Abstract：
Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.
摘要：
鉴定蛋白质-蛋白质相互作用的方法对于理解控制生物系统的分子机制至关重要。邻近依赖性标记已被证明是揭示活细胞中蛋白质-蛋白质相互作用网络的有价值的方法。来自Aquifexaeolicus（BioID2）的生物素蛋白连接酶的突变形式通过产生与蛋白质相连的生物素来支持该方法。这标记蛋白质用于捕获，提取,和识别。在这一章中,我们提出了一个专门适用于大肠杆菌的BioID2工具包，例如趋化性蛋白CheA。我们已经创建了含有BioID2的质粒作为蛋白质的表达盒(例如，CheA）在N或C末端与BioID2融合，用8×GGS接头优化。我们提供了一种在大肠杆菌细胞中表达和验证CheA-BioID2融合蛋白的方法，通过蛋白质-BioID2融合的相互作用物的体内生物素化，以及生物素化的相互作用蛋白的提取和分析。
