Simoens Laure, Jonckheere Veronique, Van Damme Petra
iRIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium.
Methods Mol Biol. 2025;2953:17-39. doi: 10.1007/978-1-0716-4694-6_2.
Since the initial use of BirA* for proximity-dependent biotin identification (BioID), researchers have explored variations of, and alternatives to, this specific biotin ligase enzyme. The application of these biotin ligase modules across diverse model systems has shown advancements in biotinylation efficiency and reduced labeling time. However, these improvements have also introduced challenges, such as increased background labeling. Bacterial BioID, a relatively recent development, is currently limited to studying bait proteins using miniTurbo- and TurboID-based fusions. So far, a comprehensive comparative analysis of the various promiscuous biotin ligases (PBLs) has been reported for only one bacterial species. In this chapter, we present a practical guide for selecting the most effective PBL tailored to the specific requirements of the bait and its corresponding model system for BioID-based interactome mapping in bacteria. Using the Salmonella enterica serovar Typhimurium type III effector protein SopB as a case study, we highlight a pioneering exploration of endogenous effector interactions directly within bacterial cells.
自从最初将BirA*用于邻近依赖性生物素识别（BioID）以来，研究人员一直在探索这种特定生物素连接酶的变体及其替代物。这些生物素连接酶模块在各种模型系统中的应用显示出生物素化效率的提高和标记时间的减少。然而，这些改进也带来了挑战，例如背景标记增加。细菌BioID是一项相对较新的进展，目前仅限于使用基于miniTurbo和TurboID的融合蛋白来研究诱饵蛋白。到目前为止，仅针对一种细菌物种报道了对各种混杂生物素连接酶（PBL）的全面比较分析。在本章中，我们提供了一份实用指南，用于选择最有效的PBL，以满足诱饵及其相应模型系统在基于BioID的细菌相互作用组图谱绘制中的特定要求。以鼠伤寒沙门氏菌III型效应蛋白SopB为例，我们重点介绍了在细菌细胞内直接对内源性效应蛋白相互作用进行的开创性探索。
