Skip to main content
SpringerLink
Log in

The proteolytic systems and heterologous proteins degradation in the methylotrophic yeastPichia pastoris

  • Industrial Microbiology
  • Mini-review
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

ThePichia pastoris expression system has been successfully used for production of various recombinant heterogeneous proteins. The productivity ofP. pastoris can be improved substantially by bioreactor cultivations. However, heterologous proteins degradation increases as well in high-cell density culture. Proteolytic degradation is a serious problem since the yeast has been employed to express recombinant proteins. In this review, some of the recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels on the cytosolic proteasome, vacuolar proteases, and proteases located within the secretory pathway inP. pastoris, are reviewed.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Achstetter T., Ehmann C., Wolf D.H. (1982). Aminopeptidase Co, a new yeast peptidase. Biochem. Biophys. Res. Comm., 109: 341–347.

    Article  CAS  PubMed  Google Scholar 

  • Achstetter T., Emter O., Ehmann C., Wolf D.H. (1984). Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast. J. Biol. Chem., 259: 13334–13343.

    CAS  PubMed  Google Scholar 

  • Ammerer G., Hunte, C.P., Rothman J.H., Saari G.C., Valls L.A., Stevens T.H. (1986). PEP4 gene ofSaccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol. Cell. Biol., 6: 2490–2499.

    CAS  PubMed  Google Scholar 

  • Bourbonnais Y., Ash J., Daigle M., Thomas D.Y. (1993). Isolation and characterization ofS. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites. EMBO J., 12: 285–294.

    CAS  PubMed  Google Scholar 

  • Brady C.P., Shimp R.L., Miles A.P., Whitmore M., Stowers A.W. (2001). High-level production and purification of P30P2MSP119, an important vaccine antigen for malaria, expressed in the methylotropic yeastPichia pastoris. Protein Expr. Purif., 23: 468–475.

    Article  CAS  PubMed  Google Scholar 

  • Brankamp R.G., Sreekrishna K., Smith P.L., Blankenship D.T., Cardin A.D. (1995). Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeastPichia pastoris. Protein Expr. Purif., 6: 813–820.

    Article  CAS  PubMed  Google Scholar 

  • Brierley R.A. (1998). Secretion of recombinant human insulin-like growth factor I (IGF-I). Methods Mol Biol, 103: 149–177.

    CAS  PubMed  Google Scholar 

  • Bussey H. (2004). Proteases and the processing of precursors to secreted proteins in yeast. Yeast, 4: 17–26.

    Article  Google Scholar 

  • López-Otín C., Overall C.M. (2002). Protease degradomics: A new challenge for proteomics. Nature Rev. Mol. Cell Biol., 7: 509–519.

    Article  Google Scholar 

  • Cereghino J.L., Cregg J.M. (2000). Heterologous protein expression in the methylotrophic yeastPichia pastoris. FEMS Microbiol. Rev., 24: 45–66.

    Article  CAS  PubMed  Google Scholar 

  • Chen D.C., Wang B.D., Chou P., Kuo T.T. (2000). Asparagine as a nitrogen source for improving the secretion of mouse a-amylase inSaccharomyces cerevisiae protease A-deficient strains. Yeast, 16: 207–217.

    Article  CAS  PubMed  Google Scholar 

  • Chung B.H., Park K.S. (1998). Simple approach to reducing proteolysis during secretory production of human parathyroid hormone inSaccharomyces cerevisiae. Biotechnol. Bioeng., 57: 245–249.

    Article  CAS  PubMed  Google Scholar 

  • Clare J.J., Rayment F.B., Ballantine S.P., Sreekrishna K., Romanos M.A. (1991a). High-level expression of tetanus toxin fragment C inPichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (NY), 9: 455–460.

    Article  CAS  Google Scholar 

  • Clare J.J., Romanos M.A., Rayment F.B., Rowedder J.E., Smith M.A., Payne M.M., Sreekrishna K., Henwood C.A. (1991b). Production of mouse epidermal growth factor in yeast: high-level secretion usingPichia pastoris strains containing multiple gene copies. Gene, 105: 205–212.

    Article  CAS  PubMed  Google Scholar 

  • Cregg J.M., Cereghino J.L., Shi J., Higgins D.R. (2000). Recombinant protein expression inPichia pastoris. Mol. Biotechnol., 16: 23–52.

    Article  CAS  PubMed  Google Scholar 

  • Cueva R., Garcia-Alvarez N., Suarez-Rendueles P. (1989). Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APE1 (LAP4) structural gene. FEBS Lett., 259: 125–129.

