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Protease-Dependent Streptomycin Sensitivity in E. coli—A System for Protease Inhibitor Selection

Bio/Technologyvolume 13pages507510 (1995) | Download Citation



We have developed a bacterial cell system in which the activity of an expressed heterologous protease confers a dominant streptomycin-sensitive (strs) phenotype on the cells. This phenotype owes its high selectivity to the fact that streptomycin (strep) resistance, which is conferred on E. coli by mutants of ribosomal protein S12, is highly recessive to strep sensitivity. Thus, when strep-resistant (strr) strains of E. coli are transformed to co-express the wild-type allele of S12 in addition to the mutant allele, their sensitivity to strep increases by a factor of 100–1000. Similarly, we found that when the same strr strains were transformed to co-express a heterologous protease and an inactive fusion of S12 with a substrate of the protease, the strep sensitivity of the cells increased 100-fold. This effect was strictly dependent on correct cleavage of the S12 precursor, required only modest levels of expression of protease and substrate, and could be competitively inhibited by co-expression of an alternative substrate gene. This system thus appears to be well-suited to the identification of protease inhibitors, either by selection from libraries of endogenously expressed random peptide-encoding genes, or by screening synthetic or natural products libraries. Protease-dependent dominant phenotypes may be more sensitive and appropriate than the more commonly used recessive phenotypes for proteases which are activating enzymes.

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  1. 1

    Birk, Y. 1987. Proteinase Inhibitors, p. 257–305. In: Hydrolytic Enzymes. A. Neuberger and K. Brocklehurst (Eds.). Elsevier, Amsterdam.

  2. 2

    Kräusslich, H.-G., Oroszlan, S. and Wimmer, E. 1989. Viral Proteinases as Targets for Chemotherapy. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  3. 3

    Nakanishi, H., Ramurthy, S., Raktabutr, A., Shen, R. and Kahn, M. 1993. Peptidomimetics of the immunoglobulin supergene family—a review. Gene 137: 51–56.

  4. 4

    Baum, E.Z., Bebernitz, G.A. and Gluzman, Y. 1990. β-galactosidase containing a human immunodeficiency vims protease cleavage site is cleaved and inactivated by human immunodeficiency virus protease. Proc. Natl. Acad. Sci. USA 87: 10023–10027.

  5. 5

    Baum, E.Z., Bebernitz,G.A. and Gluzman, Y. 1990. Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli. Proc. Natl. Acad. Sci. USA 87: 5573–5577.

  6. 6

    Block, T.M. and Grafstrom, R.H. 1990. Novel bacteriological assay for detection of potential antiviral agents. Antimicrob. Agents Chemother. 34: 2337–2341.

  7. 7

    McCall, J.O., Kadam, S. and Katz, L. 1994. A high capacity microbial screen for inhibitors of human rhinovirus protease 3C. Bio/Technology 12: 1012–1016.

  8. 8

    Powers, J.C., Odake, S., Oleksyszyn, J., Hori, H., Ueda, T., Boduszek, B. and Kam, C. 1993. Proteases—structures, mechanism and inhibitors. Agents and Actions. Supplements. 42: 3–18.

  9. 9

    Ozaki, M., Mizushima, S. and Nomura, M. 1969. Identification and functional characterization of the protein controlled by the streptomycin-resistant locus in E. coli. Nature 222: 333–339.

  10. 10

    Lando, D., Cousin, M.A. and Privat de Garilhe, M. 1973. Misreading, a fundamental aspect of the mechanism of action of several aminoglycosides. Biochemistry 12: 4528–4533.

  11. 11

    Dean, D. 1981. A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene 15: 99–102.

  12. 12

    Lisa, V., Boccardo, G., D'Agostino, G., Dellavalle, G. and d'Aquilio, M. 1981. Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathol. 71: 667–672.

  13. 13

    Dougherty, W.G. and Carrington, J.C. 1988. Expression and function of potyviral gene products. Ann. Rev. Phytopathol. 26: 123–143.

  14. 14

    Balint, R., Plooy, I. and Steele, C. 1990. The nucleotide sequence of zucchini yellow mosaic virus. Abstracts, VIII International Congress of Virology, P84-017. Genbank accession no. L31350.

  15. 15

    Post, L.E. and Nomura, M. 1980. DNA sequences from the str operon of Escherichia coli. J. Biol. Chem. 255: 4660–4666.

  16. 16

    Rose, R.E. 1988. The nucleotide sequence of pACYC184. Nucl. Acids Res. 16: 355.

  17. 17

    Weiher, H. and Schaller, H. 1982. Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element. Proc. Natl. Acad. Sci. USA 79: 1408–1412.

  18. 18

    Roberts, B. L., Markland, W., Ley, A.C., Kent, R.B., White, D.W., Guterman, S.K. and Ladner, R.C. 1992. Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc. Natl. Acad. Sci. USA 89: 2429–2433.

  19. 19

    Sambrook, J., Frisch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  20. 20

    Dower, W.J., Miller, J.F. and Ragsdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: 6127–6145.

  21. 21

    Mead, D.A., Szczesna-Skorupa, E. and Kemper, B. 1986. Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Engineering 1: 67–74.

  22. 22

    Gilbert, W., Maizels, N. and Maxam, A. 1974. Sequences of controlling regions of the lactose operon. Cold Spring Harb.Symp. Quant. Biol. 38: 845–855.

  23. 23

    Brosius, J. and Holy, A. 1984. Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc. Natl. Acad. Sci. USA 81: 6929–6933.

  24. 24

    Christie, G.E., Farnham, P.J. and Platt, T. 1981. Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. Proc. Natl. Acad. Sci. USA 78: 4180–4184.

  25. 25

    Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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    • Ingrid Plooy

    Present address: DNA Plant Technology, 6701 San Pablo Avenue, Oakland, CA, 94608


  1. Palo Alto Institute for Molecular Medicine, 2462 Wyandotte Street, Mountain View, CA, 94043

    • Robert F. Balint
    •  & Ingrid Plooy


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Correspondence to Robert F. Balint.

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