Strategies for assaying deubiquitinating enzymes.

ABSTRACT

A general method for assaying deubiquitinating enzymes (DUBs) has been developed. This new method employs an indirect enzyme assay for determining the activity of DUBs using a linear fusion of polyHis-glutathione-S-transferase-ubiquitin-ecotin (His-GST-Ub-ecotin) as a substrate. Because ecotin, a trypsin inhibitor protein from Escherichia coli, is heat stable, the activity of DUBs can be assayed indirectly by determining the ability of ecotin to inhibit trypsin after incubation of any DUB with His-GST-Ub-ecotin followed by heating at 100 degrees. In the substrate construction, His-GST fusion to Ub was used for facilitation of the substrate purification as well as for assisting the heat precipitation of His-GST-Ub and uncleaved His-GST-Ub-ecotin, as Ub itself is also heat stable. This method can also be used for assaying the proteases that process Ub-like proteins (Ubls) using the substrates, in which Ub is replaced by Ubls.