Abstract
We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a continuous assay for this protease. In order to produce the human cytomegalovirus (HCMV) protease for structural studies and accurate kinetic analysis, mutation of alanine 143 at an internal cleavage site was introduced to prevent auto-proteolysis. 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 vivo protein substrate, assembly protein precursor, was also expressed and purified for activity studies. To develop a continuous protease assay, 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 were designed. Purified HCMV A143Q protease cleaved the recombinant assembly protein precursor with Km and kcat values of 3.0 +/- 1.0 microM and 13.3 +/- 1.6 min-1. The Km for peptide substrates is at least 45-fold higher than for the natural protein substrate, but the kcat values are similar. A sensitive assay was developed using fluorescent peptide substrates, which can detect nM HCMV protease activity.
Highlights
We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a continuous assay for this protease
Proteolytic degradation of the 29-kDa protease into 16- and 13-kDa fragments started to occur as the protease concentration was increased to 2 mg/ml
These degradation fragments were previously seen in metabolically labeled E. coli lysates by Baum et al [15], and they were identified as the products of a cleavage event occurring between Ala-143 and Ala-144 at the I-site
Summary
Cloning and Expression of the HCMV Protease Catalytic Domain— DNA isolated from cultured cells infected with HCMV strain AD169 was used as the template for polymerase chain reaction amplification of the coding sequences for both the protease catalytic domain (amino acids 1–256) and assembly protein precursor. The dialyzed pool was loaded onto a 75-ml Q-Sepharose column (Pharmacia Biotech Inc.), which was equilibrated in DT buffer (1 mM DTT and 20 mM Tris-HCl, pH 8.0), followed by a 180 ml of DT buffer wash and eluted with a 675 ml of linear gradient of 0 –300 mM NaCl in DT buffer. Fractions containing HCMV protease were pooled, dialyzed against DM buffer (1 mM DTT, 20 mM Mes, pH 6.0) and loaded onto a 75-ml S-Sepharose column (Pharmacia) equilibrated with the same buffer. Each 1-ml sample containing 2 nmol of purified A143Q protease in 0.1 M phosphate buffer, pH 7.3, 1 mM EDTA, with or without denaturation
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