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:

Secretion of Active, Full– and Half–Length Human Secretory Leukocyte Protease Inhibitor by Sacchammyces Cerevisiae

Bio/Technology volume 7, pages 55–60 (1989)Cite this article

Abstract

A cDNA clone for human secretory leukocyte inhibitor (SLPI) was used to construct fusions to the secretion signal sequences of two secreted yeast proteins: invertase and α-factor. The invertase system produced both active SLPI, cleaved at the predicted signal-peptidase processing site, and an inactive form of SLPI that retains the invertase signal peptide. Both forms remained cell-associated, but it is possible to extract selectively the processed, active form of the protein without mechanical disruption of the cells. The α-factor system resulted in secretion, of several forms of active and inactive SLPI that result from the mis- or incomplete processing of the fusion polypeptide. We also report the expression and secretion of a truncated form of SLPI, using α-factor secretory signals, that contains the proposed elastase inhibitory site. This 60-amino-acid variant is a correctly processed, fully active inhibitor of leukocyte elastase but lacks the trypsin inhibitory activity of the full-length molecule.

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. Thompson, R.C. and Ohlsson, K. 1986. Isolation, properties and complete ammo-acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc. Natl. Acad. Sciences U.S.A. 83:6692–6696.

    Article  CAS  Google Scholar 

  2. Ohlsson, K., Stetler, G.L., Brewer, M.T., Rosengren, M., Hale, K.K., and Thompson, R.C. 1987. Structure, genomic organization, and tissue distribution of human secretory leukocyte protease inhibitor: A potent inhibitor of neutrophil elastase, p. 307–323 In: Pulmonary Emphysema and Proteolysis (Volume III) Academic Press, Inc., Orlando, FL.

    Google Scholar 

  3. Grutter, M., Fendnch, G., Huber, R., and Bode, W. 1988. The 2.5 Å x-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine α-chymotrypsin. EMBO J. 7:345–351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hubbard, R.C. and Crystal, R.G. 1986. Antiproteases and antioxidants Strategies for the pharmacologic prevention of lung destruction. Respiration 50(suppl 1):56–73.

    Article  CAS  PubMed  Google Scholar 

  5. Wewers, M.D., Casolaro, M.A., Sellers, S.E., Swayze, S.C., McPhaul, K.M., Wittes, J.T., and Crystal, R.G. 1987. Replacement therapy for α1-antitrypsin deficiency associated with emphysema. New Engl. J. Med. 316:1055–1062.

    Article  CAS  PubMed  Google Scholar 

  6. Weinbaum, G. and Damiano, V.V. 1987. Protease inhibitor therapy in emphysema: a promising theory with problems. Trends in Physiol. Science 8:6–7.

    Article  Google Scholar 

  7. Brake, A.J., Merryweather, J.P., Coit, D.G., Heberlein, U.A., Masiarz, F.R., Mullenbach, G.T., Urdea, M.S., Valenzuela, P., and Barr, P. 1984. α-factor directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc Natl. Acad. Sci. U.S.A. 81:4642–4646.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zsebo, K. M., Lu, H., Fieschko, J.C., Goldstein, L., Davis, J., Duker, K., Suggs, S., Lai, P., and Bitter, G. 1986. Protein secretion from Saccharomyces cerevisiae directed by the prepro-α-factor region. J. Biol. Chem. 261:5858–5865.

    CAS  PubMed  Google Scholar 

  9. Chang, C., Matteucci, M., Perry, L.J., Wulf, J.J., Chen, C.Y. and Hitzman, R.A. 1986. Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. Mol. Cell. Biology 6:1812–1819.

    Article  CAS  Google Scholar 

  10. Smith, R.A., Duncan, M. J., and Moir, D.T. 1985. Heterologous protein secretion from yeast. Science 229:1219–1229.

    Article  CAS  PubMed  Google Scholar 

  11. Taussig, R. and Carlson, M. 1983. Nucleotide sequence of the yeast SUC2 gene. Nucleic Acids Research 11:1943–1954.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kozak, M. 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic nbosomes. Cell 44: 283–292.

