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16A Bacterial/Yeast Merged Two-Hybrid SystemProtocol for Bacterial Screeningllya G. Serebriiskii, Nadia Milech, and Erica A. GolemisSummaryYeast two-hybrid systems are artificial genetic systems that allow identification and characterizationof protein–protein interactions. One common limit to the use of these techniques iswhen the intrinsic property of “bait” proteins of interest transcriptionally autoactivates reporters,eliminating the basis for interaction detection. To circumvent this problem, autoactivating baitscan be alternatively used in bacteria wherein such activation does not occur. A single-vector systemhas been developed, which can be used either in yeast or in bacteria, streamlining andexpanding capacity for protein–protein interaction screens. A concise proposal is provided for useof this system in bacteria; a companion article, chapter 15, describes use of the system in yeast.Key Words: Protein–protein interaction; transcriptional activation; two-hybrid; yeast; bacteria;library screen.1. IntroductionThe yeast two-hybrid system is a powerful tool for studying protein–proteininteractions. This genetic method, based on the reconstitution of a functionaltranscriptional activator in yeast (1), has now been used extensively both toidentify novel protein–protein interactions and to analyze known interactions.Many extensions to the original two-hybrid system have greatly expanded itsutility, enabling use of a two-hybrid paradigm to selectively study protein interactionswith RNA, DNA, peptides, and small molecules (2). Separately, a bacterialgenetic selection system analogous to the yeast two-hybrid has beendescribed (3–7). This bacteria-based system offers two significant advantagesover its yeast counterpart: (1) it permits the analysis of very large libraries (>10 8in size) and (2) it provides an alternative approach to identify interacting partnersFrom: Methods in Molecular Biology, vol. 408: Gene Function AnalysisEdited by: M. Ochs © Humana Press Inc., Totowa, NJ291