A two-hybrid dual bait system to discriminate specificity of protein interactions

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identi fy novel components of signaling networks based on interactions with define d partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to w hich observed interactions are specific. We adapted the basic two-hybrid st rategy to create a novel dual bait system designed to allow single-step scr eening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, f used to DNA binding domain B (lambda cI), Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systema tically compared LexA- and cI-fused baits and reporters. The LexA and cI ba its reporter systems are well matched for level of bait expression and sens itivity range for interaction detection and allow effective isolation of sp ecifically interacting protein pairs against a nonspecific background. Thes e reagents should prove useful to refine the selectivity of library screens , to reduce the isolation of false positives in such screens, and to perfor m directed analyses of sequence elements governing the interaction of a sin gle protein with multiple partners.