School of Life Sciences, University of Nottingham, Nottingham, UK.
Division of Biosciences, College of Health, Medicine and Life Sciences, Brunel University London, Uxbridge, UK.
Methods Mol Biol. 2024;2828:87-106. doi: 10.1007/978-1-0716-4023-4_9.
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.
方法，确定蛋白质-蛋白质相互作用是必不可少的理解分子机制控制生物系统。接近依赖标签已被证明是一种有价值的方法揭示蛋白质-蛋白质相互作用网络在活细胞中。一种突变形式的生物素蛋白连接酶从水生栖热菌（BioID2）通过产生生物素支撑着这种方法，它附着在进入接近它的蛋白质上。这为捕获、提取和鉴定蛋白质做了标记。在这一章中，我们介绍了一种特别适用于大肠杆菌的 BioID2 工具包，以趋化蛋白 CheA 为例。我们已经创建了含有 BioID2 的质粒，作为表达盒，用于将蛋白质（如 CheA）融合到 BioID2 的 N 或 C 末端，用 8×GGS 接头进行了优化。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-BioID2 融合物对相互作用因子进行体内生物素化，以及提取和分析被生物素化的相互作用蛋白。
