Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.
We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D005822:Genetic Vectors; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics*; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics*
D004279:DNA, Viral; D004792:Enzyme Precursors; D014764:Viral Proteins; D004247:DNA; D010447:Peptide Hydrolases