Willis William L, Foster Abigail, Henry Caitlin, Wu Lai Chu, Jarjour Wael
The Ohio State University Wexner Medical Center, Columbus, OH, USA.
Bio Protoc. 2020 Jun 20;10(12):e3657. doi: 10.21769/BioProtoc.3657.
Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.
转谷氨酰胺酶（TG2）催化谷氨酰基和赖氨酰基残基之间的蛋白质交联。催化活性通过转酰胺基机制发生，导致异肽键的形成。由于TG2介导的转酰胺作用在许多生物学过程中具有重要的机制意义，因此能够快速、有效地鉴定和表征候选底物的检测方法是揭示交联蛋白功能的重要第一步。在此，我们描述了一种优化且灵活的TG2交联反应和底物掺入检测方法。我们之前已将这些技术用于鉴定蛋白质高迁移率族蛋白B1（HMGB1）作为TG2底物。然而，该方法可适用于鉴定任何候选转酰胺底物。
