Expression of Recombinant Proteins in Pichia Pastoris

Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.

[1]  Mats Martinelle,et al.  Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. , 2003, Journal of biotechnology.

[2]  H. Waterham,et al.  The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1 , 1996, Molecular and cellular biology.

[3]  F. Maley,et al.  Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. , 1991, The Journal of biological chemistry.

[4]  J. Sohn,et al.  Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae , 2000, Applied Microbiology and Biotechnology.

[5]  Ming-Shiun Tsai,et al.  Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris. , 2002, Protein expression and purification.

[6]  J. O'callaghan,et al.  Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris , 2002, Journal of Industrial Microbiology and Biotechnology.

[7]  William C. Raschke,et al.  Recent Advances in the Expression of Foreign Genes in Pichia pastoris , 1993, Bio/Technology.

[8]  C. Schein,et al.  Formation of Soluble Recombinant Proteins in Escherichia Coli is Favored by Lower Growth Temperature , 1988, Bio/Technology.

[9]  R. Pearson,et al.  Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. , 1991, Methods in enzymology.

[10]  A. Milon,et al.  Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris , 1996, FEBS letters.

[11]  Joseph Shiloach,et al.  Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris. , 2003, Biotechnology and bioengineering.

[12]  Sreelatha T. Reddy,et al.  High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris. , 2002, Protein expression and purification.

[13]  A. Murasugi,et al.  Production of recombinant human bile salt-stimulated lipase in Pichia pastoris. , 2001, Protein expression and purification.

[14]  A. Daugulis,et al.  A rational approach to improving productivity in recombinant Pichia pastoris fermentation. , 2001, Biotechnology and bioengineering.

[15]  K. Palmu,et al.  Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme. , 1998, Protein expression and purification.

[16]  J. Cregg,et al.  Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. , 2002, Current opinion in biotechnology.

[17]  J J Clare,et al.  Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. , 1991, Gene.

[18]  A. Subramaniam,et al.  Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. , 1997, Gene.

[19]  R. Contreras,et al.  Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase. , 1997, European journal of biochemistry.

[20]  K. Faber,et al.  The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins). , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Cregg,et al.  Introduction to Pichia pastoris. , 1998, Methods in molecular biology.

[22]  R. Armitage,et al.  Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. , 1997, Gene.

[23]  M. Popp,et al.  Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris. , 1997, Protein expression and purification.

[24]  J. Lagarias,et al.  The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Brierley,et al.  Secretion of recombinant human insulin-like growth factor I (IGF-I). , 1998, Methods in molecular biology.

[26]  R. B. Trimble,et al.  Overview of N- and O-linked oligosaccharide structures found in various yeast species. , 1999, Biochimica et biophysica acta.

[27]  X. Liu,et al.  Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris , 1997, Applied Microbiology and Biotechnology.

[28]  O. Rossanese,et al.  A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris , 1998, Yeast.

[29]  M. Gautier,et al.  Optimization of the wheat puroindoline‐a production in Pichia pastoris , 2001, Journal of applied microbiology.

[30]  R. Beavis,et al.  Structural studies of alpha-N-acetylgalactosaminidase: effect of glycosylation on the level of expression, secretion efficiency, and enzyme activity. , 1998, Archives of biochemistry and biophysics.

[31]  T. Takao,et al.  Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation. , 2001, FEMS yeast research.

[32]  N. Meinander,et al.  Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. , 2002, Biotechnology and bioengineering.

[33]  M. Nimtz,et al.  Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease. , 1999, Glycobiology.

[34]  Youtao Song,et al.  Secretion of Glycosylated α-Lactalbumin in Yeast Pichia pastoris , 2002 .

[35]  T. Creighton.  Methods in Enzymology , 1968, The Yale Journal of Biology and Medicine.

[36]  R. Schmid,et al.  Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. , 2001, Journal of biotechnology.

[37]  V. Storme,et al.  High–Level Secretion and Very Efficient Isotopic Labeling of Tick Anticoagulant Peptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris , 1994, Bio/Technology.