    Article  CAS  PubMed  Google Scholar 

  • Egel-Mitani M., Flygenring H.P., Hansen M.T. (1990). A novel aspartyl protease allowing KEX2-independent MF alpha pro-pheromone processing in yeast. Yeast, 6: 127–137.

    Article  CAS  PubMed  Google Scholar 

  • Flores M.V., Cuellas A., Voget C.E. (1999). The proteolytic system of the yeastKluyveromyces lactis. Yeast, 15: 1437–1448.

    Article  CAS  PubMed  Google Scholar 

  • Germain D., Dumas F., Vernet T., Bourbonnais Y., Thomas D.Y., Boileau G. (1992). The pro-region of the Kex2 endoprotease ofSaccharomyces cerevisiae is removed by self-processing. FEBS Lett., 299: 283–286.

    Article  CAS  PubMed  Google Scholar 

  • Gleeson M.A., White C.E., Meininger D.P., Komives E.A. (1998). Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol. Biol., 103: 81–94.

    CAS  PubMed  Google Scholar 

  • Goodrick J.C., Xu M., Finnegan R., Schilling B.M., Schiavi S., Hoppe H., Wan N.C. (2001). High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutivePichia pastoris expression system. Biotechnol. Bioeng., 74: 492–497.

    Article  CAS  PubMed  Google Scholar 

  • Gustavsson M., Lehtio J., Denman S., Teeri T.T., Hult K., Martinelle M. (2001). Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed inPichia pastoris. Protein Eng., 14: 711–715.

    Article  CAS  PubMed  Google Scholar 

  • Hilt W., Wolf D.H. (1992). Stress-induced proteolysis in yeast. Mol. Microbiol. 6, 2437–2442.

    CAS  PubMed  Google Scholar 

  • Hilt W., Wolf D.H. (1995). Proteasomes of the yeastS. cerevisiae: genes, structure and functions. Mol. Biol. Reports, 21: 3–10.

    Article  CAS  Google Scholar 

  • Hirsch H.H., Schiffer H.H., Miiller H., Wolf D.H. (1992). Biogenesis of the yeast vacuole(lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide. Eur. J. Biochem., 203: 641–653.

    Article  CAS  PubMed  Google Scholar 

  • Holmquist M., Tessier D.C., Cygler M. (1997). High-level production of recombinantGeotrichum candidum lipases in yeastPichia pastoris. Protein Expr. Purif., 11: 35–40.

    Article  CAS  PubMed  Google Scholar 

  • Hong F., Meinander N.Q., Jonsson L.J. (2002). Fermentation strategies for improved heterologous expression of laccase inPichia pastoris. Biotechnol. Bioeng., 79: 438–449.

    Article  CAS  PubMed  Google Scholar 

  • Inan M., Chiruvolu V., Eskridge K.M., Vlasuk G.P., Dickerson K., Brown S., Meagher M.M. (1999). Optimization of temperature-glycerol-pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production byPichia pastoris. Enzyme Microb. Technol., 24: 438–445.

    Article  CAS  Google Scholar 

  • Jahic M., Gustavsson M., Jansen A.K., Martinelle M., Enfors S.O. (2003a). Analysis and control of proteolysis of a fusion protein inPichia pastoris fed-batch processes. J. Biotechnol., 102: 45–53.

    Article  CAS  PubMed  Google Scholar 

  • Jahic M., Knoblechner J., Charoenrat T., Enfors S.O., Veide A. (2006). InterfacingPichia pastoris cultivation with expanded bed adsorption. Biotechnol. Bioeng., 93: 1040–1049.

    Article  CAS  PubMed  Google Scholar 

  • Jahic M., Wallberg F., Bollok M., Garcia P., Enfors S.O. (2003b). Temperature limited fed-batch technique for control of proteolysis inPichia pastoris bioreactor cultures. Microb. Cell Fact., 2: 6.

    Article  PubMed  Google Scholar 

  • Jones E.W. (1991). Three proteolytic systems in the yeastSaccharomyces cerevisiae. J. Biol. Chem., 266: 7963–7966.

    CAS  PubMed  Google Scholar 

  • Jonsson L.J., Saloheimo M., Penttila M. (1997). Laccase from the white-rot fungusTrametes versicolor: cDNA cloning of Icc1 and expression inPichia pastoris. Curr. Genet., 32: 425–430.

    Article  CAS  PubMed  Google Scholar 

  • Jung G., Ueno H., Hayashi R. (1999). Carboxypeptidase Y: structural basis for protein sorting and catalytic triad. J. Biochem. (Tokyo), 126: 1–6.

