Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

Keiko Doi-Kawano, Yoshiaki Kouzuma, Nobuyuki Yamasaki, Makoto Kimura

研究成果: ジャーナルへの寄稿記事

14 引用 (Scopus)

抄録

Sunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg30-Leu-Gln-Arg-Thr34, Thr37) in the middle region as compared with other phytocystatins. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg30-Leu-Gln-Arg-Thr34) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile1-Pro2) as well as the N-terminal glycine residues Gly3 and/or Gly4 play an important role in manifesting the inhibitory activity toward papain.

元の言語英語
ページ(範囲)911-916
ページ数6
ジャーナルJournal of Biochemistry
124
発行部数5
DOI
出版物ステータス出版済み - 1 1 1998

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Cysteine Proteinase Inhibitors
Mutagenesis
Helianthus
Cloning
Molecular Cloning
Seed
Seeds
Papain
Complementary DNA
Amino Acids
Cathepsin H
Cystatins
Cathepsin L
Cathepsin B
Column chromatography
Ion Exchange Chromatography
Mutant Proteins
Protein Sorting Signals
Sepharose
Glycine

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

これを引用

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds. / Doi-Kawano, Keiko; Kouzuma, Yoshiaki; Yamasaki, Nobuyuki; Kimura, Makoto.

:: Journal of Biochemistry, 巻 124, 番号 5, 01.01.1998, p. 911-916.

研究成果: ジャーナルへの寄稿記事

Doi-Kawano, Keiko ; Kouzuma, Yoshiaki ; Yamasaki, Nobuyuki ; Kimura, Makoto. / Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds. :: Journal of Biochemistry. 1998 ; 巻 124, 番号 5. pp. 911-916.
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abstract = "Sunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg30-Leu-Gln-Arg-Thr34, Thr37) in the middle region as compared with other phytocystatins. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg30-Leu-Gln-Arg-Thr34) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile1-Pro2) as well as the N-terminal glycine residues Gly3 and/or Gly4 play an important role in manifesting the inhibitory activity toward papain.",
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