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 …