    Article  Google Scholar 

  13. Stetler, G.L. and Thorner, J. 1984. Molecular cloning of hormone responsive genes from the yeast Saccharomyces cerevisiae. Proc. Acad. Sci. U.S.A. 81:1144–1148.

    Article  CAS  Google Scholar 

  14. Van Arsdell, S.W., Stetler, G.L., and Thorner, J. 1987. Yeast repeated element sigma contains a hormone-inducible promoter. Mol. Cell. Biology 7:749–759.

    Article  CAS  Google Scholar 

  15. Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. 1984. Isolation of the utative structural gene for the lysine arginine cleaving endopeptidase required for processing of yeast prepro-α factor. Cell 37:1075–1089.

    Article  CAS  PubMed  Google Scholar 

  16. Julius, D., Blair, L., Brake, A., Sprague, G., and Thorner, J. 1983. Yeast α-factor is processed from a larger precursor polypeptide: the essential role of a membrane bound dipeptidyl aminopeptidase. Cell 32:839–852.

    Article  CAS  PubMed  Google Scholar 

  17. Stetler, G., Brewer, M.T., and Thompson, R. C. 1986. Isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. Nucleic Acids Res. 14:7883–7896.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kurjan, J. and Herskowitz, I. 1982. Structure of a yeast pheromone gene (MFα1) a putative α-factor precursor containing four tandem copies of mature α-factor. Cell 30:933–943.

    Article  CAS  PubMed  Google Scholar 

  19. De Baetselier, A., Crahay, J., Delcour, J., and Hanotier, J. 1986. A simple and rapid recovery procedure of heterologous proteins synthesized by yeasts and localized in the periplasmic space. Yeast 2:s453.

    Google Scholar 

  20. Rothman, J.H., Hunter, C.P., Valls, L.A., and Stevens, T.H. 1986. Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene. Proc. Natl. Acad. Sci. U.S.A. 83:3248–3252.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kunkel, T.A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sciences. U.S.A. 82:488–492.

    Article  CAS  Google Scholar 

  22. Rudolph, H., Koenig Rauseo, I., and Hinnen, A. 1985. One-step gene replacement in yeast by cotransformation. Gene 36:87–95.

    Article  CAS  PubMed  Google Scholar 

  23. Davis, R.W., Botstein, D., and Roth, J.R. 1980. Advanced Bacterial Genetics: A Manual for Genetic Engineering. Cold Spring Harbor Laboratory, New York.

  24. Struhl, K., Stinchcomb, D.T., Scherer, S., and Davis, R.W. 1979. High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 76:1035–1039.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sherman, F., Fink, G.R., and Lawrence, C.W. 1979. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory, New York

  26. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Hewick, R.M., Hunkapiller, N.W., Hood, L.E., and Dreyer, W.J. 1981. A gas liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256:7990–7997.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Szewczyk, B. and Kozloff, L.M. 1985. A method for the efficient blotting of strongly basic proteins from sodium dodecy sulfate polyacrylamide gels to nitrocellulose Analytical Biochem. 150:403–407.

    Article  CAS  Google Scholar 

  30. Sanger, F. and Coulson, A.R. 1975. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94:441–448.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. G. L. Stetler

    Present address: Somatogen, Inc., 350 Interlocken Parkway, Suite 100, Broomfield, CO, 80020

  2. G. L. Stetler: Corresponding author.

Authors and Affiliations

  1. Synergen, 1885 33rd Street, Boulder, CO, 80301

    C. Forsyth, T. Gleason, J. Wilson & R. C. Thompson

Authors
  1. G. L. Stetler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Forsyth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. Gleason
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R. C. Thompson
    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

Stetler, G., Forsyth, C., Gleason, T. et al. Secretion of Active, Full– and Half–Length Human Secretory Leukocyte Protease Inhibitor by Sacchammyces Cerevisiae. Nat Biotechnol 7, 55–60 (1989). https://doi.org/10.1038/nbt0189-55

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0189-55

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