Advertisement

Bioreactor Production of scFv Fragments in Pichia pastoris

  • Stephan HellwigEmail author
  • Georg Melmer
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The pubmed search term “pastoris[Title] AND (express[Title] OR produced[Title] OR expression[Title] OR production[Title])” yielded 877 hits in December 2008, dated from 1987 to 2009. At the same time, the search term “pastoris[Title] AND (bioreactor[Title] OR fed-batch[Title] OR continuous[Title] OR fermentations[Title] OR large-scale[Title] OR fermentation[Title] OR pilot[Title])” returned 92 hits –published between 1990 and 2009. This analysis is somewhat superficial and ostentatious, but it suggests that the majority of researchers publishing on Pichia use it as a tool for rather than an object of their work. This is not to say that the majority should change their focus, but in fact researchers sometimes face difficulties when the need to obtain useful amounts of a target protein produced in Pichia calls for scale-up from the benchtop protocols to a bioreactor-based process. This chapter attempts to provide a reliable protocol for AOX1-driven bioreactor production of secreted scFvs or other proteins.

Keywords

Methanol Concentration Oxygen Transfer Rate Folding Rate Feed Pump AOX1 Promoter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Bahrami A, Shojaosadati SA, Khalilzadeh R, Farahani EV (2008) Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris. Biotechnol Lett 30(6):1081–1085PubMedCrossRefGoogle Scholar
  2. Brankamp RG, Sreekrishna K, Smith PL, Blankenship DT, Cardin AD (1995) Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris. Protein Expr Purif 6(6):813–820PubMedCrossRefGoogle Scholar
  3. Brierley RA, Bussineau C, Kosson R, Melton A, Siegel RS (1990) Fermentation development of recombinant Pichia pastoris expressing the heterologous gene: bovine lysozyme. Ann N Y Acad Sci 589:350–362PubMedCrossRefGoogle Scholar
  4. Cai H, Chen L, Wan L, Zeng L, Yang H, Li S, Li Y, Cheng J, Lu X (2009) High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in Pichia pastoris. Appl Microbiol Biotechnol 82(1):41–48PubMedCrossRefGoogle Scholar
  5. Chang SW, Shieh CJ, Lee GC, Akoh CC, Shaw JF (2006) Optimized growth kinetics of Pichia pastoris and recombinant Candida rugosa LIP1 production by RSM. J Mol Microbiol Biotechnol 11(1–2):28–40PubMedCrossRefGoogle Scholar
  6. Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105(2):205–212PubMedCrossRefGoogle Scholar
  7. Cupit PM, Whyte JA, Porter AJ, Browne MJ, Holmes SD, Harris WJ, Cunningham C (1999) Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris. Lett Appl Microbiol 29(5):273–277PubMedCrossRefGoogle Scholar
  8. 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(1):18–26PubMedCrossRefGoogle Scholar
  9. d'Anjou MC, Daugulis AJ (2001) A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol Bioeng 72(1):1–11PubMedCrossRefGoogle Scholar
  10. Eldin P, Pauza ME, Hieda Y, Lin G, Murtaugh MP, Pentel PR, Pennell CA (1997) High-level secretion of two antibody single chain Fv fragments by Pichia pastoris. J Immunol Methods 201(1):67–75PubMedCrossRefGoogle Scholar
  11. Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94(2):353–361PubMedCrossRefGoogle Scholar
  12. Guarna MM, Cote HC, Amandoron EA, MacGillivray RT, Warren RA, Kilburn DG (1996) Engineering factor X fusions for expression in Pichia pastoris. Ann N Y Acad Sci 799:397–400PubMedCrossRefGoogle Scholar
  13. Hellwig S, Emde F, Raven NP, Henke M, van Der Logt P, Fischer R (2001) Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. Biotechnol Bioeng 74(4):344–352PubMedCrossRefGoogle Scholar
  14. Hellwig S, Robin F, Drossard J, Raven NP, Vaquero-Martin C, Shively JE, Fischer R (1999) Production of carcinoembryonic antigen (CEA) N-A3 domain in Pichia pastoris by fermentation. Biotechnol Appl Biochem 30(Pt 3):267–275PubMedGoogle Scholar