[38]  R. Salesse,et al.  High-level secretion of biologically active recombinant porcine follicle-stimulating hormone by the methylotrophic yeast Pichia pastoris. , 1998, Biochemical and biophysical research communications.

[39]  M. de Hoop,et al.  Import of proteins into peroxisomes and other microbodies. , 1992, The Biochemical journal.

[40]  C. Hew,et al.  Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. , 2001, Protein expression and purification.

[41]  T. Kuo,et al.  Asparagine as a nitrogen source for improving the secretion of mouse α‐amylase in Saccharomyces cerevisiae protease A‐deficient strains , 2000, Yeast.

[42]  S.J.Higgins,et al.  Protein Expression A Practical Approach , 2004 .

[43]  J. Cregg,et al.  PER3, a Gene Required for Peroxisome Biogenesis in Pichia pastoris, Encodes a Peroxisomal Membrane Protein Involved in Protein Import (*) , 1995, The Journal of Biological Chemistry.

[44]  V. Venturi,et al.  Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris. , 2002, FEMS yeast research.

[45]  Wenhui Zhang,et al.  Design of Methanol Feed Control in Pichia pastoris Fermentations Based upon a Growth Model , 2002, Biotechnology progress.

[46]  M. Jolivet,et al.  Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris : evidence of hyper O‐glycosylation , 2001, Biotechnology and applied biochemistry.

[47]  V. Renugopalakrishnan,et al.  Retroengineering bacteriorhodopsins: Design of smart proteins by bionanotechnology , 2003 .

[48]  Alain Milon,et al.  Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris. , 2002, Protein expression and purification.

[49]  S. Sasaki,et al.  Complete amino acid sequence of amelogenin in developing bovine enamel. , 1984, Biochemical and biophysical research communications.

[50]  M. Yu,et al.  Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli. , 1990, European journal of biochemistry.

[51]  H. Waterham,et al.  The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals , 1994, The Journal of cell biology.

[52]  R. Takors,et al.  Human Chymotrypsinogen B Production with Pichiapastoris by Integrated Development of Fermentation and Downstream Processing. Part 1. Fermentation , 2001, Biotechnology progress.

[53]  J. Cregg,et al.  Use of site-specific recombination to regenerate selectable markers , 1989, Molecular and General Genetics MGG.

[54]  R. Dwek,et al.  Oligosaccharides of recombinant mouse gelatinase B variants. , 1998, Biochimica et biophysica acta.

[55]  H. Waterham,et al.  Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. , 1997, Gene.

[56]  Byung-Kwon Choi,et al.  Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[57]  D. Kilburn,et al.  O-Glycosylation of a Recombinant Carbohydrate-Binding Module Mutant Secreted by Pichia pastoris , 2003, Journal of Molecular Microbiology and Biotechnology.

[58]  Pingzuo Li,et al.  Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins. , 2001, Protein expression and purification.

[59]  T. Jeffries,et al.  A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. , 1998, Gene.

[60]  F. Soubrier,et al.  Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity. , 1996, The Biochemical journal.

[61]  C. Scorer,et al.  Foreign gene expression in yeast: a review , 1992, Yeast.

[62]  J. Cregg,et al.  Heterologous protein expression in the methylotrophic yeast Pichia pastoris. , 2000, FEMS microbiology reviews.

[63]  H. Michel,et al.  Expression of functional mouse 5‐HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization , 1995, FEBS letters.

[64]  J. Tschopp,et al.  Size distribution and general structural features of N‐linked oligosaccharides from the methylotrophic yeast, Pichia pastoris , 1989, Yeast.

[65]  D. Pope,et al.  Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens , 1978, Journal of bacteriology.

[66]  J. Cregg,et al.  Expression of foreign genes in the yeast Pichia pastoris. , 2001, Genetic engineering.

[67]  S L Morrison,et al.  Effect of glycosylation on antibody function: implications for genetic engineering. , 1997, Trends in biotechnology.

[68]  M. Hirose,et al.  Characterization of N‐linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris , 2002, Yeast.

[69]  J. Tschopp,et al.  High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia Pastoris , 1987, Bio/Technology.

