Use of the Yeast Two-Hybrid System for Identifying the Cascade of Protein Interactions Resulting in Apoptotic Cell Death

Use of the yeast two-hybrid system allows rapid identification of interacting protein or proteins for a specific target protein. The technique is readily applied and allows immediate isolation of a complementary DNA (cDNA) encoding the interacting protein. Two areas of interest are addressed using the yeast two-hybrid system. The first involves protein interactions between known protein interactors and the second involves the identification of novel protein partners for specific target proteins. Novel protein interactions are identified by screening cDNA libraries with a target protein of interest. The target protein is fused in the same reading frame with a DNA binding domain. The DNA binding domain is from GAL4 or LexA. Several advantages of using the yeast two-hybrid system are that protein purification is not required to identify the novel interactor, that antibody production against the unknown protein is not required, and that the cDNA of the novel interactor is isolated at the time of interaction identification. The choice of two-hybrid system vectors may depend on the availability of an appropriate cDNA library or the choice of vector to be used in the generation of a new cDNA library.