Skip to main content
SpringerLink
Log in

Protein secretion in Pichia pastoris and advances in protein production

  • Mini-Review
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Bernales S, Papa FR, Walter P (2006) Intracellular signaling by the unfolded protein response. Annu Rev Cell Dev Biol 22:487–508

    Article  CAS  Google Scholar 

  • Braren I, Greunke K, Umland O, Deckers S, Bredehorst R, Spillner E (2007) Comparative expression of different antibody formats in mammalian cells and Pichia pastoris. Biotechnol Appl Biochem 47:205–214

    Article  CAS  Google Scholar 

  • Brodsky JL (1998) Translocation of proteins across the endoplasmic reticulum membrane. Int Rev Cytol 178:277–328

    Article  CAS  Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  Google Scholar 

  • Chung BK, Selvarasu S, Andrea C, Ryu J, Lee H, Ahn J, Lee DY (2010) Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement. Microb Cell Fact 9:50

    Article  Google Scholar 

  • Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323

    CAS  Google Scholar 

  • Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52

    Article  CAS  Google Scholar 

  • Cudna RE, Dickson AJ (2003) Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 81:56–65

    Article  CAS  Google Scholar 

  • Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  CAS  Google Scholar 

  • Damasceno LM, Pla I, Chang HJ, Cohen L, Ritter G, Old LJ, Batt CA (2004) An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif 37:18–26

    Article  CAS  Google Scholar 

  • Damasceno LM, Anderson KA, Ritter G, Cregg JM, Old LJ, Batt CA (2007) Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris. Appl Microbiol Biotechnol 74:381–389

    Article  CAS  Google Scholar 

  • De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouze P, Van de Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566

    Article  Google Scholar 

  • Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306

    Article  CAS  Google Scholar 

  • Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392

    Article  CAS  Google Scholar 

  • Eckart MR, Bussineau CM (1996) Quality and authenticity of heterologous proteins synthesized in yeast. Curr Opin Biotechnol 7:525–530

    Article  CAS  Google Scholar 

  • Ellis SB, Brust PF, Koutz PJ, Waters AF, Harpold MM, Gingeras TR (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5:1111–1121

    CAS  Google Scholar 

  • Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750

    Article  CAS  Google Scholar 

  • Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E, Veenhuis M, van der Klei I (2005) New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - A comparison. FEMS Yeast Res 5:1079–1096

    Article  CAS  Google Scholar 

  • Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, Callewaert N (2010) The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 9:49

    Article  Google Scholar 

  • Ha SH, Park JJ, Kim JW, Jeong JW, Noh KS, Jeon YJ, Kin HS, Kim HB (2001) Molecular cloning and high-level expression of G2 protein of hantaan (HTN) virus 76–118 strain in the yeast Pichia pastoris KM71. Virus Genes 22:167–173

    Article  CAS  Google Scholar 

  • Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV (2006) Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res 23:790–797

    Article  CAS  Google Scholar 

  • Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Lin H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443

    Article  CAS  Google Scholar 

  • Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6:1099–1108

    Article  CAS  Google Scholar 

  • Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10:3787–3799

    CAS  Google Scholar 

  • Higgins DR, Cregg JM (1998) Introduction to Pichia pastoris. Methods Mol Biol 103:1–15

    CAS  Google Scholar 

  • Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367–375

    Article  CAS  Google Scholar 

  • Huang CJ, Damasceno LM, Anderson KA, Zhang S, Old LJ, Batt CA (2011) A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol 90:235–247

    Article  CAS  Google Scholar 

  • Inan M, Aryasomayajula D, Sinha J, Meagher MM (2006) Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnol Bioeng 93:771–778

    Article  CAS  Google Scholar 

  • Jacobs PP, Geysens S, Vervecken W, Contreras R, Callewaert N (2009) Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4:58–70

    Article  CAS  Google Scholar 

  • Khandekar SS, Silverman C, Wells-Marani J, Bacon AM, Birrell H, Brigham-Burke M, DeMarini DJ, Jonak ZL, Camilleri P, Fishman-Lobell J (2001) Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells. Protein Expr Purif 23:301–310

    Article  CAS  Google Scholar 

  • Koganesawa N, Aizawa T, Masaki K, Matsuura A, Nimori H, Bando H, Kawano K, Nitta K (2001) Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. Protein Eng 14:705–710

    Article  CAS  Google Scholar 

  • Kowalski JM, Parekh RN, Mao J, Wittrup KD (1998) Protein folding stability can determine the efficiency of escape from endoplasmic reticulum quality control. J Biol Chem 273:19453–19458

    Article  CAS  Google Scholar 

  • Kuberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Puhler A, Schwab H, Glieder A, Pichler H (2011) High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 154:312–320

    Article  Google Scholar 

  • Kurtzman CP (2009) Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J Ind Microbiol Biotechnol 36:1435–1438

    Article  CAS  Google Scholar 

  • Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi BK, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215

    Article  CAS  Google Scholar 

  • Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    Article  CAS  Google Scholar 

  • Mansur M, Cabello C, Hernandez L, Pais J, Varas L, Valdes J, Terrero Y, Hidalgo A, Plana L, Besada V, Garcia L, Lamazares E, Castellanos L, Martonez E (2005) Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol Lett 27:339–345

    Article  CAS  Google Scholar 

  • Marx H, Mecklenbrauker A, Gasser B, Sauer M, Mattanovich D (2009) Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res 9:1260–1270

    Article  CAS  Google Scholar 

  • Mattanovich D, Graf A, Stadlmann J, Dragosites M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B (2009) Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 8:29

