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 A 600 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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
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.
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.
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.
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.
Geiser, J. R. (2005) Recombinational cloning vectors for regulated expression in Saccharomyces cerevisiae. BioTechniques 38, 378–389.
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. 107–120
Acknowledgments
The author would like to thank Phil Hieter, Stuart Tugendreich and Teri Melese for their advice. This work was supported by grant 017392 from the National Cancer Institute of Canada, with funds from the Canadian Cancer Society.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Balgi, A.D., Roberge, M. (2009). Screening for Chemical Inhibitors of Heterologous Proteins Expressed in Yeast Using a Simple Growth-Restoration Assay. In: Clemons, P., Tolliday, N., Wagner, B. (eds) Cell-Based Assays for High-Throughput Screening. Methods in Molecular Biology, vol 486. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-545-3_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-545-3_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-544-6
Online ISBN: 978-1-60327-545-3
eBook Packages: Springer Protocols