The gene encoding the C-terminal protease domain (27 kDa) of the nuclear inclusion protein a of turnip mosaic potyvirus C5 was cloned and expressed as a fusion protein with glutathione S-transferase inEscherichia coliXL1-blue. Two forms of the protease (27 and 25 kDa) were purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography and exhibited the specific proteolytic activity when a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu, or anin vitrotranslation product of the polyprotein containing the cleavage site between the nuclear inclusion protein b and the capsid protein, was used as a substrate. The purified proteases showed aKmof 1.15 ± 0.16 mMand aVmaxof 0.74 ± 0.091 μmol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser223and Gly224near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp81or Cys151, two putative active site residues in the 27-kDa protease, to Asn or Ser, respectively, prevented the generation of the 25-kDa protein and diminished the proteolytic activity of the protease drastically, suggesting that the 27-kDa protease cleaves itself between Ser223and Gly224to generate the 25-kDa protein.
Bibliographical noteFunding Information:
We thank Dr. S. K. Green and Dr. H. G. Park for kindly providing TuMV-C5 strain. We also thank Professor G. An and Professor S. K. Jang for comments on the correction of the manuscript. This work was supported partly by the HAN project from Ministry of Science and Technology, Korea.