Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA.
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2023;2591:151-169. doi: 10.1007/978-1-0716-2803-4_10.
Archaea can be used as microbial platforms to discover new types of deubiquitinase-like (DUB-like) enzymes and to produce ubiquitin/ubiquitin-like (Ub/Ubl) protein conjugates as substrates for DUB/DUB-like activity assays. Here we outline how to use archaea to synthesize, purify, and assay the activity of DUB-like enzymes with unusual properties, including catalytic activity in hypersaline conditions, organic solvents, and high temperatures. We also outline the application of archaea in forming Ub/Ubl isopeptide linkages that include the covalent attachments of diverse archaeal and eukaryotic Ub/Ubls to target proteins. Archaea form these Ub/Ubl-linked protein conjugates in vivo, and the resulting products are found to serve as useful DUB substrates for in vitro assays.
古菌可用作微生物平台，用于发现新型去泛素化酶（DUB-like）和生产泛素/泛素样（Ub/Ubl）蛋白缀合物，作为 DUB/DUB-like 活性测定的底物。本文概述了如何利用古菌合成、纯化和测定具有特殊性质的 DUB-like 酶的活性，包括在高盐、有机溶剂和高温条件下的催化活性。我们还概述了古菌在形成 Ub/Ubl 异肽键中的应用，包括各种古菌和真核生物的 Ub/Ubl 与靶蛋白的共价连接。古菌在体内形成这些 Ub/Ubl 连接的蛋白缀合物，所得产物可作为体外测定的有用 DUB 底物。
