Application of Archaea in Deubiquitinase-Like Enzyme Discovery and Activity Assay

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.