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

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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.

Original languageEnglish
Pages (from-to)911-916
Number of pages6
JournalJournal of Biochemistry
Volume124
Issue number5
DOIs
Publication statusPublished - Jan 1 1998

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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