Deubiquitinating enzyme purification, assay inhibitors, and characterization.
Despite the identification of numerous deubiquitinating enzymes (DUBs) in recent years, the large majority of this class of enzymes has not been well characterized. This chapter describes biochemical methods that can be used to characterize the function and substrate specificity of DUBs. Methods described will include: fluorescence assay using ubiquitin-amidomethylcoumarin (AMC); a high-performance liquid chromatography assay using ubiquitin ethyl ester or ubiquitin fusion peptides as model substrates to monitor DUB activity; and the purification of a recombinant human DUB, isopeptidase T, in E. coli using low-temperature expression as well as ion-exchange and affinity chromatography.
Russell
Nathaniel S
NS
Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory University School of Medicine, Atlanta, GA, USA.
Wilkinson
Keith D
KD
D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011480:Protease Inhibitors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025801:Ubiquitin / Q000737:chemistry*
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D025801:Ubiquitin; D010450:Endopeptidases; C091939:ubiquitin isopeptidase