    CAS  Google Scholar 

  • Kang H.A., Choi E.S., Hong W.K., Kim J.Y., Ko S.M., Sohn J.H., Rhee S.K. (2000). Proteolytic stability of recombinant human serum albumin secreted in the yeastSaccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 53: 575–582.

    Article  CAS  PubMed  Google Scholar 

  • Klionsky D.J., Banta L.M., Emr S.D. (1988). Intracellular sorting and processing of a yeast vacuolar hydrolase: Proteinase A propeptide contains vacuolar targeting information. Mol. Cell. Biol., 8: 2105–2116.

    CAS  PubMed  Google Scholar 

  • Knop M., Schiffer H.H., Rupp S., Wolf D.H. (1993). Vacuolar/lysosomal proteolysis: proteases, substrates, mechanisms. Curr. Opin. Cell. Biol., 5: 990–996.

    Article  CAS  PubMed  Google Scholar 

  • Koganesawa N., Aizawa T., Shimojo H., Miura K., Ohnishi A., Demura M., Hayakawa Y., Nitta K., Kawano K. (2002). Expression and purification of a small cytokine growth-blocking peptide from armywormPseudaletia separata by an optimized fermentation method using the methylotrophic yeastPichia pastoris. Protein Expres. Purif., 25: 416–425.

    Article  CAS  Google Scholar 

  • Kuwae S., Ohyama M., Ohya T., Ohi H., Kobayashi K. (2005). Production of recombinant human antithrombin byPichia pastoris. J. Biosci. Bioeng., 99: 264–271.

    Article  CAS  PubMed  Google Scholar 

  • Li Z., Xiong F., Lin Q., d’Anjou M., Daugulis A.J., Yang D.S., Hew C.L. (2001). Low-temperature increases the yield of biologically active herring antifreeze protein inPichia pastoris. Protein Expr. Purif., 21: 438–445.

    Article  PubMed  Google Scholar 

  • Lin H., Kim T., Xiong F., Yang X.M. (2007). Enhancing the production of fc fusion protein in fed-batch fermentation ofPichia pastoris by design of experiments. Biotechnol. Progr., 23: 621–625.

    Article  CAS  Google Scholar 

  • Macauley-Patrick S., Fazenda M.L., McNeil B., Harvey L.M. (2005). Heterologous protein production using thePichia pastoris expression system. Yeast, 22: 249–270.

    Article  CAS  PubMed  Google Scholar 

  • Maeda H., Chatani E., Koyama T., Sugiura M., Izumi H., Hayashi R. (2004). Indiscriminate glycosylation of procarboxypeptidase Y expressed inPichia pastoris. Carbohyd. Res., 339: 1041–1045.

    Article  CAS  Google Scholar 

  • Moehle C.M., Dixon C.K., Jones E.W. (1989). Processing pathway for protease B ofSaccharomyces cerevisiae. J. Cell Biol., 108: 309–324.

    Article  CAS  PubMed  Google Scholar 

  • Moehle C.M., Tizard R., Lemmon S.K., Smart J., Jones E.W. (1987). Protease B of the lysosomelike vacuole of the yeastSaccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol. Cell. Biol., 7: 4390–4399.

    CAS  PubMed  Google Scholar 

  • Nakagawa T., Murakami K., Nakayama K. (1993). Identification of an isoform with an extremely large Cys-rich region of PC6, a Kex2-like processing endoprotease. FEBS Lett., 327: 165–171.

    Article  CAS  PubMed  Google Scholar 

  • Nakayama K., Watanabe T., Nakagawa T., Kim W.S., Nagahama M., Hosaka M., Hatsuzawa K., Kondoh-Hashiba K., Murakami K. (1992). Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites. J. Biol. Chem., 267: 16335–16340.

    CAS  PubMed  Google Scholar 

  • Nebes V., Jones E.W. (1991). Activation of the proteinase B precursor of the yeastSaccharomyces cerevisiae by autocatalysis and by an internal sequence. J. Biol. Chem., 266: 22851–22857.

    CAS  PubMed  Google Scholar 

  • Nothwehr S.F., Roberts C.J., Stevens T.H. (1993). Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues. J. Cell Biol., 121: 1197–1209.

    Article  CAS  PubMed  Google Scholar 

  • Ramos C., Winther J.R., Kielland-Brandt M.C. (1994). Requirement of the propeptide forin vivo formation of active yeast carboxypeptidase Y. J. Biol. Chem., 269: 7006–7012.

