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

Virology. 1995 Jun 20;210(1):194-201. doi: 10.1006/viro.1995.1331.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 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 / enzymology
  • Polymerase Chain Reaction
  • Potyvirus / enzymology*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Rhinovirus / enzymology
  • Tobacco / virology
  • Viral Proteins / chemistry
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism*

Substances

  • Recombinant Proteins
  • Viral Proteins
  • Endopeptidases
  • nuclear inclusion protein a, mosaic viruses