Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited Km and Kcat, values of 0.3 mM and 0.143 sec(-1) at pH 5.3 and was inhibited by pepstatin A.