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

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.