Expression and molecular characterization of an enzymatically active recombinant human spumaretrovirus protease.

The human foamy virus (HFV) protease (PR) was cloned into a modified thioredoxin fusion vector that carried a His-tag in the centrally located surface loop of the E. coli trxA protein, bacterially expressed as a soluble fusion protein, and subsequently purified by affinity chromatography. By using HFV Gag protein substrates, the purified recombinant HFV PR was enzymatically active whereas the corresponding active site PR mutant Asp/Ala was inactive. Incubation of synthetic peptides containing residues that flank the putative cleavage site with the recombinant HFV PR and subsequent matrix-assisted laser desorption ionization mass spectrometry of the cleavage products identified the proteolytic processing site of the HFV Gag precursor p74 and revealed that the peptide sequence RAVNTVTQ was cleaved between the Asn and Thr bond.