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 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.
Pinko
C
C
Molecular Biology/Biochemistry Group, Agouron Pharmaceuticals, Inc., San Diego, California 92121, USA.
Margosiak
S A
SA
Vanderpool
D
D
Gutowski
J C
JC
Condon
B
B
Kan
C C
CC
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009842:Oligopeptides; D011994:Recombinant Proteins; D010450:Endopeptidases