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Efficient Extracellular Expression of a Foreign Protein in Streptomyces Using Secretory Protease Inhibitor (SSI) Gene Fusions

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Abstract

Using the gene for a secreted protease inhibitor, Streptomyces subtilisin inhibitor (SSI), we have established a secretory expression system for the products of foreign genes in Streptomyces. A sex pheromone peptide, cADl, of Enterococcus faecalis was expressed and secreted as a stable complete fusion protein with SSI in amounts comparable to the native SSI in S. lividans 66. Recombinant cADl, isolated from the fusion protein by chemical digestion with BrCN, has the same amino acid composition and sequence, retention time on reverse phase HPLC, molecular mass, and biological activity as authentic cADl. This system should be useful for the efficient expression of other foreign gene products, and as a model for heterologous gene expression in Streptomyces.

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References

  1. Gray, G., Selzer, G., Buell, G., Shaw, P., Escanez, S., Hofer, S., Voegeli, P., and Thompson, C.J. 1984. Synthesis of bovine growth hormone by Streptomyces lividans. Gene 32:21–30.

    Article  CAS  Google Scholar 

  2. Munoz, A., Perez-Aranda, A., and Barbero, J.L. 1985. Cloning and expression of human interleukin 2 in Streptomyces lividans using the Escherichia coli consensus promoter. Biochem. Biophys. Res. Comm. 133:511–519.

    Article  CAS  Google Scholar 

  3. Ali, N.A., and Dale, J.W. 1986. Secretion by Streptomyces lividans of a cloned Gram-negative beta-lactamase. FEMS Microbiol. Lett. 33:277–280.

    Article  CAS  Google Scholar 

  4. Pulido, Diego., Vara, J.A. and Jimenez, A. 1986. Cloning and expression in biologically active form of the gene for human interferon α2 in Streptomyces. Gene 45:167–174.

    Article  CAS  Google Scholar 

  5. Noack, D., Geuther, R., Tonew, M., Breitling, R. and Behnke, D. 1988. Expression and secretion of interferon-α1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene. Gene 68:53–62.

    Article  CAS  Google Scholar 

  6. Obata, S., Taguchi, S., Kumagai, I., and Miura, K. 1989. Molecular cloning and nucleotide sequence determination of the gene encoding Streptomyces Subtilisin Inhibitor (SSI). J. Biochem. 105:367–371.

    Article  CAS  Google Scholar 

  7. Obata, S., Furukubo, S., Kumagai, I., Takahashi, H. and Miura, K. 1989. High-level expression in Streptomyces Iividans 66 of a gene encoding Streptomyces Subtilisin Inhibitor from Streptomyces albognseolus S-3253. J. Biochem. 105:372–376.

    Article  CAS  Google Scholar 

  8. Mori, M., Sakagami, Y., Narita, M., Isogai, A., Fujino, M., Kitada, C., Craig, R.A., Clewell, D.B., and Suzuki, A. 1985. Isolation and structure of the bacterial sex pheromone, cAD1, that induces plasmid transfer in Streptococcus faecalis. FEBS Lett. 178:97–100.

    Article  Google Scholar 

  9. 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 

  10. Ouchterlony, O. 1967. Handbook of Experimental Immunology, Weir, D. M. (Ed.). Blackwell Scientific, Oxford, Edinburgh, p. 676.

    Google Scholar 

  11. Murao, S., Sato, S., and Muto, N. 1972. Isolation of alkaline protease inhibitor from Streptomyces albogiseolus S-3253. Agr. Biol. Chem. 36:1737–1744.

    Article  CAS  Google Scholar 

  12. Hiromi, K., Akasaka, K., Mitsui, Y., Tonomura, B. and Murao, S. ed. 1985. Protein Protease Inhibitor-The case of Streptomyces Subtilisin (SSI) Inhibitor Chapters 7, 9 (SSI). Elsevier Science Publishers B. V., Amsterdam.

    Google Scholar 

  13. Crestfield, A.M., Moore, S. and Stein, W.H. 1963. The preparation and enzymatic hydolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622–627.

    CAS  PubMed  Google Scholar 

  14. Gross, E., and Witkop, B. 1961. Selective cleavage of the methionyl peptide bonds in ribonuclease with cyanogen bromide. J. Amer. Chem. Soc 83:1510–1511.

    Article  CAS  Google Scholar 

  15. Dunny, G.M., Craig, R.L., Carron, R.B., and Clewell, D.B. 1979. Plasmid transfer in Streptococcus faecalis: Production of multiple sex pheromone by recipients. Plasmid 2:454–465.

    Article  CAS  Google Scholar 

  16. Nagai, K., Thogerson, H.C. 1984. Generation of β-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309:810–812.

    Article  CAS  Google Scholar 

  17. Hattori, M., and Sakaki, Y. 1986. Dideoxy sequencing using denatured plasmid templates. Anal. Biochem. 152:232–238.

    Article  CAS  Google Scholar 

  18. Morinaga, Y., Franceschini, T., Inouye, S., Inouye, M. 1984. Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA. BioTechnology 2:636–639.

    CAS  Google Scholar 

  19. Hunter, I.S. 1985. DNA Cloning: A Practical Approach, Vol. 2. Glover, D. M. (Ed.) IRL Press.

    Google Scholar 

  20. Bensadoun, A. and Weinstein, D. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70:241–250.

    Article  CAS  Google Scholar 

  21. L'Italien, J.J. and Strickler, J.E. 1982. Application of high-performance liquid chromatographic peptide purification to protein microsequencing by solid-phase Edman degradation. Anal. Biochemistry 127:198–212.

    Article  CAS  Google Scholar 

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Author notes

  1. Seiichi Taguchi

    Present address: Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba, 278, Japan

  2. Kin-ichiro Miura: Corresponding author.

Authors and Affiliations

  1. Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Tokyo, 113, Japan

    Izumi Kumagai & Kin-ichiro Miura

  2. Department of Agricultural Chemistry, The University of Tokyo, 1-1-1 Yayoi, Tokyo, 113, Japan

    Jiro Nakayama & Akinori Suzuki

Authors
  1. Seiichi Taguchi
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  2. Izumi Kumagai
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  3. Jiro Nakayama
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  4. Akinori Suzuki
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  5. Kin-ichiro Miura
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Taguchi, S., Kumagai, I., Nakayama, J. et al. Efficient Extracellular Expression of a Foreign Protein in Streptomyces Using Secretory Protease Inhibitor (SSI) Gene Fusions. Nat Biotechnol 7, 1063–1066 (1989). https://doi.org/10.1038/nbt1089-1063

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  • DOI: https://doi.org/10.1038/nbt1089-1063

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