Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris

Bio/Technology volume 5, pages 479–485 (1987)Cite this article

Abstract

The methylotrophic industrial yeast, Pi–chia pastoris, has been developed as a host system for the large–scale production of heterologous proteins. As an example of the systems, the synthesis of hepatitis B surface antigen (HBsAg) is described. Expression of the HBsAg gene is controlled by a methanol–regulated promoter derived from the primary alcohol oxidase gene, AOX1, of P. pastoris. The highest level of HBsAg is observed in a novel mutant strain of P. pastoris in which the AOX1 structural gene is deleted and replaced by a construction composed of the AOX1 promoter–HBsAg gene expression cassette. When grown on methanol, 2–3% of the soluble protein in this strain is HBsAg. Characterization of the recombinant HBsAg indicates that virtually all of the P. pastoris–synthesized HBsAg exists in a form which is similar to human serum–derived HBsAg 22 nm particles. Scale–up of the HBsAg production process from shake–flask to high cell–density fermentor cultures is described as well. A single culture of the P. pastoris HBsAg expression strain in a volume of 240 liters and at a density of 60 grams/liter dry weight of cells produced 0.4 grams/liter, or a total of 90 grams of HBsAg 22 nm particles, an amount sufficient for approximately 9 million doses of vaccine.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Hitzeman, R.A., Hagie, F.E., Levine, H.L., Goeddel, D.V., Ammerer, G. and Hall, B.D. 1981. Expression of a human gene for interferon in yeast. Nature 293: 717–722.

    Article  CAS  PubMed  Google Scholar 

  2. Chen, C.Y., Opperman, H. and Hitzeman, R.A. 1984. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae. Nucl. Acids Res. 12: 8951–8970.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Innis, M.A., Holland, M.J., McCabe, P.C., Cole, G.E., Wittman, V.P., Tal, R., Watt, K.W.K., Gelfand, D.H., Holland, J.P. and Meade, J.H. 1985. Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228: 21–26.

    Article  CAS  PubMed  Google Scholar 

  4. Lemontt, J.F., Wei, C.-M. and Dackowski, W.R. 1985. Expression of active human uterine tissue plasminogen activator in yeast. DNA 4: 419–428.

    Article  CAS  PubMed  Google Scholar 

  5. Mellor, J., Dobson, M.J., Roberts, N.A., Kingsman, A.J. and Kingsman, S.M. 1985. Factors affecting heterologous gene expression in Saccharomyces cerevisiae. Gene 33: 215–226.

    Article  CAS  PubMed  Google Scholar 

  6. Smith, R.A., Duncan, M.J. and Moir, D.T. 1985. Heterologous protein secretion from yeast. Science 229: 1219–1224.

    Article  CAS  PubMed  Google Scholar 

  7. Etcheverry, T., Forrester, W. and Hitzeman, R. 1986. Regulation of the chelatin promoter during the expression of human serum albumin or yeast phosphoglycerate kinase in yeast. Bio/Technology 4: 726–730.

    CAS  Google Scholar 

  8. Green, R., Schaber, M.D., Shields, D. and Kramer, R. 1986. Secretion of somatostatin by Saccharomyces cerevisiae: correct processing of an α-factor-somatostatin hybrid. J. Biol. Chem. 261: 7558–7565.

    CAS  PubMed  Google Scholar 

  9. Wen, D. and Schlesinger, M.J. 1986. Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 83: 3639–3643.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Primrose, S.B., Derbyshire, P., Jones, I.M., Nugent, M. and Tacon, W. 1983. Hereditary instability of recombinant DNA molecules. Soc. Gen. Microbiol. 10: 63–67.

    CAS  Google Scholar 

  11. Loison, G., Nguyen-Juilleret, M., Alouani, S. and Marquet, M. 1986. Plasmid-transformed ura3 fur1 double-mutants of S. cerevisiae: an autoselection system applicable to the production of foreign proteins. Bio/Technology 4: 433–444.

    CAS  Google Scholar 

  12. Srienc, F., Campbell, J.L. and Bailey, J.E. 1986. Analysis of unstable recombinant Saccharomyces cerevisiae population growth in selective medium. Biotechnol. Bioeng. 28: 996–1006.

