Recombinant human cytomegalovirus protease with a C-terminal (His)(6) extension: Purification, autocatalytic release of the mature enzyme, and biochemical characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-a mino-acid polypeptide with a hexahistidinyl tag at the C terminus was expre ssed in Escherichia coli. The first 262 amino acids of the recombinant prot ein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteol ysis at that site. The hexahistidinyl tag was placed downstream of amino ac id 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at a mino acids 256-257 constitutes a site naturally cleaved by the protease dur ing capsid maturation. The 268-amino-acid polypeptide with the (His), tag w as expressed at high levels in E. coli as inclusion bodies. After solubiliz ation of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage o f the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids includin g the (His), tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C te rminus. Approximately 40 mg of purified CMV PR was obtained per liter of ce ll culture using this strategy. The enzymatic activity of CMV PR purified f rom inclusion bodies and refolded to an active enzyme was similar to the en zymatic activity of CMV PR expressed as a soluble protein in E. coli. In ad dition, the refolded CMV PR could be crystallized for X-ray diffraction. (C ) 1998 Academic Press.