Yeast two-hybrid system for studying protein-protein interactions--stage 4: Isolation of library plasmid insert and...

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. Stage 4, described here, outlines a series of first-order and subsequent control experiments designed to establish whether an interacting protein is likely to be biologically significant. The number of positive interactions obtained will vary drastically from bait to bait. Subsequent processing methods will depend on the number initially obtained and on the preference of the individual investigator.