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      Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.

      Amino Acid Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Endopeptidases, Escherichia coli, Gene Expression, Hepatovirus, enzymology, Humans, Kinetics, Molecular Sequence Data, Molecular Weight, Open Reading Frames, Plants, Toxic, Plasmids, Poliovirus, Polymerase Chain Reaction, Potyvirus, Recombinant Proteins, chemistry, isolation & purification, metabolism, Rhinovirus, Tobacco, virology, Viral Proteins

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          The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.

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