Using the Yeast Two-Hybrid System to Identify Interacting Proteins
The yeast two-hybrid system is a powerful technique for studying protein-protein interactions. Two proteins are separately fused to the independent DNA-binding and transcriptional activation domains of the Gal4p transcription factor. If the proteins interact, they reconstitute a functional Gal4p that activates expression of reporter gene(s). In this way, two individual proteins may be tested for their ability to interact, and a transcriptional readout can be measured to detect this interaction. Furthermore, novel interacting partners can be found by screening a single protein or domain against a library of other proteins using this system. It is this latter feature—the ability to search for interacting proteins without any prior knowledge of the identity of such proteins—that is the most powerful application of the two-hybrid technique.
Key WordsTwo-hybrid methodology protocol library screening
- 12.Pierce Chemical Co., P.O. Box 117, Rockford IL 61105. http://www.piercenet.com.
- 14.BD Biosciences Clontech, 1020 E Meadow Circle, Palo Alto, CA 94303. MATCHMAKER Library User Manual (PT1020-1) version PR2562. http:// www.clontech.com.
- 15.Zhu, L., Gunn, D., and Kuchibhatla, S. (1997) The Yeast Two-Hybrid System (Bartel, P. L. and Fields, S., eds.) Oxford University Press, New York, NY, pp. 73–96.Google Scholar
- 16.Bai, C. and Elledge, S.J. in ref. 15, pp. 11–28.Google Scholar
- 17.Sambrook, J. and Russell, D.W. (2001) Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar