Ouellette Scot P, Karimova Gouzel, Davi Marilyne, Ladant Daniel
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.
Curr Protoc Mol Biol. 2017 Apr 3;118:20.12.1-20.12.24. doi: 10.1002/cpmb.36.
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.
细菌双杂交（BACTH，即“基于百日咳博德特氏菌腺苷酸环化酶的双杂交”）技术是一种在体内分析蛋白质-蛋白质相互作用的简单快速的遗传学方法。在这个系统中，将感兴趣的蛋白质与来自百日咳博德特氏菌腺苷酸环化酶催化结构域的两个互补片段进行基因融合，并在缺乏腺苷酸环化酶的大肠杆菌菌株中共同表达。杂交蛋白的结合恢复了环磷酸腺苷（cAMP）的合成，进而触发分解代谢操纵子（如乳糖操纵子或麦芽糖调节子）的表达。由于BACTH使用cAMP第二信使，嵌合蛋白之间的结合可以在远离转录机制的地方发生。因此，这项技术特别适用于研究涉及可能不溶于细胞质和/或可能仅在膜平面上结合的整合膜蛋白或膜相关蛋白的相互作用。本单元描述了用BACTH遗传系统表征蛋白质-蛋白质相互作用以及寻找已知蛋白质潜在相互作用伙伴的基本程序。© 2017约翰威立父子公司版权所有。
