Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Y. Kouzuma, H. Inanaga, K. Doi-Kawano, N. Yamasaki, Makoto Kimura

Research output: Contribution to journalArticle

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Abstract

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.

Original languageEnglish
Pages (from-to)161-166
Number of pages6
JournalJournal of Biochemistry
Volume128
Issue number2
DOIs
Publication statusPublished - Jan 1 2000

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Cystatins
Cysteine Proteinase Inhibitors
Helianthus
Cloning
Molecular Cloning
Seed
Seeds
Complementary DNA
Papain
Peptide Hydrolases
Proteolysis
Amino Acids
Gene Library
Stoichiometry
Escherichia coli
Amino Acid Sequence
Clone Cells
Processing
Helianthus annuus SMC protein

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds. / Kouzuma, Y.; Inanaga, H.; Doi-Kawano, K.; Yamasaki, N.; Kimura, Makoto.

In: Journal of Biochemistry, Vol. 128, No. 2, 01.01.2000, p. 161-166.

Research output: Contribution to journalArticle

Kouzuma, Y. ; Inanaga, H. ; Doi-Kawano, K. ; Yamasaki, N. ; Kimura, Makoto. / Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds. In: Journal of Biochemistry. 2000 ; Vol. 128, No. 2. pp. 161-166.
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