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Serebriiskii I , Khazak V , Golemis EA
A two-hybrid dual bait system to discriminate specificity of protein interactions
Journal of Biological Chemistry. 1999 Jun 11;274(24) :17080-17087
PMID: ISI:000080780400060   
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Abstract
Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined 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 which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening 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, fused 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 systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baits reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.
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Times Cited: 30 English Article 204TL J BIOL CHEM