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Title
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Expression and characterization of recombinant hepatitis A virus 3C proteinase.
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Authors
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B.A.Malcolm,
S.M.Chin,
D.A.Jewell,
J.R.Stratton-Thomas,
K.B.Thudium,
R.Ralston,
S.Rosenberg.
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Ref.
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Biochemistry, 1992,
31,
3358-3363.
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PubMed id
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Abstract
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The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy
expression vector in Escherichia coli under control of the tac promoter. The
resulting plasmid construction produced 3C proteinase as a soluble and active
enzyme constituting approximately 10% of total cellular proteins. The enzyme was
purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC
reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was
developed, and synthetic peptides derived from the predicted cleavage sites of
the HAV polyprotein were tested for proteolysis of the enzyme. The peptide
representing the 2B/2C cleavage site was cleaved most efficiently with a Km and
kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed
mutagenesis was then used to identify the cysteine at position 172 as the active
site nucleophile. Finally, the purified enzyme showed the expected
endoproteinase activity on the P1 precursor protein generated by in vitro
transcription/translation.
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