Screening for Chemical Inhibitors of Heterologous Proteins Expressed in Yeast Using a Simple Growth-Restoration Assay

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 486)

Summary

Overexpression of heterologous proteins in the yeast Saccharomyces cerevisiae often inhibits its growth, while inhibitors of the overexpressed proteins can restore growth. These simple observations form the basis of a technically easy, inexpensive, scalable, and widely applicable assay to identify inhibitors of such proteins. An expression plasmid for the inducible expression of a gene of interest is introduced into a yeast strain rendered more sensitive to chemicals by deletion of efflux pumps. Protein expression is induced, cells are exposed to test chemicals, and growth is measured by A600 reading. The chemicals that relieve growth inhibition are subjected to secondary assays to establish their selectivity toward the protein of interest. This assay has been used successfully to identify inhibitors of proteins of viral, microbial, and mammalian origin.

Key words

Chemical inhibitor Drug screening Growth restoration Saccharomyces cerevisiae 

References

  1. 1.
    Tugendreich, S., Perkins, E., Couto, J., Barthmeier, P., Sun, D., Tulac, S.,(2001) A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays. Genome Res. 11, 1899–1912.PubMedGoogle Scholar
  2. 2.
    Kurtz, S., Luo, G., Hahnenberger, K. M., Brooks, C., Gecha, O., Ingalls, K.,(1995) Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus. Antimicrob. Agents Chemother. 39, 2204–2209.PubMedCrossRefGoogle Scholar
  3. 3.
    Perkins, E., Sun, D., Nguyen, A., Tulac, S., Francesco, M., Tavana, H.,(2001) Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast. Cancer Res. 61, 4175–4183.PubMedGoogle Scholar
  4. 4.
    Vottero, E., Balgi, A., Woods, K., Tugendreich, S., Melese, T., Andersen, R. J.,(2006) Inhibitors of human indoleamine 2,3-dioxygenase identified with a target-based screen in yeast. Biotechnol. J. 1, 282–288.PubMedCrossRefGoogle Scholar
  5. 5.
    Rogers, B., Decottignies, A., Kolaczkowski, M., Carvajal, E., Balzi, E., and Goffeau, A. (2001) The pleiotropic drug ABC transporters from Saccharomyces cerevisiae. J. Mol. Microbiol. Biotechnol. 3, 207–214.PubMedGoogle Scholar
  6. 6.
    Geiser, J. R. (2005) Recombinational cloning vectors for regulated expression in Saccharomyces cerevisiae. BioTechniques 38, 378–389.PubMedCrossRefGoogle Scholar
  7. 7.
    Gietz, D. and Woods, R. (2006) Yeast transformation by the Li/SS carrier DNA/PEG method, in Methods in Molecular Biology: Yeast Protocols (Xiao, W., ed.). Humana Press, Totowa, NJ, pp. 107–120Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of British ColumbiaVancouverCanada

Personalised recommendations