[70]  Shann-Tzong Jiang,et al.  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. , 2002, Journal of agricultural and food chemistry.

[71]  Wenhui Zhang,et al.  Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement , 2003, Journal of Industrial Microbiology and Biotechnology.

[72]  J. D. Yang,et al.  Single-chain Fv with manifold N-glycans as bifunctional scaffolds for immunomolecules. , 1998, Protein engineering.

[73]  J. P. van Dijken,et al.  The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. , 1983, Advances in microbial physiology.

[74]  B. Svensson,et al.  Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic bacillus alpha-amylase produced in Pichia pastoris. , 2001, Protein expression and purification.

[75]  K. Okabayashi,et al.  Secretion of a variant of human single‐chain urokinase‐type plasminogen activator without an N‐glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product , 1996, Yeast.

[76]  K. Hult,et al.  Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain. , 2001, Protein expression and purification.

[77]  A. Murasugi,et al.  Production of native recombinant human midkine in the yeast, Pichia pastoris. , 2003, Protein expression and purification.

[78]  J. O'callaghan,et al.  Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. , 2002, Microbiology.

[79]  T. Teeri,et al.  Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. , 2000, Biotechnology and bioengineering.

[80]  T. Aizawa,et al.  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. , 2002, Protein expression and purification.

[81]  J. Cregg,et al.  Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations , 1997 .

[82]  P. Karplus,et al.  Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. , 2000, Archives of biochemistry and biophysics.

[83]  J. W. Whittaker,et al.  Expression of recombinant galactose oxidase by Pichia pastoris. , 2000, Protein expression and purification.

[84]  E. Komives,et al.  Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388. , 1995, Protein engineering.

[85]  J. Cregg,et al.  Structural comparison of the Pichia pastoris alcohol oxidase genes , 1989, Yeast.

[86]  J. Hoeffler,et al.  Small vectors for expression based on dominant drug resistance with direct multicopy selection. , 1998, Methods in molecular biology.

[87]  M. Chapman,et al.  A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen. , 2000, Protein expression and purification.

[88]  M. Alting-Mees,et al.  Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. , 2003, Protein expression and purification.

[89]  A. Wu,et al.  Expression, purification, and characterization of a two domain carcinoembryonic antigen minigene (N-A3) in pichia pastoris. The essential role of the N-domain. , 1998, Anticancer research.

[90]  John D. Chung,et al.  Design of metabolic feed controllers: Application to high‐density fermentations of Pichia pastoris , 2000, Biotechnology and bioengineering.

[91]  M. Meagher,et al.  High cell-density fermentation. , 1998, Methods in molecular biology.

[92]  Michael A. Romanos,et al.  Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High–level Foreign Gene Expression , 1994, Bio/Technology.

[93]  R. Stoltenburg,et al.  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 , 2002, Yeast.

[94]  W. McCombie,et al.  High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris , 1988, Journal of basic microbiology.

[95]  K. G. Han,et al.  Soluble expression of cloned phage K11 RNA polymerase gene in Escherichia coli at a low temperature. , 1999, Protein expression and purification.

[96]  M. Oobatake,et al.  Thermal denaturation of a recombinant mouse amelogenin: Circular dichroism and differential scanning calorimetric studies , 2005, Proteins.

[97]  R. Contreras,et al.  Use of HDEL‐tagged Trichoderma reesei mannosyl oligosaccharide 1,2‐α‐D‐mannosidase for N‐glycan engineering in Pichia pastoris , 2001, FEBS letters.

[98]  Nigel Jenkins,et al.  Getting the glycosylation right: Implications for the biotechnology industry , 1996, Nature Biotechnology.

[99]  J. Cregg,et al.  An efficient screen for peroxisome-deficient mutants of Pichia pastoris , 1992, Journal of bacteriology.

[100]  Wenhui Zhang,et al.  Improved Production of Recombinant Ovine Interferon‐τ by Mut+ Strain of Pichiapastoris Using an Optimized Methanol Feed Profile , 2003, Biotechnology progress.

[101]  J. Boonstra,et al.  Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts , 2000, Applied and Environmental Microbiology.