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17Engineering Cys2His2 Zinc Finger Domains Usinga Bacterial Cell-Based Two-Hybrid Selection SystemStacey Thibodeau-Beganny and J. Keith JoungSummarySynthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identifiedfrom large randomized libraries using selection methodologies such as phage display. It hasbeen previously demonstrated that a bacterial cell-based two-hybrid system is at least as effectiveas phage display for selecting zinc fingers with desired specificities from these libraries. In thischapter the authors provide updated and detailed protocols for performing zinc finger selectionsusing the bacterial two-hybrid system.Key Words: Artificial transcription factor; bacterial two-hybrid; Cys2His2 zinc finger; zincfinger nuclease; gene therapy; gene targeting; protein engineering; synthetic biology.1. IntroductionArtificial Cys2His2 zinc finger domains (C2H2 ZFs) with engineered DNAbindingspecificities have shown promise for applications in both biologicalresearch and gene therapy (1–9). Selection-based methods for altering the specificitiesof individual C2H2 ZFs typically involve randomization of amino acidresidues in the DNA-recognition helix to generate large libraries followed by useof a selection method to identify variants with desired DNA-binding specificities.Early studies utilized phage display for the selection method (2,10,11) butmore recent studies have demonstrated that a bacterial cell-based two-hybrid(B2H) system works as well as phage display, and might be, in certain cases,more effective (12,13). In addition, the B2H system is somewhat easier to usethan phage display because it directly selects for proteins in an in vivo, cellularcontext whereas phage display requires multiple rounds of in vitro selection.In this chapter, detailed methods are described for using the B2H system toidentify individual C2H2 ZFs with desired DNA-binding specificities fromrandomized libraries more than 10 8 in size. Although, similar protocols haveFrom: Methods in Molecular Biology, vol. 408: Gene Function AnalysisEdited by: M. Ochs © Humana Press Inc., Totowa, NJ317