Ubiquitin vinyl methyl ester, (HA-tag)

The ubiquitin cascade in its many forms includes a large family of cysteine proteases known as deubiquitinylating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but with little evidence for specific biological activity.

HA-tagged ubiquitin molecules derivatised by chemical ligation at the C-terminus with thiol-reactive functional groups can be used as potent, irreversible inhibitors of DUBs through their covalent modification of the active site and, consequently, can act as specific probes for enzymes with DUB activity. Such probes have been utilized for the identification of DUBs and associated components from cell lysates by proteomic methods, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure.

The nature of the electrophilic group used as the C-terminal derivative, for example vinyl sulfone (-VS), vinyl methylester (-VME), 2-bromoethyl (-Br) or 2-chloroethyl (-Cl), may determine the subset of DUB enzymes detected. The HA peptide sequence (YPYDVPDYA), derived from the influenza hemagglutinin protein, facilitates sensitive identification or purification of HA-Ub-VME modified DUBs through recognition by HA-reactive antibodies in free or agarose-immobilised.