SINGLE-CHAIN RECOMBINANT HUMAN CYTOMEGALOVIRUS PROTEASE - ACTIVITY AGAINST ITS NATURAL PROTEIN SUBSTRATE AND FLUOROGENIC PEPTIDE SUBSTRATES

We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a con tinuous assay for this protease. In order to produce the human cytomeg alovirus (HCMV) protease for structural studies and accurate kinetic a nalysis, mutation of alanine 143 at an internal cleavage site was intr oduced to prevent autoproteolysis. The resulting soluble 29-kDa A143Q protease was purified to homogeneity as a stable single-chain protein by hydrophobic interaction and ionic-exchange chromatography. The in v ivo protein substrate, assembly protein precursor, was also expressed and purified for activity studies. To develop a continuous protease as say, fluorescent synthetic peptide substrates similar to the cleavage sequence P5 to P5' of the maturation site containing anthranilic acid and nitrotyrosine as a resonance energy transfer donor-acceptor pair w ere designed. Purified HCMV A143Q protease cleaved the recombinant ass embly protein precursor with K-m and k(cat) values of 3.0 +/- 1.0 mu M and 13.3 +/- 1.6 min(-1). The K-m for peptide substrates is at least 45-fold higher than for the natural protein substrate, but the k(cat) values are similar. A sensitive assay was developed using fluorescent peptide substrates, which can detect nM HCMV protease activity.