Abstract
The two-hybrid system has gained wide popularity in the last decade because of the relative ease and speed with which novel interacting proteins can be isolated. Because the two-hybrid approach is based on the interaction of hybrid proteins in a living yeast cell, it offers numerous advantages in comparison to traditional biochemical methods, including the avoidance of costly protein purifications or development of antibodies, and, consequently, great reduction in time required to identify novel protein partners. On the other hand, because two-hybrid screens rely on the indirect readout of transcriptional activation of reporter genes, they are intrinsically prone to distinct classes of artifacts related to the biasing of the transcriptional response. While two-hybrid assays have been quite successful at identifying real interactions (one estimate of success rate is ∼70% for usable baits; [1]), the vast majority of library screens also identify at least one protein that is presumed to be a false positive. The first part of this chapter discusses what classes of protein tend to isolate or be isolated as false positives, and the second part presents simple approaches to limit the occurrence of false positive.
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References
Serebriiskii, I. and Golemis, E. A. http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html.>
Mendelsohn, A. R. and Brent, R. (1999) Protein interaction methods—toward an endgame. Science 284, 1948–1950.
Fields, S. and Uetz, P. http://weber.u.washington.edu/~sfields/projects/YPLM?>
Gyuris, J., Golemis, E. A., Chertkov, H., and Brent, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803.
Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205–214.
Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S. (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582.
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H., and Elledge, S. J. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555–569.
El Housni, H., Vandenbroere, I., Perez-Morga, D., Christophe, D., and Pirson, I. (1998) A rare case of false positive in a yeast two-hybrid screening: the selection of rearranged bait constructs that produce a functional gal4 activity. Anal. Biochem. 262, 94–96.
Shaywitz, D. A., Espenshade, P. J., Gimeno, R. E., and Kaiser, C. A. (1997) COPII subunit interactions in the assembly of the vesicle coat. J. Biol. Chem. 272, 25,413–25,416.
Estojak, J., Brent, R., and Golemis, E. A. (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell. Biol. 15, 5820–5829.
Bartel, P., Chien, C.-t., Sternglanz, R., and Fields, S. (1993) Elimination of false positives that arise in using the two-hybrid screen. Biotechniques 14, 920–924.
Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interaction. Nature 340, 245, 246.
Serebriiskii, I., Estojak, J., and Golemis, E. A. (2000) Approaches to detecting false positives in yeast two-hybrid systems. Biotechniques 28, 328–330, 332-336.
Duttweiler, H. M. (1996) A highly sensitive and non-lethal beta-galactosidase plate assay for yeast. TIG 12, 340–341.
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, 1425–1436.
Harper, J. W., Adami, G., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816.
Finley, R. and Brent, R. (1994) Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc. Natl. Acad. Sci. USA 91, 12,980–12,984.
Brent, R. and Finley, R. L. J. (1997) Understanding gene and allele function with two-hybrid methods. Annu. Rev. Genet. 31, 663–704.
Petermann, R., Mossier, B. M., Aryee, D. N., and Kovar, H. (1998) A recombination based method to rapidly assess specificity of two-hybrid clones in yeast. Nucleic Acids Res. 26, 2252, 2253.
Jiang, R. and Carlson, M. (1996) Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10, 3105–3115.
Inouye, C., Dhillon, N., Durfee, T., Zambryski, P. C., and Thorner, J. (1997) Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions. Genetics 147, 479–492.
Xu, C. W., Mendelsohn, A. R., and Brent, R. (1997) Cells that register logical relationships among proteins. Proc. Natl. Acad. Sci. USA 94, 12,473–12,478.
Serebriiskii, I., Khazak, V., and Golemis, E. A. (1999) A two-hybrid dual bait system to discriminate specificity of protein interactions. J. Biol. Chem. 274, 17,080–17,087.
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Serebriiskii, I.G., Golemis, E.A. (2001). Two-Hybrid System and False Positives. In: MacDonald, P.N. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 177. Humana Press. https://doi.org/10.1385/1-59259-210-4:123
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DOI: https://doi.org/10.1385/1-59259-210-4:123
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