Expression, purification, and identification of a novel self-cleavage site of the Nla C-terminal 27-kDa protease of turnip mosaic potyvirus C5.
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 in Escherichia coli XL1-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 an in vitro translation 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 a Km of 1.15 +/- 0.16 mM and a Vmax of 0.74 +/- 0.091 mumol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser223 and Gly224 near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp81 or 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 Ser223 and Gly224 to generate the 25-kDa protein.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017800:Potyvirus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses