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:

Continuous Production of a Novel Lysozyme via Secretion from the Yeast, Pichia pastoris

Bio/Technology volume 7, pages 160–164 (1989)Cite this article

Abstract

We have cloned a cDNA for bovine lysozyme c2, a novel lysozyme characteristic of the cow stomach and expressed its protein product by secretion using its native signal sequence in the methylotrophic yeast, Pichia pastoris. A semi–continuous fermentation process used for production resulted in extremely high cell densities and product concentrations. During fed–batch fermentation, bovine lysozyme concentrations exceeded 550 mg/liter, and cell densities exceeded 120 g dry cells/liter. In continuous culture, bovine lysozyme and cell concentrations remained constant at approximately 350 mg/liter and 100 g dry cells/liter, respectively. The recombinant product is a single biologically active species that constitutes >80% of the protein in media from expressing cells; the signal sequence of the pre–lysozyme is accurately processed.

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. Cregg, J.M., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, P.A., Davis, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G., and Thill, G.P. 1987. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/Technology 5:479–485.

    CAS  Google Scholar 

  2. Tschopp, J.F., Sverlow, G., Kosson, R., Craig, W., and Grinna, L. 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast Pichia pastoris. Bio/Technology 5:1305–1308.

    CAS  Google Scholar 

  3. 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  Google Scholar 

  4. Tschopp, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A., and Gingeras, T.R. 1987. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucl. Acids Res. 15:3859–3876.

    Article  CAS  Google Scholar 

  5. Dobson, D.E., Prager, E.M., and Wilson, A.C. 1984. Stomach lysozymes of ruminants. I. Distribution and catalytic properties. J. Biol. Chem. 259:11607–11616.

    CAS  PubMed  Google Scholar 

  6. Jolles, P., Schoentgen, F., Jolles, J., Dobson, D.E., Prager, E.M. and Wilson, A.C. 1984. Stomach lysozymes of ruminants. II. Amino acid sequence of cow lysozyme 2 and immunological comparisons with other lysozymes. J. Biol. Chem. 259:11617–11625.

    CAS  PubMed  Google Scholar 

  7. Weisman, L.S., Krummel, B.M., and Wilson, A.C. 1986. Evolutionary shift in the site of cleavage of prelysozyme. J. Biol. Chem. 261:2309–2313.

    CAS  PubMed  Google Scholar 

  8. Yoshimura, K., Toibana, A., and Nakahama, K. 1988. Human lysozyme: Sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 150:794–801.

    Article  CAS  Google Scholar 

  9. Digan, M.E., Tschopp, J., Grinna, L., Lair, S.V., Craig, W.S., Velicelebi, G., Siegel, R., Davis, G.R., and Thill, G.P. 1988. Secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris. Dev. Ind. Microbiol. 29 59–65.

    CAS  Google Scholar 

  10. 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  Google Scholar 

  11. 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, p. 1–18. In: Biological Research on Industrial Yeasts, Vol. II. Stewart, G. G., Russell, I., Klein, R. D., and Hiebsch, R. R. (Eds.). CRC Press, Boca Raton, Florida.

    Google Scholar 

  12. Brierley, R.A., Bussineau, C., Kosson, R., Melton, A., and Siegel, R.S. 1989. Fermentation development of the yeast Pichia pastoris expressing the heterologous gene: bovine lysozyme. Ann. N.Y. Acad, Sci. In press.

  13. Gubler, U. and Hoffman, B.J. 1983. A simple and very efficient method for generating cDNA libraries. Gene 25:263–269.

    Article  CAS  Google Scholar 

  14. Lapeyre, B. and Amalric, F. 1985. A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein. Gene 37:215–220.

    Article  CAS  Google Scholar 

  15. Huynh, T.V., Young, R.A., and Davis, R.W. 1984. Constructing and screening cDNA libraries in λgt10 and λgt11, p. 49–78. In: DNA Cloning Techniques: A Practical Approach. Glover, D. (Ed.) IRL Press, Oxford.

    Google Scholar 

  16. Wood, W.I., Capon, D.J., Simonsen, C.C., Eaton, D.L., Gitschier, J., Keyt, B., Seeburg, P.H., Smith, D.H., Hollingshead, P., Wion, K.L., Delwart, E., Tuddenham, E.G.D., Vehar, G.A., and Lawn, R.M. 1984. Expression of active human factor VIII from recombinant DNA clones. Nature 312:330–337.

    Article  CAS  Google Scholar 

  17. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

    Article  CAS  Google Scholar 

  18. Karanthanasis, S. M13 DNA sequencing using reverse transcriptase. BRL Focus 4:3–6.

  19. Zoller, M.J. and Smith, M. 1983. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Meth. Enzymol. 100:468–500.

    Article  CAS  Google Scholar 

  20. Hobden, A.N., Read, M.J., Dykes, C.W., and Harford, S. 1985. M13 clones carrying point mutations: Identification by solution hybridization. Anal. Biochem. 144 75–78.

    Article  CAS  Google Scholar 

  21. Ito, H., Murata, K., and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations or thiol compounds. Agric. Biol. Chem. 48:341–347.

    Google Scholar 

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

    Article  CAS  Google Scholar 

  23. 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  Google Scholar 

  24. 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  Google Scholar 

  25. Shugar, D. 1952. Measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochim. Biophys. Acta 8:302–309.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. M. E. Digan: Corresponding author.

Authors and Affiliations

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

    M. E. Digan, S. V. Lair, R. A. Brierley, R. S. Siegel, M. E. Williams, S. B. Ellis, P. A. Kellaris, S. A. Provow, W. S. Craig, G. Veliçelebi, M. M. Harpold & G. P. Thill

Authors
  1. M. E. Digan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. V. Lair
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. A. Brierley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. S. Siegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. E. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S. B. Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. P. A. Kellaris
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S. A. Provow
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. W. S. Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. G. Veliçelebi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. M. M. Harpold
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. 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

Digan, M., Lair, S., Brierley, R. et al. Continuous Production of a Novel Lysozyme via Secretion from the Yeast, Pichia pastoris. Nat Biotechnol 7, 160–164 (1989). https://doi.org/10.1038/nbt0289-160

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0289-160

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