved with the optimal expression of specific classes of proteins will become better defined.