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 p rotein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an iso lated virus particle and expressed as a fusion protein with glutathione S-t ransferase in Escherichia coli XL1-blue. The 27-kDa protease was purified f rom the fusion protein by glutathione affinity chromatography and Mono S ch romatography. The purified protease exhibited the specific proteolytic acti vity 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 an d between nuclear inclusion protein b and capsid protein, respectively. The K-m and k(cat) values were about 0,2 mM and 0.071 s(-1), respectively, whi ch were approximately five-fold lower than those obtained for the NIa prote ase of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa proteas e is different in the binding affinity as wed as in the catalytic power fro m the TuMN NIa protease, In contrast to the NIa proteases from TuMV and tob acco etch virus, the TVMV NIa protease was not autocatalytically cleaved in to smaller proteins, indicating that the C-terminal truncation is not a com mon phenomenon occurring in all potyviral NIa proteases, These results sugg est that the TVMV NIa protease has a unique biochemical property distinct f rom those of other potyviral proteases.