Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds
Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds
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DOI：
10.1093/oxfordjournals.jbchem.a022207
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发表时间：
1998-11-01
影响因子：
2.7
通讯作者：
Kimura, M
中科院分区：
文献类型：
--
作者：
Doi-Kawano, K;Kouzuma, Y;Kimura, M
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 (Arg(30)-Leu-Gln-Arg-Thr(34), Thr(37)) in the middle region as compared with other phytocystatins [Kouzuma ct al, (1996) J. Biochem. 119, 1106-1113], 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 (Arg(30)-Leu-Gln-Arg-Thr(34)) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile(1)-Pro(2)) as well as the N-terminal glycine residues Gly(3) and/or Gly(4) play an important role in manifesting the inhibitory activity toward papain.
