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15A Bacterial/Yeast Merged Two-Hybrid SystemProtocol for Yeast Screening With Single or Parallel BaitsNadezhda Y. Tikhmyanova, Eugene A. Izumchenko, Ilya G. Serebriiskii,and Erica A. GolemisSummaryThe yeast two-hybrid system is a useful tool for identifying new protein–protein interactions,and for the dissection of previously identified interactions. An important issue in protein-interactionstudies is frequently that of determining whether a protein associates specifically with one proteinor domain of interest, or has a more promiscuous interaction profile. To help address thisissue, the authors have created a new two-hybrid system, which can be used either in bacteria orin yeast to counterscreen against “decoy” baits in parallel with a primary screen, hence improvingthe power and specificity of the method. Protocols of this system for use in yeast are provided;a companion article, Serebriiski et al., describes alternative use of this system in bacteria.Key Words: Decoy bait; false-positive; protein–protein interaction; yeast two hybrid; bacterialtwo hybrid system; proteomics, library screening.1. IntroductionAs it was first introduced in 1989 as a means to study protein–protein interactions(1), the two-hybrid system has evolved into a robust technology. Thenumber of novel interactions detected through use of this system now numberin thousands, based on the work of many individual investigators, and increasingly,the output from high-throughput proteomics projects (e.g., refs. 2–7).These efforts have begun to yield genome-wide protein interaction linkagemaps, and together with other protein interaction detection technologies such astandem affinity purification-mass spectrometry (8,9) are key components supportingthe nascent field of systems biology (e.g., refs. 10 and 11). On a smallerscale, the two-hybrid system is a useful way for an individual scientist to gainsome insight into the function of a poorly understood protein, by identifyingFrom: Methods in Molecular Biology, vol. 408: Gene Function AnalysisEdited by: M. Ochs © Humana Press Inc., Totowa, NJ257
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15A Bacterial/Yeast Merged Two-Hybrid SystemProtocol for Yeast Screening With Single or Parallel BaitsNadezhda Y. Tikhmyanova, Eugene A. Izumchenko, Ilya G. Serebriiskii,and Erica A. GolemisSummaryThe yeast two-hybrid system is a useful tool for identifying new protein–protein interactions,and for the dissection of previously identified interactions. An important issue in protein-interactionstudies is frequently that of determining whether a protein associates specifically with one proteinor domain of interest, or has a more promiscuous interaction profile. To help address thisissue, the authors have created a new two-hybrid system, which can be used either in bacteria orin yeast to counterscreen against “decoy” baits in parallel with a primary screen, hence improvingthe power and specificity of the method. Protocols of this system for use in yeast are provided;a companion article, Serebriiski et al., describes alternative use of this system in bacteria.Key Words: Decoy bait; false-positive; protein–protein interaction; yeast two hybrid; bacterialtwo hybrid system; proteomics, library screening.1. IntroductionAs it was first introduced in 1989 as a means to study protein–protein interactions(1), the two-hybrid system has evolved into a robust technology. Thenumber of novel interactions detected through use of this system now numberin thousands, based on the work of many individual investigators, and increasingly,the output from high-throughput proteomics projects (e.g., refs. 2–7).These efforts have begun to yield genome-wide protein interaction linkagemaps, and together with other protein interaction detection technologies such astandem affinity purification-mass spectrometry (8,9) are key components supportingthe nascent field of systems biology (e.g., refs. 10 and 11). On a smallerscale, the two-hybrid system is a useful way for an individual scientist to gainsome insight into the function of a poorly understood protein, by identifyingFrom: Methods in Molecular Biology, vol. 408: Gene Function AnalysisEdited by: M. Ochs © Humana Press Inc., Totowa, NJ257