    Article  Google Scholar 

  • Ng DT, Brown JD, Walter P (1996) Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 134:269–278

    Article  CAS  Google Scholar 

  • Outchkourov NS, Stiekema WJ, Jongsma MA (2002) Optimization of the expression of equistatin in Pichia pastoris. Protein Expr Purif 24:18–24

    Article  CAS  Google Scholar 

  • Parekh R, Forrester K, Wittrup D (1995) Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae. Protein Expr Purif 6:537–545

    Article  CAS  Google Scholar 

  • Poppenberger B, Berthiller F, Bachmann H, Lucyshyn D, Peterbauer C, Mitterbaurer R, Schuhmacher R, Krska R, Glossi J, Adam G (2006) Heterologous expression of Arabidopsis UDP-glucosyltransferases in Saccharomyces cerevisiae for production of zearalenone-4-O-glucoside. Appl Environ Microbiol 72:4404–4410

    Article  CAS  Google Scholar 

  • Prinz B, Schultchen J, Rydzewski R, Holz C, Boettner M, Stahl U, Lang C (2004) Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. J Struct Funct Genomics 5:29–44

    Article  CAS  Google Scholar 

  • Raemaekers RJ, de Muro L, Gatehouse JA, Fordham-Skelton AP (1999) Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide. Eur J Biochem 265:394–403

    Article  CAS  Google Scholar 

  • Resina D, Maurer M, Cos O, Arnau C, Carnicer M, Marx H, Gasser B, Valero F, Mattanovich D, Ferrer P (2009) Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. Nat Biotechnol 25:396–403

    CAS  Google Scholar 

  • Schmidt FR (2004) Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol 65:363–372

    Article  CAS  Google Scholar 

  • Shi L, Wang D, Chan W, Cheng L (2007) Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 54:220–226

    Article  CAS  Google Scholar 

  • Shukla AK, Haase W, Reinhart C, Michel H (2007) Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells. Int J Biochem Cell Biol 39:931–942

    Article  CAS  Google Scholar 

  • Sreekrishna K, Potenz RH, Cruze JA, McCombie WR, Parker KA, Nelles L, Mazzaferro PK, Holden KA, Harrison RG, Wood PJ (1988) High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris. J Basic Microbiol 28:265–278

    Article  CAS  Google Scholar 

  • Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190:55–62

    Article  CAS  Google Scholar 

  • Stadlmayr G, Mecklenbrauker A, Rothmuller M, Maurer M, Sauer M, Mattanovich D, Gasser B (2010) Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 150:519–529

    Article  CAS  Google Scholar 

  • Sunga AJ, Cregg JM (2004) The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris. Gene 330:39–47

    Article  CAS  Google Scholar 

  • Swanton E, Bulleid NJ (2003) Protein folding and translocation across the endoplasmic reticulum membrane. Mol Membr Biol 20:99–104

    Article  CAS  Google Scholar 

  • Trujillo LE, Arrieta JG, Dafhnis F, Garcia J, Valdes J, Tambara Y, Perez M, Hernandez L (2001) Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris. Enzyme Microb Technol 28:139–144

    Article  CAS  Google Scholar 

  • Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15:3859–3876

    Article  CAS  Google Scholar 

  • Vad R, Nafstad E, Dahl LA, Gabrielsen OS (2005) Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol 116:251–260

    Article  CAS  Google Scholar 

  • Valdes I, Hermida L, Zulueta A, Martin J, Silva R, Alvarez M, Guzman MG, Guillen G (2007) Expression in Pichia pastoris and immunological evaluation of a truncated dengue envelope protein. Mol Biotechnol 35:23–30

    Article  CAS  Google Scholar 

  • Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K, Contreras R (2004) In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol 70:2639–2646

    Article  CAS  Google Scholar 

  • Walsh G (2010) Biopharmaceutical benchmarks. Nat Biotech 28:917–924

    Article  CAS  Google Scholar 

  • Wamalwa BM, Zhao G, Sakka M, Shiundu PM, Kimura T, Sakka K (2007) High-level heterologous expression of Bacillus halodurans putative xylanase xyn11a (BH0899) in Kluyveromyces lactis. Biosci Biotechnol Biochem 71:688–693

    Article  CAS  Google Scholar 

  • Wu J, Kaufman RJ (2006) From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ 13:374–384

    Article  CAS  Google Scholar 

  • Yokoyama S (2003) Protein expression systems for structural genomics and proteomics. Curr Opin Chem Biol 7:39–43

    Article  CAS  Google Scholar 

  • Zhang W, Zhao HL, Xue C, Xiong XH, Yao XQ, Li XY, Chen HP, Liu ZM (2006) Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins. Biotechnol Prog 22:1090–1095

    Article  CAS  Google Scholar 

  • Zhu T, Guo M, Tang Z, Zhang M, Zhuang Y, Chu J, Zhang S (2009) Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris. J Appl Microbiol 107:954–963

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Chung-Jr Huang

    Present address: Cell Science and Technology, AMGEN Inc, Thousand Oaks, CA, USA

Authors and Affiliations

  1. Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, MG, Brazil

    Leonardo M. Damasceno

  2. Department of Microbiology, Cornell University, Ithaca, NY, USA

    Chung-Jr Huang & Carl A. Batt

Authors
  1. Leonardo M. Damasceno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chung-Jr Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carl A. Batt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carl A. Batt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Damasceno, L.M., Huang, CJ. & Batt, C.A. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93, 31–39 (2012). https://doi.org/10.1007/s00253-011-3654-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3654-z

Keywords

Search

Navigation