Quantitative analysis of USP activity in vitro.
Deubiquitinating enzymes
Protein purification
Protein–protein interaction
Quantitative enzymatic activity studies
Ubiquitin
Ubiquitin-specific protease
Journal
Methods in enzymology
ISSN: 1557-7988
Titre abrégé: Methods Enzymol
Pays: United States
ID NLM: 0212271
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
10
3
2019
pubmed:
10
3
2019
medline:
12
11
2019
Statut:
ppublish
Résumé
Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.
Identifiants
pubmed: 30850056
pii: S0076-6879(18)30519-6
doi: 10.1016/bs.mie.2018.12.023
pii:
doi:
Substances chimiques
Ubiquitin
0
Ubiquitin-Specific Proteases
EC 3.4.19.12
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
281-319Informations de copyright
© 2019 Elsevier Inc. All rights reserved.