title: Analysis of Membrane Protein Interactions with a Bacterial Adenylate Cyclase-Based Two-Hybrid (BACTH) Technique.
abstract: The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-based Two-Hybrid") technique is a simple and fast genetic approach to analyze protein-protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic domain of Bordetella pertussis adenylate cyclase and co-expressed in strains of Escherichia coli deficient in adenylate cyclase. Association of the hybrid proteins restores synthesis of cyclic AMP (cAMP), which then triggers the expression of catabolic operons such as the lactose operon or the maltose regulon. As BACTH uses a cAMP second messenger, the association between the chimeric proteins can take place at a distance from the transcription machinery. This technique is therefore particularly appropriate for studying interactions involving integral-membrane or membrane-associated proteins that may not be soluble in the cytoplasm, and/or that may only associate in the plane of the membrane. This unit describes the basic procedures to characterize protein-protein interactions with the BACTH genetic system and to search for potential partners of known proteins. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France.|Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota.
authors.forename: Scot P
authors.initials: SP
authors.lastname: Ouellette
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France.
authors.forename: Gouzel
authors.initials: G
authors.lastname: Karimova
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France.
authors.forename: Marilyne
authors.initials: M
authors.lastname: Davi
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases
keywords: Escherichia coli; cAMP signaling; library screening; membrane protein; protein interaction assay; two-hybrid technique