Using the Yeast Two-Hybrid System to Identify ProteinProtein Interactions

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.