Yeast Two-Hybrid Assay for Studying Protein-Protein Interactions

  • Ahmed Osman
Part of the Methods in Molecular Biology™ book series (MIMB, volume 270)


Protein-protein interactions occur in a wide variety of biological processes and essentially control the cell fate from division to death. Today, the identification of proteins that interact with a protein of interest is a focus of intensive research and is an essential element of the rapidly growing field of proteomics. Yeast two-hybrid assays represent a versatile tool to study protein interactions in vivo. GAL4-based assay, for example, uses yeast transcription factor GAL4 for detection of protein interactions by transcriptional activation. Some transcription factors (such as GAL4) possess a characteristic phenomenon that the transactivation function can be restored when the factor’s DNA-binding domain (DBD) and its transcription-activation domain (AD) are brought together by two interacting, heterologous proteins. GAL4-yeast two-hybrid assay uses two expression vectors, one uses GAL4-DBD and the other uses GAL4-AD. DNA sequences encoding the two proteins of interest (or a protein and a complementary DNA library) can be cloned in the GAL4-DBD and GAL4-AD vectors to form the bait and the target of the interaction trap, respectively. A selection of host cells with different reporter genes and different growth selection markers provides a means to detect and confirm protein-protein interactions and highlight the flexibility of these assays to fit different applications. This chapter presents an outline for the GAL4-based yeast two-hybrid system with a detailed description of the vectors, host cells, and methods for detection and verifying protein interactions.

Key Words

ADE2 Auxotrophic markers GAL4 GAL4-AD GAL4-BD HIS3 LacZ LacZ assays LEU2 MEL1 pADH TRP1 yeast plasmid preparation yeast transformation yeast two-hybrid 


  1. 1.
    Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230), 245–246.PubMedCrossRefGoogle Scholar
  2. 2.
    Rine, J. (1991) Gene overexpression in studies of Saccharomyces cervisiae, in Guide to Yeast Genetics and Molecular Biology, Vol. 194 (Guthrie C., ed.), Academic Press, New York, pp. 239–250.CrossRefGoogle Scholar
  3. 3.
    Kingsman, A. J., Clarke, L., Mortimer, R. K., et al. (1979) Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region. Gene 7(2), 141–152.PubMedCrossRefGoogle Scholar
  4. 4.
    Storms, R. K., Holowachuck, E. W., and Friesen, J. D. (1981) Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene. Mol. Cell Biol. 1(9), 836–842.PubMedGoogle Scholar
  5. 5.
    Castanon, M. J., Spevak, W., Adolf, G. R., et al. (1988) Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae. Gene 66(2), 223–234.PubMedCrossRefGoogle Scholar
  6. 6.
    Hashimoto, H., Kikuchi, Y., Nogi, Y., et al. (1983) Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol. Gen. Genet. 191(1), 31–38.PubMedCrossRefGoogle Scholar
  7. 7.
    Flick, J. S. and Johnston, M. (1990) Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. Mol. Cell Biol. 10(9), 4757–4769.PubMedGoogle Scholar
  8. 8.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, Plainview, NY.Google Scholar
  9. 9.
    Franco, G. R., Valadao, A. F., Azevedo, V., et al. (2000) The Schistosoma gene discovery program: state of the art. Int. J. Parasitol. 30(4), 453–463.PubMedCrossRefGoogle Scholar
  10. 10.
    Oliveira, G. and Johnston, D. A. (2001) Mining the schistosome DNA sequence database. Trends Parasitol. 17(10), 501–503.PubMedCrossRefGoogle Scholar
  11. 11.
    Ito, H., Fukuda, Y., Murata, K., et al. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153(1), 163–168.PubMedGoogle Scholar
  12. 12.
    Schiestl, R. H. and Gietz, R. D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16(5–6), 339–346.PubMedCrossRefGoogle Scholar
  13. 13.
    Hill, J., Donald, K. A., Griffiths, D. E., et al. (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 19(20), 5791.PubMedCrossRefGoogle Scholar
  14. 14.
    Breeden, L. and Nasmyth, K. (1985) Regulation of the yeast HO gene. Cold Spring Harb. Symp. Quant. Biol. 50, 643–650.PubMedGoogle Scholar
  15. 15.
    Giacomini, A., Corich, V., Ollero, F. J., et al. (1992) Experimental conditions may affect reproducibility of the beta-galactosidase assay. FEMS Microbiol. Lett. 79(1–3), 87–90.PubMedGoogle Scholar
  16. 16.
    Miller, A. L., Frost, R. G., and O’Brien, J. S. (1977) Purified human liver acid beta-D-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide. Biochem. J. 165(3), 591–594.PubMedGoogle Scholar
  17. 17.
    Hoffman, C. S. and Winston, F. (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57(2–3), 267–272.PubMedCrossRefGoogle Scholar
  18. 18.
    De Sampaio, G., Bourdineaud, J. P., and Lauquin, G. J. (1999) A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting. Mol. Microbiol. 34(2), 247–256.PubMedCrossRefGoogle Scholar
  19. 19.
    Osman, A., Niles, E. G., and LoVerde, P. T. (2001) Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. J. Biol. Chem. 276(13), 10,072–10,082.PubMedCrossRefGoogle Scholar
  20. 20.
    Freebern, W. J., Osman, A., Niles, E. G., et al. (1999) Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression. J. Biol. Chem. 274(8), 4577–4585.PubMedCrossRefGoogle Scholar
  21. 21.
    Li, B. and Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. FASEB J. 7(10), 957–963.PubMedGoogle Scholar
  22. 22.
    Bartel, P., Chien, C. T., Sternglanz, R., et al. (1993) Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14(6), 920–924.PubMedGoogle Scholar
  23. 23.
    James, P., Halladay, J., and Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144(4), 1425–1436.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa,NJ 2004

Authors and Affiliations

  • Ahmed Osman
    • 1
  1. 1.Department of MicrobiologySchool of Medicine and Biomedical Sciences, SUNY at BuffaloBuffalo

Personalised recommendations