    Article  CAS  PubMed  Google Scholar 

  13. Shuster, J.R. 1987. Regulated gene expression of heterologous proteins in yeasts. In: Biological Research on Industrial Yeasts. Stewart, G.G., Russell, I., Klein, R.D., and Hiebsch, R.R. (eds.). CRC Press, Boca Raton, Florida.

    Google Scholar 

  14. Parulekar, S.J., Semones, G.B., Rolf, M.J., Lievense, J.C. and Lim, H.C. 1986. Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae. Biotechnol. Bioeng. 28: 700–710.

    Article  CAS  PubMed  Google Scholar 

  15. Ellis, S.B., Brust, P.F., Koutz, P.J., Waters, A.F., Harpold, M.M. and Gingeras, T.R. 1985. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol. Cell. Biol. 5: 1111–1121.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cregg, J.M. 1987. Genetics of methylotrophic yeasts. In: Proceedings of the Fifth International Symposium on Microbial Growth on Cl Compounds. Duine, J. A. and van Verseveld, H.W. (eds.). Gunk Publishing Co., Dordrect, Netherlands (in press).

    Chapter  Google Scholar 

  17. Couderc, R. and Baratti, J. 1980. Oxidation of methanol by the yeast Pichia pastoris. Purification and properties of alcohol oxidase. Agric. Biol. Chem. 44: 2279–2289.

    CAS  Google Scholar 

  18. Wegner, E.H. 1983. Biochemical conversions by yeast fermentation at high-cell densities. U.S. patent 4414329.

  19. Tiollais, P., Pourcel, C. and Dejean, A. 1985. The hepatitis B virus. Nature 317: 489–495.

    Article  CAS  PubMed  Google Scholar 

  20. Charnay, P., Gervais, M., Louise, A., Galibert, F. and Tiollais, P. 1980. Biosynthesis of hepatitis B virus surface antigen in Escherichia coli. Nature 286: 893–895.

    Article  CAS  PubMed  Google Scholar 

  21. Edman, J.C., Hallewell, R.A., Valenzuela, P., Goodman, H.M. and Rutter, W.J. 1981. Synthesis of hepatitis B surface and core antigens in E. coli. Nature 291: 503–506.

    Article  CAS  PubMed  Google Scholar 

  22. Fujisawa, Y., Ito, Y., Sasada, R., Ono, Y., Igarashi, K., Marumoto, R., Kikuchi, M. and Sugino, Y. 1983. Direct expression of hepatitis B surface antigen gene in E. coli. Nucl. Acids Res. 11: 3581–3591.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G. and Hall, B.D. 1982. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 298: 347–350.

    Article  CAS  PubMed  Google Scholar 

  24. Hitzeman, R.A., Chen, C.Y., Hagie, F.E., Patzer, E.J., Liu, C.C., Estell, D.A., Miller, J.V., Yaffe, A., Kleid, D.G., Levinson, A.D. and Oppermann, H. 1983. Expression of hepatitis B virus surface antigen in yeast. Nucl. Acids Res. 11: 2745–2763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Miyanohara, A., Toh-E, A., Nozaki, C., Hamada, F., Ohtomo, N. and Matsubara, K. 1983. Expression of hepatitis B surface antigen gene in yeast. Proc. Natl. Acad. Sci. USA 80: 1–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. De Wilde, M., Cabezon, T., Harford, N., Rutgers, T., Simoen, E. and Van Wijnendaele, F. 1985. Production in yeast of hepatitis B surface antigen by r-DNA technology, p.99–107. In: Congress, Joint IABS/WHO Symposium on Standardization and Control of Biologicals produced by Recombinant DNA Technology, Vol. 59, Karger S., Basel.

  27. Lee, G.M., Song, K.B., Rhee, S.K. and Han, M.H. 1986. Plasmid maintenance and growth of recombinant Saccharomyces cerevisiae producing hepatitis B virus surface antigen. Biotechnol. Lett. 8: 385–390.

    Article  CAS  Google Scholar 

  28. Ting, L.-P., Chang, H.-K. and Chiang, C.-S. 1986. Expression of hepatitis B surface antigen by yeast actin gene promoter. Biochem. Biophys. Res. Commun. 136: 1063–1070.

