Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin.
Curr Protoc. 2023 Mar;3(3):e702. doi: 10.1002/cpz1.702.
Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.
蛋白质经常在高级复合物中发挥作用。定义蛋白质-蛋白质相互作用对于全面了解它们的活性和调节至关重要。邻近生物素化已成为一种非常特异的方法，可以在活细胞或生物体中捕获瞬时和稳定的相互作用。邻近生物素化利用与诱饵蛋白融合的杂乱生物素化酶，导致邻近内源性蛋白的生物素化。在非常严格的条件下，生物素化的相互作用体被纯化，并通过质谱鉴定，以获得高可信度的候选结合伴侣列表。AirID 是最近描述的一种专门为邻近标记设计的生物素连接酶。该方案详细介绍了使用 I 型干扰素反应期间形成的蛋白质复合物作为示例的 AirID 邻近生物素化。它涵盖了 AirID 融合蛋白的构建和验证以及生物素化相互作用体的富集和鉴定。我们描述了一种使用分割 AirID 的变体。在这种情况下，AirID 被分割成两个无活性的片段，只有在诱饵蛋白的二聚化时才会恢复连接酶活性。这允许选择性地检测与诱饵的同源或异源二聚体形式相互作用的蛋白质。该方案考虑了设计策略、优化和不同生物素连接酶的特性，以确定每个实验问题的最佳条件。我们还讨论了常见的陷阱以及如何解决它们。这些方法使邻近生物素化成为定义蛋白质相互作用组的有力工具。 © 2023 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：AirID 融合蛋白的构建和功能验证备选方案：分割 AirID 融合蛋白的构建和功能验证支持方案：生物素化蛋白的 Western blot 基本方案 2：生物素化、富集和蛋白质相互作用体的鉴定。
