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J Biol Chem, Vol. 274, Issue 24, 17080-17087, June 11, 1999
From the Division of Basic Science, Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
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 (
A Two-hybrid Dual Bait System to Discriminate Specificity of
Protein Interactions
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 baitr 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.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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