Yeast two-hybrid system for studying protein-protein interactions--stage 1: Construction and characterization of a...

An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the yeast two-hybrid system, uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term two-hybrid derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. In stage 1 of the method, detailed in this protocol, characterization of a novel bait is described, with attention to controls that increase the chance of the bait functioning in a two-hybrid screen.