    Article  CAS  PubMed  Google Scholar 

  29. Valenzuela, P., Coit, D. and Kuo, C.H. 1985. Synthesis and assembly in yeast of hepatitis B surface antigen particles containing the polyalbumin receptor. Bio/Technology 3: 317–320.

    CAS  Google Scholar 

  30. Valenzuela, P., Gray, P., Quiroga, M., Zaldivar, J., Goodman, H.M. and Rutter, W.J. 1979. Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen. Nature 280: 815–819.

    Article  CAS  PubMed  Google Scholar 

  31. Cregg, J.M., Barringer, K.J., Hessler, A.Y. and Madden, K.R. 1985. Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5: 3376–3385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rothstein, R.J. 1983. One-step gene disruption in yeast. Methods in Enzymology 101: 202–210.

    Article  CAS  PubMed  Google Scholar 

  33. Cregg, J.M. and Madden, K.R. 1987. Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Biological Research on Industrial Yeasts. Stewart, G.G., Russell, I., Klein, R.D., and Hiebsch, R.R. (eds.). CRC Press, Boca Raton, Florida (in press).

    Google Scholar 

  34. Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.

    Article  CAS  PubMed  Google Scholar 

  35. Wampler, D.E., Lehman, E.D., Boger, J., McAleer, W.J. and Scolnick, E.M. 1985. Multiple chemical forms of hepatitis B surface antigen produced in yeast. Proc. Natl. Acad. Sci. USA 82: 6830–6834.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shih, J.W.-K. and Gerin, J.L. 1977. Proteins of hepatitis B surface antigen: Amino acid compositions of the major polypeptides. J. Virol. 21: 1219–1222.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Peterson, D.L. 1981. Isolation and characterization of the major protein and glycoprotein of hepatitis B surface antigen. J. Biol. Chem. 256: 6975–6983.

    CAS  PubMed  Google Scholar 

  38. Stibbe, W. and Gerlich, W.H. 1983. Structural relationships between minor and major proteins of hepatitis B surface antigen. J. Virol. 46: 626–628.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gerin, J.L., Purcell, R., Hoggan, M., Holland, P. and Chanock, R. 1969. Biophysical properties of Australia antigen. J. Virol. 4: 763–768.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Gerin, J.L., Holland, P.V. and Purcell, R.H. 1971. Australia antigen: large-scale purification from human serum and biochemical studies of its proteins. J. Virol. 7: 569–576.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. McAleer, W.J., Buynak, E.B., Maigetter, R.Z., Wampler, D.E., Miller, W.J. and Hilleman, M.R. 1984. Human hepatitis B vaccine from recombinant yeast. Nature 307: 178–180.

    Article  CAS  PubMed  Google Scholar 

  42. Grunstein, M. and Hogness, D.S. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961–3965.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

  44. Morrissey, J.H. 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117: 307–310.

    Article  CAS  PubMed  Google Scholar 

  45. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275.

    CAS  PubMed  Google Scholar 

  47. Hirschman, S.Z., Gerber, M. and Garfinkel, E. 1974. Purification of naked intranuclear particles from human liver infected by hepatitis B virus. Proc. Natl. Acad. Sci. USA 71: 3345–3349.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. J. Cruze and R. Torregrossa: Research and Development Division, Phillips Petroleum Company, Bartlesville, Oklahoma 74004.

  2. J. M. Cregg: Corresponding author.

Authors and Affiliations

  1. The Salk Institute Biotechology/Industrial Associates, Inc., La Jolla, California, 92037

    J. M. Cregg, J. F. Tschopp, C. Stillman, R. Siegel, M. Akong, W. S. Craig, R. G. Buckholz, K. R. Madden, P. A. Kellaris, G. R. Davis, B. L. Smiley, J. Cruze, R. Torregrossa, G. Velicȩlebi & G. P. Thill

Authors
  1. J. M. Cregg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. F. Tschopp
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Stillman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Siegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Akong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. W. S. Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R. G. Buckholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. K. R. Madden
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. P. A. Kellaris
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. G. R. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. B. L. Smiley
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. J. Cruze
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. R. Torregrossa
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. G. Velicȩlebi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. G. P. Thill
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cregg, J., Tschopp, J., Stillman, C. et al. High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris. Nat Biotechnol 5, 479–485 (1987). https://doi.org/10.1038/nbt0587-479

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0587-479

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing