Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

Abstract

The protease (3Cpro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3Cpro has been achieved by fusing the protein to the carboxy-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3Cpro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3Cpro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3Cpro. Sufficient protease 3Cpro has been purified for initial attempts at crystallization. © 1992.