Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Jul;56(7):2125–2132. doi: 10.1128/aem.56.7.2125-2132.1990

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.

R Hiramatsu 1, T Yamashita 1, J Aikawa 1, S Horinouchi 1, T Beppu 1
PMCID: PMC184571  PMID: 2117879

Abstract

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.

Full text

PDF
2125

Images in this article

2129Image
on p.2129
2130Image
on p.2130

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bitter G. A., Egan K. M. Expression of interferon-gamma from hybrid yeast GPD promoters containing upstream regulatory sequences from the GAL1-GAL10 intergenic region. Gene. 1988 Sep 30;69(2):193–207. doi: 10.1016/0378-1119(88)90430-1. [DOI] [PubMed] [Google Scholar]
  2. Bitter G. A., Egan K. M., Koski R. A., Jones M. O., Elliott S. G., Giffin J. C. Expression and secretion vectors for yeast. Methods Enzymol. 1987;153:516–544. doi: 10.1016/0076-6879(87)53076-2. [DOI] [PubMed] [Google Scholar]
  3. Brake A. J., Merryweather J. P., Coit D. G., Heberlein U. A., Masiarz F. R., Mullenbach G. T., Urdea M. S., Valenzuela P., Barr P. J. Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4642–4646. doi: 10.1073/pnas.81.15.4642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elliott S., Giffin J., Suggs S., Lau E. P., Banks A. R. Secretion of glycosylated human erythropoietin from yeast directed by the alpha-factor leader region. Gene. 1989 Jun 30;79(1):167–180. doi: 10.1016/0378-1119(89)90102-9. [DOI] [PubMed] [Google Scholar]
  5. Fieschko J. C., Egan K. M., Ritch T., Koski R. A., Jones M., Bitter G. A. Controlled expression and purification of human immune interferon from high-cell-density fermentations of Saccharomyces cerevisiae. Biotechnol Bioeng. 1987 Jun;29(9):1113–1121. doi: 10.1002/bit.260290911. [DOI] [PubMed] [Google Scholar]
  6. Fuller R. S., Brake A., Thorner J. Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1434–1438. doi: 10.1073/pnas.86.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goeddel D. V., Heyneker H. L., Hozumi T., Arentzen R., Itakura K., Yansura D. G., Ross M. J., Miozzari G., Crea R., Seeburg P. H. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature. 1979 Oct 18;281(5732):544–548. doi: 10.1038/281544a0. [DOI] [PubMed] [Google Scholar]
  8. Hashimoto H., Kikuchi Y., Nogi Y., Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol Gen Genet. 1983;191(1):31–38. doi: 10.1007/BF00330886. [DOI] [PubMed] [Google Scholar]
  9. Hayakawa T., Toibana A., Marumoto R., Nakahama K., Kikuchi M., Fujimoto K., Ikehara M. Expression of human lysozyme in an insoluble form in yeast. Gene. 1987;56(1):53–59. doi: 10.1016/0378-1119(87)90157-0. [DOI] [PubMed] [Google Scholar]
  10. Hiramatsu R., Aikawa J., Horinouchi S., Beppu T. Secretion by yeast of the zymogen form of Mucor rennin, an aspartic proteinase of Mucor pusillus, and its conversion to the mature form. J Biol Chem. 1989 Oct 5;264(28):16862–16866. [PubMed] [Google Scholar]
  11. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  12. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnston S. A., Zavortink M. J., Debouck C., Hopper J. E. Functional domains of the yeast regulatory protein GAL4. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6553–6557. doi: 10.1073/pnas.83.17.6553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laughon A., Driscoll R., Wills N., Gesteland R. F. Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):268–275. doi: 10.1128/mcb.4.2.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miyajima A., Otsu K., Schreurs J., Bond M. W., Abrams J. S., Arai K. Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. cerevisiae: mutagenesis of the potential glycosylation sites. EMBO J. 1986 Jun;5(6):1193–1197. doi: 10.1002/j.1460-2075.1986.tb04346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nogi Y., Fukasawa T. Nucleotide sequence of the transcriptional initiation region of the yeast GAL7 gene. Nucleic Acids Res. 1983 Dec 20;11(24):8555–8568. doi: 10.1093/nar/11.24.8555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Penttilä M. E., André L., Lehtovaara P., Bailey M., Teeri T. T., Knowles J. K. Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene. 1988;63(1):103–112. doi: 10.1016/0378-1119(88)90549-5. [DOI] [PubMed] [Google Scholar]
  18. Smith R. A., Duncan M. J., Moir D. T. Heterologous protein secretion from yeast. Science. 1985 Sep 20;229(4719):1219–1224. doi: 10.1126/science.3939723. [DOI] [PubMed] [Google Scholar]
  19. Tonouchi N., Shoun H., Uozumi T., Beppu T. Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus. Nucleic Acids Res. 1986 Oct 10;14(19):7557–7568. doi: 10.1093/nar/14.19.7557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yamashita T., Tonouchi N., Uozumi T., Beppu T. Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells. Mol Gen Genet. 1987 Dec;210(3):462–467. doi: 10.1007/BF00327198. [DOI] [PubMed] [Google Scholar]
  21. Zsebo K. M., Lu H. S., Fieschko J. C., Goldstein L., Davis J., Duker K., Suggs S. V., Lai P. H., Bitter G. A. Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region. J Biol Chem. 1986 May 5;261(13):5858–5865. [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES