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.
Parks
T D
TD
Department of Microbiology, Oregon State University, Corvallis 97331-3804, USA.
Howard
E D
ED
Wolpert
T J
TJ
Arp
D J
DJ
Dougherty
W G
WG
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004926:Escherichia coli; D015870:Gene Expression; D006507:Hepatovirus / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010947:Plants, Toxic; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology; D016133:Polymerase Chain Reaction; D017800:Potyvirus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses