Quantitative analysis of USP activity in vitro.

ABSTRACT

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.