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

ABSTRACT: 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.

SUBMITTER: Hepowit NL

PROVIDER: S-EPMC9920728 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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

Hepowit Nathaniel L NL Maupin-Furlow Julie A JA

Methods in molecular biology (Clifton, N.J.) 20230101

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 ...[more]

PMID: 36350548

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