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 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 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.
Serebriiskii
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I
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Khazak
V
V
Golemis
E A
EA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D008869:Microtubule-Associated Proteins; D008957:Models, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D019204:GTP-Binding Proteins; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins; D018631:ras Proteins; D000099160:LexA Repressor Protein; C016649:phage repressor proteins