Yeast Two-Hybrid System for Studying Protein-Protein Interactions--Stage 3: Screen for Interacting Proteins

Abstract

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. Once the bait strain has been made and characterized (stage 1) and the library strain has been transformed and frozen in aliquots (stage 2), the next step is to mate the two strains. Stage 3 of the method, described here, details the mating and screening of the cDNA library for positive interactors.