    CAS  PubMed  Google Scholar 

  • Roberts C.J., Pohlig G., Rothman J.H., Stevens T.H. (1989). Structure, biosynthesis and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole. J. Cell Biol., 108: 1363–1373.

    Article  CAS  PubMed  Google Scholar 

  • Shelness G.S., Blobel G. (1990). Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein. J. Biol. Chem., 265: 9512–9519.

    CAS  PubMed  Google Scholar 

  • Shi X., Karkut T., Chamankhah M., Alting-Mees M., Hemmingsen S.M., Hegedus D. (2003). Optimal conditions for the expression of a single-chain antibody (scFv) gene inPichia pastoris. Protein Expr. Purif., 28: 321–330.

    Article  CAS  PubMed  Google Scholar 

  • Shiba Y., Fukui F., Ichikawa K., Serizawa N., Yoshikawa H. (1998). Process development for high-level secretory production of carboxypeptidase Y bySaccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 50: 34–41.

    Article  CAS  PubMed  Google Scholar 

  • Shilton B.H., Li Y., Tessier D., Thomas D.Y., Cygler M. (1996). Crystallization of a soluble form of the Kex1p serine carboxypeptidase fromSaccharomyces cerevisiae. Protein Sci., 5: 395–397.

    CAS  PubMed  Google Scholar 

  • Sinha J., Plantz B.A., Zhang W., Gouthro M., Schlegel V., Liu C.P., Meagher M.M. (2003). Improved production of recombinant ovine interferon-tau by mut(+) strain ofPichia pastoris using an optimized methanol feed profile. Biotechnol. Prog., 19: 794–802.

    Article  CAS  PubMed  Google Scholar 

  • Sorensen S.O., van den Hazel H.B., Kielland-Brandt M.C., Winther J.R. (1994). pH-dependent processing of yeast procarboxypeptidase Y by proteinase Ain vivo andin vitro. Eur. J. Biochem., 220: 19–27.

    Article  CAS  PubMed  Google Scholar 

  • Spormann D.O., Heim J., Wolf D.H. (1991). Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme. Eur. J. Biochem., 197: 399–405.

    Article  CAS  PubMed  Google Scholar 

  • Sreekrishna K., Brankamp R.G., Kropp K.E., Blankenship D.T., Tsay J.T., Smith P.L., Wierschke J.D., Subramaniam A., Birkenberger L.A. (1997). Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeastPichia pastoris. Gene, 190: 55–62.

    Article  CAS  PubMed  Google Scholar 

  • van den Hazel H.B., Kielland-Brandt M.C., Winther J.R. (1995). Random substitution of large parts of the propeptide of yeast proteinase A. J. Biol. Chem., 270: 8602–8609.

    Article  PubMed  Google Scholar 

  • Van Den Hazel H.B., Kielland-Brandt M.C., Winther J.R. (1996). Review: biosynthesis and function of yeast vacuolar proteases. Yeast, 12: 1–16.

    Article  Google Scholar 

  • Werten M.W., van den Bosch T.J., Wind R.D., Mooibroek H., de Wolf F.A. (1999). High-yield secretion of recombinant gelatins byPichia pastoris. Yeast, 15: 1087–1096.

    Article  CAS  PubMed  Google Scholar 

  • Woolford C.A., Daniels L.B., Park F.J., Jones E.W., van Arsdell J.N., Innis M.A. (1986). The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation ofSaccharomyces cerevisiae vacuolar hydrolases. Mol. Cell Biol., 6: 2500–2510.

    CAS  PubMed  Google Scholar 

  • Yasuhara T., Nakai T., Ohashi A. (1994). Aminopeptidase Y, a new aminopeptidase fromSaccharomyces cerevisiae. J. Biol. Chem., 269: 13644–13650.

    CAS  PubMed  Google Scholar 

  • Zhou X.S., Zhang Y.X. (2002). Decrease of proteolytic degradation of recombinant hirudin produced byPichia pastoris by controlling the specific growth rate. Biotechnol. Lett., 24: 1449–1453.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Engineering, School of Pharmaceutics, Jiangsu University, 212013, Zhenjiang, P.R. China

    Yewang Zhang & Ruijiang Liu

  2. School of Biochemical Engineering, Jiangxi Agricultural University, 330045, Nanchang, P.R. China

    Xiaoyu Wu

Authors
  1. Yewang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruijiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaoyu Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yewang Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, Y., Liu, R. & Wu, X. The proteolytic systems and heterologous proteins degradation in the methylotrophic yeastPichia pastoris . Ann. Microbiol. 57, 553–560 (2007). https://doi.org/10.1007/BF03175354

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03175354

Key words

Search

Navigation