Skip to main content
SpringerLink
Log in

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization

  • Biotechnological Products and Process Engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Agaphonov MO, Plotnikova TA, Fokina AV, Romanova NV, Packeiser AN, Kang HA, Ter-Avanesyan MD (2007) Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway. FEMS Yeast Res 7:1145–1152

    Article  CAS  Google Scholar 

  • Antebi A, Fink GR (1992) The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol Biol Cell 3:633–654

    Article  CAS  Google Scholar 

  • Bates S, MacCallum DM, Bertram G, Munro CA, Hughes HB, Buurman ET, Brown AJ, Odds FC, Gow NA (2005) Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence. J Biol Chem 280:23408–23415

    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 

  • Chang SW, Lee GC, Shaw JF (2006) Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem 54:815–822

    Article  CAS  Google Scholar 

  • Cortés JC, Katoh-Fukui R, Moto K, Ribas JC, Ishiguro J (2004) Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis. Eukaryot Cell 3:1124–1135

    Article  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 

  • Dürr G, Strayle J, Plemper R, Elbs S, Klee SK, Catty P, Wolf DH, Rudolph HK (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell 9:1149–1162

    Article  Google Scholar 

  • Dux MP, Inan M (2006) Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris. Yeast 23:613–621

    Article  CAS  Google Scholar 

  • Gasser B, Maurer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttilä M, Mattanovich D (2007) Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics 8:179

    Article  Google Scholar 

  • 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 2:6

    Article  Google Scholar 

  • Ko JH, Hahm MS, Kang HA, Nam SW, Chung BH (2002) Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene. Protein Expr Purif 25:488–493

    Article  CAS  Google Scholar 

  • 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 23:621–625

    Article  CAS  Google Scholar 

  • Melnick LM, Turner BG, Puma P, Price-Tillotson B, Salvato KA, Dumais DR, Moir DT, Broeze RJ, Avgerinos GC (1990) Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast. J Biol Chem 265:801–807

    CAS  PubMed  Google Scholar 

  • Murphy KP Jr, Gagne P, Pazmany C, Moody MD (1998) Expression of human interleukin-17 in Pichia pastoris: purification and characterization. Protein Expr Purif 12:208–214

    Article  CAS  Google Scholar 

  • Plantz BA, Sinha J, Villarete L, Nickerson KW, Schlegel VL (2006) Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl Microbiol Biotechnol 72:297–305

    Article  CAS  Google Scholar 

  • Schenk J, Balazs K, Jungo C, Urfer J, Wegmann C, Zocchi A, Marison IW, von Stockar U (2008) Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain. Biotechnol Bioeng 99:368–377

    Article  CAS  Google Scholar 

  • Sinclair G, Choy FY (2002) Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 26:96–105

    Article  CAS  Google Scholar 

  • Smith RA, Duncan MJ, Moir DT (1985) Heterologous protein secretion from yeast. Science 229:1219–1224

    Article  CAS  Google Scholar 

  • Subramanian GM, Fiscella M, Lamousé-Smith A, Zeuzem S, McHutchison JG (2007) Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nat Biotechnol 25:1411–1419

    Article  CAS  Google Scholar 

  • Surribas A, Stahn R, Montesinos JL, Enfors SO, Valero F, Jahic M (2007) Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies. J Biotechnol 130:291–299

    Article  CAS  Google Scholar 

  • Uccelletti D, Farina F, Mancini P, Palleschi C (2004) KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis. J Biotechnol 109:93–101

    Article  CAS  Google Scholar 

  • Wang J, Nguyen V, Glen J, Henderson B, Saul A, Miller LH (2005) Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media. Biotechnol Bioeng 90:838–847

    Article  CAS  Google Scholar 

  • Werten MW, van den Bosch TJ, Wind RD, Mooibroek H, de Wolf FA (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1087–1096

    Article  CAS  Google Scholar 

  • Woo JH, Liu YY, Mathias A, Stavrou S, Wang Z, Thompson J, Neville DM Jr (2002) Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expr Purif 25:270–282

    Article  CAS  Google Scholar 

  • Woo JH, Liu YY, Stavrou S, Neville DM Jr (2004) Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris. Appl Environ Microbiol 70:3370–3376

    Article  CAS  Google Scholar 

  • Woo JH, Liu JS, Kang SH, Singh R, Park SK, Su Y, Ortiz J, Neville DM Jr, Willingham MC, Frankel AE (2008) GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials. Protein Expr Purif 58:1–11

    Article  CAS  Google Scholar 

  • Xiao AF, Zhou XS, Zhou L, Zhang YX (2006) Detection of intracellular reactive oxygen species by flow cytometry in Pichia pastoris fermentation. Chinese J Biotechnol 22:273–277

    Article  CAS  Google Scholar 

  • Xue C, Zhao HL, Liu ZM (2005) A simple and practical densimetric scanning protocol with computer programs. Lett Biotechnol 16:284–286

    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 

  • Zhang W, Inan M, Meagher MM (2007) Rational design and optimization of fed-batch and continuous fermentations. Methods Mol Biol 389:43–64

    Article  CAS  Google Scholar 

  • Zhao HL, Xue C, Wang Y, Li XY, Xiong XH, Yao XQ, Liu ZM (2007) Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation. J Biotechnol 131:245–252

    Article  CAS  Google Scholar 

  • Zhao HL, Xue C, Wang Y, Duan QF, Xiong XH, Yao XQ, Liu ZM (2008) Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon-alpha2b fusion protein. Yeast 25:279–286

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai district, Beijing, 100071, People’s Republic of China

    Hong Liang Zhao, Chong Xue, Yang Wang, Xue Qin Yao & Zhi Min Liu

Authors
  1. Hong Liang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chong Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xue Qin Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhi Min Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhi Min Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhao, H.L., Xue, C., Wang, Y. et al. Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization. Appl Microbiol Biotechnol 81, 235–241 (2008). https://doi.org/10.1007/s00253-008-1666-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1666-0

Keywords

Search

Navigation