Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease

Abstract

ABSTRACT 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 K m and K cat values of 0.3 mM and 0.143 sec −1 at pH 5.3 and was inhibited by pepstatin A.