Abstract
The yeast two-hybrid system is currently one of the most standardized protein interaction mapping techniques. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors. The protein of interest (bait) is fused to a DNA-binding domain, and complementary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined resulting in transcription from a reporter gene. Here, we describe protocols to test interactions between two individual proteins and to look for novel interacting partners by screening a single protein or domain against a library of other proteins using a GAL4 based yeast two-hybrid system.
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References
Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246
Legrain P, Wojcik J, Gauthier JM (2001) Protein–protein interaction maps: a lead towards cellular functions. Trends Genet 17:346–352
Koegl M, Uetz P (2007) Improving yeast two-hybrid screening systems. Brief Funct Genomic Proteomic 6:302–312
Guo D, Rajamaki ML, Valkonen J (2008) Protein–protein interactions: the yeast two-hybrid system. Methods Mol Biol 451:421–439
Fashena SJ, Serebriiskii IG, Golemis EA (2000) LexA-based two-hybrid systems. Methods Enzymol 328:14–26
Chien CT et al (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 U S A 88:9578–9582
Bartel PL et al (1993) Using the two-hybrid system to detect protein–protein interactions. In: Hartley DA (ed) Cellular interactions in development: A practical approach. Oxford University Press, Oxford, pp 153–179
Harper J et al (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1cyclin-dependent kinases. Cell 75:805–816
Durfee T et al (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7:555–569
James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436
Guthrie C, Fink GR (eds) (1991) Guide to yeast genetics and molecular biology, vol 169. Academic, San Diego
Ausubel FM et al (eds) (1999) Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology, 4th edn. Wiley, New York
Guarente L (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101:181–191
Acknowledgments
We are grateful to Sharon White for critical reading the manuscript and making many helpful suggestions. This research was supported by a grant from the Spanish Ministerio de Ciencia y Tecnología (AGL2010-22287-C02-02/AGR) and Fondo Europeo de Desarrollo Regional (FEDER).
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Rodríguez-Negrete, E., Bejarano, E.R., Castillo, A.G. (2014). Using the Yeast Two-Hybrid System to Identify Protein–Protein Interactions. In: Jorrin-Novo, J., Komatsu, S., Weckwerth, W., Wienkoop, S. (eds) Plant Proteomics. Methods in Molecular Biology, vol 1072. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-631-3_18
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DOI: https://doi.org/10.1007/978-1-62703-631-3_18
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Publisher Name: Humana Press, Totowa, NJ
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