Expression, Purification, and Identification of a Novel Self-Cleavage Site of the NIa C-Terminal 27-kDa Protease of Turnip Mosaic Potyvirus C5

DO HYUNG KIM, YU SHIN PARK, SUNG SOO KIM, JISUN LEW, HONG GIL NAM, KWAN YONG CHOI

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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 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.

Original languageEnglish (US)
Article number70024
Pages (from-to)517-525
Number of pages9
JournalVirology
Volume213
Issue number2
DOIs
StatePublished - Jan 1 1995

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Potyvirus
Brassica napus
Peptide Hydrolases
Intranuclear Inclusion Bodies
Nuclear Proteins
Proteins
Polyproteins
Capsid Proteins
Glutathione Transferase
Affinity Chromatography
Point Mutation
Glutathione
Chromatography
Catalytic Domain
Peptides
Genes

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Expression, Purification, and Identification of a Novel Self-Cleavage Site of the NIa C-Terminal 27-kDa Protease of Turnip Mosaic Potyvirus C5. / KIM, DO HYUNG; PARK, YU SHIN; KIM, SUNG SOO; LEW, JISUN; NAM, HONG GIL; CHOI, KWAN YONG.

In: Virology, Vol. 213, No. 2, 70024, 01.01.1995, p. 517-525.

Research output: Contribution to journalArticle

KIM, DO HYUNG ; PARK, YU SHIN ; KIM, SUNG SOO ; LEW, JISUN ; NAM, HONG GIL ; CHOI, KWAN YONG. / Expression, Purification, and Identification of a Novel Self-Cleavage Site of the NIa C-Terminal 27-kDa Protease of Turnip Mosaic Potyvirus C5. In: Virology. 1995 ; Vol. 213, No. 2. pp. 517-525.
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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 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.",
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