  15. Henry A, Masters CL, Beyreuther K, Cappai R (1997) Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization. Protein Expr Purif 10(2):283–291PubMedCrossRefGoogle Scholar
  16. Invitrogen (2002) Pichia fermentation process guidelines, Version B. Available from Invitrogen. http://tools.invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf. Cited 5 Mar 2002
  17. Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO (2006) Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnol Prog 22(6):1465–1473PubMedGoogle Scholar
  18. Jahic M, Wallberg F, Bollok M, Garcia P, Enfors SO (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microb Cell Fact 2(1):6PubMedCrossRefGoogle Scholar
  19. Jungo C, Schenk J, Pasquier M, Marison IW, von Stockar U (2007) A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris. J Biotechnol 131(1):57–66PubMedCrossRefGoogle Scholar
  20. Katakura Y, Zhang W, Zhuang G, Omasa T, Kishimoto M, Goto Y, Suga K (1998) Effect of methanol concentration on the production of human ß2-glycoprotein I Doman V by a recombinant Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. J Ferm Bioeng 86(5):482–487CrossRefGoogle Scholar
  21. Khatri NK, Hoffmann F (2006) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93(5):871–879PubMedCrossRefGoogle Scholar
  22. Lin H, Kim T, Xiong F, Yang X (2007) Enhancing the production of Fc fusion protein in fed-batch fermentation of Pichia pastoris by design of experiments. Biotechnol Prog 23(3):621–625PubMedCrossRefGoogle Scholar
  23. Loewen MC, Liu X, Davies PL, Daugulis AJ (1997) Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris. Appl Microbiol Biotechnol 48(4):480–486PubMedCrossRefGoogle Scholar
  24. Miller KD, Weaver-Feldhaus J, Gray SA, Siegel RW, Feldhaus MJ (2005) Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expr Purif 42(2):255–267PubMedCrossRefGoogle Scholar
  25. Panjideh H, Coelho V, Dernedde J, Fuchs H, Keilholz U, Thiel E, Deckert PM (2008) Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants. Bioprocess Biosyst Eng 31(6):559–568PubMedCrossRefGoogle Scholar
  26. Phithakrotchanakoon C, Daduang R, Thamchaipenet A, Wangkam T, Srikhirin T, Eurwilaichitr L, Champreda V (2009) Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance. Appl Microbiol Biotechnol 82(1):131–140PubMedCrossRefGoogle Scholar
  27. Ren F, Li BC, Zhang NN, Cao M, Dan WB, Zhang SQ (2008) Expression, purification and characterization of anti-BAFF antibody secreted from the yeast Pichia pastoris. Biotechnol Lett 30(6):1075–1080PubMedCrossRefGoogle Scholar
  28. 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(1):55–62PubMedCrossRefGoogle Scholar
  29. Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Fuke M, Holden K, Phelps D, Wood P et al (1989) High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry 28(9):4117–4125PubMedCrossRefGoogle Scholar
  30. 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(9):3859–3876PubMedCrossRefGoogle Scholar
  31. Wan L, Cai H, Yang H, Lu Y, Li Y, Li X, Li S, Zhang J, Li Y, Cheng J, Lu X (2008) High-level expression of a functional humanized single-chain variable fragment antibody against CD25 in Pichia pastoris. Appl Microbiol Biotechnol 81(1):33–41PubMedCrossRefGoogle Scholar
  32. Woo JH, Liu YY, Neville DM Jr (2006) Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion. J Biotechnol 121(1):75–85PubMedCrossRefGoogle Scholar
  33. Wu D, Hao YY, Chu J, Zhuang YP, Zhang SL (2008) Inhibition of degradation and aggregation of recombinant human consensus interferon-alpha mutant expressed in Pichia pastoris with complex medium in bioreactor. Appl Microbiol Biotechnol 80(6):1063–1071PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Leitung Integrated Production PlatformsFraunhofer IMEAachenGermany

Personalised recommendations