EXPRESSION, PURIFICATION, AND IDENTIFICATION OF A NOVEL SELF-CLEAVAGE SITE OF THE NLA C-TERMINAL 27-KDA PROTEASE OF TURNIP MOSAIC POTYVIRUS C5

Abstract

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 XL 1-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-G ln-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 K-m of 1.15 +/- 0.16 mM and a V-max of 0.74 +/- 0.091 mu mol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser(223) and Gly(224) near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp(81) or Cys(151), 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 Ser(223) and Gly(224) t, generate the 25-kDa protein. (C) 1995 Academic Press, Inc.