Skip to main content
SpringerLink
Log in

Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Although the fission yeast Schizosaccharomyces pombe has been used for high-level heterologous protein production, the productivity of secreted human serum transferrin (hTF) has been low, presumably, because the protein harbors twenty disulfide bonds and two N-glycosylation sites. In the present study, we found that overexpression of endogenous putative protein disulfide isomerase (PDI) improved productivity. Whole genome sequence analysis of S. pombe revealed five putative PDI genes and overexpression of two of them, SPAC17H9.14c and SPBC3D6.13c (SpPdi2p or SpPdi3p, respectively), significantly improved the productivity of secreted hTF. GFP-fused SpPdi2p and SpPdi3p were found to localize to the endoplasmic reticulum. Co-overexpression of SpPdi2p or SpPdi3p with hTF coupled with modifications to the growth medium reported in our previous study were able to increase the level of secreted hTF approximately 30-fold relative to conventional conditions.

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
Fig. 5

Similar content being viewed by others

References

  • Appenzeller-Herzog C, Ellgaard L (2008) The human PDI family: versatility packed into a single fold. Biochim Biophys Acta 1783:535–548

    Article  CAS  Google Scholar 

  • Ballou CE, Ballou L, Ball G (1994) Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties. Proc Natl Acad Sci USA 91:9327–9331

    Article  CAS  Google Scholar 

  • Bao WG, Huo KK, Li YY, Fukuhara H (2000) Protein disulphide isomerase genes of Kluyveromyces lactis. Yeast 16:329–341

    Article  CAS  Google Scholar 

  • Brazer SC, Williams HP, Chappell TG, Cande WZ (2000) A fission yeast kinesin affects Golgi membrane recycling. Yeast 16:149–166

    Article  CAS  Google Scholar 

  • Broker M, Ragg H, Karges HE (1987) Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim Biophys Acta 908:203–213

    CAS  Google Scholar 

  • Bush DA, Horisberger M, Horman I, Wursch P (1974) The wall structure of Schizosaccharomyces pombe. J Gen Microbiol 81:199–206

    CAS  Google Scholar 

  • Cooper JP, Nimmo ER, Allshire RC, Cech TR (1997) Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385:744–747

    Article  CAS  Google Scholar 

  • Farquhar R, Honey N, Murant SJ, Bossier P, Schultz L, Montgomery D, Ellis RW, Freedman RB, Tuite MF (1991) Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae. Gene 108:81–89

    Article  CAS  Google Scholar 

  • Ferrari DM, Soling HD (1999) The protein disulphide-isomerase family: unravelling a string of folds. Biochem J 339:1–10

    Article  CAS  Google Scholar 

  • Frand AR, Kaiser CA (1999) Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum. Mol Cell 4:469–477

    Article  CAS  Google Scholar 

  • Frand AR, Kaiser CA (2000) Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum. Mol Biol Cell 11:2833–2843

    CAS  Google Scholar 

  • Freedman RB, Hirst TR, Tuite MF (1994) Protein disulphide isomerase: building bridges in protein folding. Trends Biochem Sci 19:331–336

    Article  CAS  Google Scholar 

  • Giga-Hama Y (1997) Fission yeast Schizosaccharomyces pombe:an attractive host for heterologous protein production. Springer-Verlag, Berlin

    Google Scholar 

  • Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem 30:235–244

    CAS  Google Scholar 

  • Giga-Hama Y, Tohda H, Takegawa K, Kumagai H (2007) Schizosaccharomyces pombe minimum genome factory. Biotechnol Appl Biochem 46:147–155

    Article  CAS  Google Scholar 

  • Goldberger RF, Epstein CJ, Anfinsen CB (1963) Acceleration of reactivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver. J Biol Chem 238:628–635

    CAS  Google Scholar 

  • Hayano T, Hirose M, Kikuchi M (1995) Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell. FEBS Lett 377:505–511

    Article  CAS  Google Scholar 

  • Idiris A, Bi K, Tohda H, Kumagai H, Giga-Hama Y (2006a) Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins. Yeast 23:83–99

    Article  CAS  Google Scholar 

  • Idiris A, Tohda H, Bi KW, Isoai A, Kumagai H, Giga-Hama Y (2006b) Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 73:404–420

    Article  CAS  Google Scholar 

  • Idiris A, Tohda H, Sasaki M, Okada K, Kumagai H, Giga-Hama Y, Takegawa K (2009) Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway. Appl Microbiol Biotechnol (this issue)

  • Ikeda S, Nikaido K, Araki K, Yoshitake A, Kumagai H, Isoai A (2004) Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe: development of a fed-batch fermentation and purification process. J Biosci Bioeng 98:366–373

    CAS  Google Scholar 

  • Isoai A, Kimura H, Reichert A, Schorgendorfer K, Nikaido K, Tohda H, Giga-Hama Y, Mutoh N, Kumagai H (2002) Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells. Biotechnol Bioeng 80:22–32

    Article  CAS  Google Scholar 

  • Kettner K, Blomberg A, Rodel G (2004) Schizosaccharomyces pombe ER oxidoreductin-like proteins SpEro1a p and SpEro1b p. Yeast 21:1035–1044

    Article  CAS  Google Scholar 

  • Kim SJ, Choi YS, Kim HG, Park EH, Lim CJ (2006) Cloning, characterization and regulation of a protein disulfide isomerase from the fission yeast Schizosaccharomyces pombe. Mol Biol Rep 33:187–196

    Article  CAS  Google Scholar 

  • Mukaiyama H, Giga-Hama Y, Tohda H, Takegawa K (2009) Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe. Appl Microbiol Biotechnol 88:155–164

    Google Scholar 

  • Nakamura T, Nakamura-Kubo M, Hirata A, Shimoda C (2001) The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1 + -encoding syntaxin-like protein. Mol Biol Cell 12:3955–3972

    CAS  Google Scholar 

  • Nakamura-Kubo M, Nakamura T, Hirata A, Shimoda C (2003) The fission yeast spo14+ gene encoding a functional homologue of budding yeast Sec12 is required for the development of forespore membranes. Mol Biol Cell 14:1109–1124

    Article  CAS  Google Scholar 

  • Okada H, Sekiya T, Yokoyama K, Tohda H, Kumagai H, Morikawa Y (1998a) Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl Microbiol Biotechnol 49:301–308

    Article  CAS  Google Scholar 

  • Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998b) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64:555–563

    CAS  Google Scholar 

  • Pidoux AL, Armstrong J (1992) Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe. EMBO J 11:1583–1591

    CAS  Google Scholar 

  • Robinson AS, Hines V, Wittrup KD (1994) Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae. Biotechnology (N Y) 12:381–384

    Article  CAS  Google Scholar 

  • Russel P (1989) Gene cloning and expression in fission yeast. Academic, San Diego

    Google Scholar 

  • Sakoh-Nakatogawa M, Nishikawa S, Endo T (2009) Roles of protein-disulfide isomerase-mediated disulfide bond formation of yeast Mnl1p in endoplasmic reticulum-associated degradation. J Biol Chem 284:11815–11825

    Article  CAS  Google Scholar 

  • Sander P, Grunewald S, Reilander H, Michel H (1994) Expression of the human D2S dopamine receptor in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: a comparative study. FEBS Lett 344:41–46

    Article  CAS  Google Scholar 

  • Shusta EV, Raines RT, Pluckthun A, Wittrup KD (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol 16:773–777

    Article  CAS  Google Scholar 

  • Tachibana C, Stevens TH (1992) The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase. Mol Cell Biol 12:4601–4611

    CAS  Google Scholar 

  • Tachikawa H, Takeuchi Y, Funahashi W, Miura T, Gao XD, Fujimoto D, Mizunaga T, Onodera K (1995) Isolation and characterization of a yeast gene, MPD1, the overexpression of which suppresses inviability caused by protein disulfide isomerase depletion. FEBS Lett 369:212–216

    Article  CAS  Google Scholar 

  • Tachikawa H, Funahashi W, Takeuchi Y, Nakanishi H, Nishihara R, Katoh S, Gao XD, Mizunaga T, Fujimoto D (1997) Overproduction of Mpd2p suppresses the lethality of protein disulfide isomerase depletion in a CXXC sequence dependent manner. Biochem Biophys Res Commun 239:710–714

    Article  CAS  Google Scholar 

  • Takegawa K, Tohda H, Sasaki M, Idiris A, Ohashi T, Mukaiyama H, Giga-Hama Y, Kumagai H (2009) Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system. Biotechnol Appl Biochem 53:227–235

    Article  CAS  Google Scholar 

  • Tommasino M, Contorni M, Cavalieri F (1992) HPV16 E7 phosphorylation in fission yeast: characterization and biological effects. Gene 111:93–98

    Article  CAS  Google Scholar 

  • Wang Q, Chang A (1999) Eps1, a novel PDI-related protein involved in ER quality control in yeast. EMBO J 18:5972–5982

    Article  CAS  Google Scholar 

  • Wang Q, Chang A (2003) Substrate recognition in ER-associated degradation mediated by Eps1, a member of the protein disulfide isomerase family. EMBO J 22:3792–3802

    Article  CAS  Google Scholar 

  • Zhao Y, Lieberman HB (1995) Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes. DNA Cell Biol 14:359–371

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We dedicate this work to the memory of the late Yuko Giga-Hama, a mentor to so many of us. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by the Ministry of Economy, Trade and Industry (METI) of Japan, as supported by the New Energy and Industrial Technology Development Organization (NEDO).

Author information

Authors and Affiliations

  1. ASPEX Division, Research Center, Asahi Glass Co., Ltd., 1150 Hazawa-cho, Kanagawa-ku, Yokohama, 221-8755, Japan

    Hiroyuki Mukaiyama & Hideki Tohda

  2. Laboratory of Applied Microbiology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581, Japan

    Hiroyuki Mukaiyama & Kaoru Takegawa

Authors
  1. Hiroyuki Mukaiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hideki Tohda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kaoru Takegawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kaoru Takegawa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mukaiyama, H., Tohda, H. & Takegawa, K. Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe . Appl Microbiol Biotechnol 86, 1135–1143 (2010). https://doi.org/10.1007/s00253-009-2393-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-009-2393-x

Keywords

Search

Navigation