Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA.
Molecular Biology Institute, University of California, Los Angeles, CA, USA.
Methods Mol Biol. 2020;2071:323-346. doi: 10.1007/978-1-4939-9857-9_18.
BioID is an in vivo biotinylation system developed to examine the proximal and interacting proteins of a bait protein within a subcellular compartment. This approach has been exploited in Toxoplasma for protein-protein interaction studies and proteomic characterizations of intracellular compartments. The BioID method requires constructing a translational fusion between a protein of interest and the promiscuous biotin ligase BirA∗ (a mutant of the E. coli protein BirA) which enables trafficking of the protein to the correct intracellular compartment and association with its partners. Proximity labelling occurs upon addition of biotin to the media and the biotinylated target proteins are then be purified using stringent conditions via streptavidin chromatography. In this chapter, we describe the methodology to fuse BirA∗ (or the newer variant BioID2) to a bait protein using endogenous gene tagging in Toxoplasma and then identify the proximal and interacting proteins using in vivo biotinylation, streptavidin purification and mass spectrometric analysis.
BioID 是一种体内生物素化系统，用于研究细胞内隔室中诱饵蛋白的近端和相互作用蛋白。该方法已在弓形虫中用于蛋白质-蛋白质相互作用研究和细胞内隔室的蛋白质组学特征分析。BioID 方法要求在感兴趣的蛋白质和广谱生物素连接酶 BirA*（大肠杆菌蛋白 BirA 的突变体）之间构建翻译融合，从而使蛋白质能够运送到正确的细胞内隔室并与其伴侣结合。在向培养基中添加生物素后，会发生邻近标记，然后通过链霉亲和素层析在严格条件下使用生物素化的靶蛋白进行纯化。在本章中，我们描述了使用弓形虫中的内源基因标记将 BirA*（或较新的变体 BioID2）融合到诱饵蛋白中的方法，然后使用体内生物素化、链霉亲和素纯化和质谱分析来鉴定近端和相互作用蛋白。
