Molecular cloning, expression, and purification of nuclear inclusion A protease from tobacco vein mottling virus.
The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.
Hwang
D C
DC
Division of Molecular Life Sciences and Center for Biofunctional Molecules, Pohang University of Science and Technology, Korea.
Kim
D H
DH
Kang
B H
BH
Song
B D
BD
Choi
K Y
KY
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005450:Fluorescamine / Q000737:chemistry; D017930:Genes, Reporter; D007181:Inclusion Bodies, Viral / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D019176:Potyviridae / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005450:Fluorescamine; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses