EXPRESSION AND MOLECULAR CHARACTERIZATION OF AN ENZYMATICALLY ACTIVE RECOMBINANT HUMAN SPUMARETROVIRUS PROTEASE

The human foamy virus (HFV) protease (PR) was cloned into a modified t hioredoxin fusion vector that carried a His-tag in the centrally locat ed surface loop of the E. coli trxA protein, bacterially expressed as a soluble fusion protein, and subsequently purified by affinity chroma tography. By using HFV Gag protein substrates, the purified recombinan t HFV PR was enzymatically active whereas the corresponding active sit e PR mutant Asp/Ala was inactive. Incubation of synthetic peptides con taining residues that flank the putative cleavage site with the recomb inant HFV PR and subsequent matrix-assisted laser desorption ionizatio n mass spectrometry of the cleavage products identified the proteolyti c processing site of the HPV Gag precursor p74 and revealed that the p eptide sequence RAVNTVTQ was cleaved between the Asn and Thr bond. (C) 1997 Academic Press.