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. 1997 May;63(5):1808–1813. doi: 10.1128/aem.63.5.1808-1813.1997

Evaluation of a novel subtilisin inhibitor gene and mutant derivatives for the expression and secretion of mouse tumor necrosis factor alpha by Streptomyces lividans.

E Lammertyn 1, L Van Mellaert 1, S Schacht 1, C Dillen 1, E Sablon 1, A Van Broekhoven 1, J Anné 1
PMCID: PMC168474  PMID: 9143114

Abstract

In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.

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Selected References

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  1. Akita M., Sasaki S., Matsuyama S., Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem. 1990 May 15;265(14):8164–8169. [PubMed] [Google Scholar]
  2. Chen M., Nagarajan V. Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis. J Bacteriol. 1994 Sep;176(18):5796–5801. doi: 10.1128/jb.176.18.5796-5801.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Engler-Blum G., Meier M., Frank J., Müller G. A. Reduction of background problems in nonradioactive northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal Biochem. 1993 May 1;210(2):235–244. doi: 10.1006/abio.1993.1189. [DOI] [PubMed] [Google Scholar]
  4. Fass S. H., Engels J. W. Influence of specific signal peptide mutations on the expression and secretion of the alpha-amylase inhibitor tendamistat in Streptomyces lividans. J Biol Chem. 1996 Jun 21;271(25):15244–15252. doi: 10.1074/jbc.271.25.15244. [DOI] [PubMed] [Google Scholar]
  5. Fornwald J. A., Donovan M. J., Gerber R., Keller J., Taylor D. P., Arcuri E. J., Brawner M. E. Soluble forms of the human T cell receptor CD4 are efficiently expressed by Streptomyces lividans. Biotechnology (N Y) 1993 Sep;11(9):1031–1036. doi: 10.1038/nbt0993-1031. [DOI] [PubMed] [Google Scholar]
  6. Gennity J., Goldstein J., Inouye M. Signal peptide mutants of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):233–269. doi: 10.1007/BF00763167. [DOI] [PubMed] [Google Scholar]
  7. Gilbert M., Morosoli R., Shareck F., Kluepfel D. Production and secretion of proteins by streptomycetes. Crit Rev Biotechnol. 1995;15(1):13–39. doi: 10.3109/07388559509150530. [DOI] [PubMed] [Google Scholar]
  8. Hall M. N., Gabay J., Schwartz M. Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli. EMBO J. 1983;2(1):15–19. doi: 10.1002/j.1460-2075.1983.tb01373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heremans H., Van Damme J., Dillen C., Dijkmans R., Billiau A. Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice. J Exp Med. 1990 Jun 1;171(6):1853–1869. doi: 10.1084/jem.171.6.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Inouye S., Soberon X., Franceschini T., Nakamura K., Itakura K., Inouye M. Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3438–3441. doi: 10.1073/pnas.79.11.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Izard J. W., Kendall D. A. Signal peptides: exquisitely designed transport promoters. Mol Microbiol. 1994 Sep;13(5):765–773. doi: 10.1111/j.1365-2958.1994.tb00469.x. [DOI] [PubMed] [Google Scholar]
  12. Mitsui Y., Satow Y., Watanabe Y., Hirono S., Iitaka Y. Crystal structures of Streptomyces subtilisin inhibitor and its complex with subtilisin BPN'. Nature. 1979 Feb 8;277(5696):447–452. doi: 10.1038/277447a0. [DOI] [PubMed] [Google Scholar]
  13. Puziss J. W., Fikes J. D., Bassford P. J., Jr Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol. 1989 May;171(5):2303–2311. doi: 10.1128/jb.171.5.2303-2311.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Puziss J. W., Harvey R. J., Bassford P. J., Jr Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP. J Bacteriol. 1992 Oct;174(20):6488–6497. doi: 10.1128/jb.174.20.6488-6497.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Strickler J. E., Berka T. R., Gorniak J., Fornwald J., Keys R., Rowland J. J., Rosenberg M., Taylor D. P. Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression. J Biol Chem. 1992 Feb 15;267(5):3236–3241. [PubMed] [Google Scholar]
  16. Taguchi S., Maeno M., Momose H. Extracellular production system of heterologous peptide driven by a secretory protease inhibitor of Streptomyces. Appl Microbiol Biotechnol. 1992 Mar;36(6):749–753. doi: 10.1007/BF00172187. [DOI] [PubMed] [Google Scholar]
  17. Taguchi S., Misawa S., Yoshida Y., Momose H. Microbial secretion of biologically active human transforming growth factor alpha fused to the Streptomyces protease inhibitor. Gene. 1995 Jul 4;159(2):239–243. doi: 10.1016/0378-1119(95)00107-h. [DOI] [PubMed] [Google Scholar]
  18. Ueda Y., Tsumoto K., Watanabe K., Kumagai I. Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans. Gene. 1993 Jul 15;129(1):129–134. doi: 10.1016/0378-1119(93)90708-b. [DOI] [PubMed] [Google Scholar]
  19. Van Mellaert L., Dillen C., Proost P., Sablon E., DeLeys R., Van Broekhoven A., Heremans H., Van Damme J., Eyssen H., Anné J. Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans. Gene. 1994 Dec 2;150(1):153–158. doi: 10.1016/0378-1119(94)90876-1. [DOI] [PubMed] [Google Scholar]
  20. Ward J. M., Janssen G. R., Kieser T., Bibb M. J., Buttner M. J., Bibb M. J. Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet. 1986 Jun;203(3):468–478. doi: 10.1007/BF00422072. [DOI] [PubMed] [Google Scholar]

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