Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds

Yoshiaki Kouzuma, Keiko Tsukigata, Hideko Inanaga, Keiko Doi-Kawano, Nobuyuki Yamasaki, Makoto Kimura

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.

Original languageEnglish
Pages (from-to)969-972
Number of pages4
JournalBioscience, Biotechnology and Biochemistry
Volume65
Issue number4
DOIs
Publication statusPublished - Apr 1 2001

Fingerprint

Cystatins
Cysteine Proteinase Inhibitors
Helianthus
Cloning
Molecular Cloning
Seed
Seeds
Complementary DNA
Papain
Amino Acids
Peptides
Escherichia coli
Open Reading Frames
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Rate constants
Proteins
Peptide Hydrolases
Nucleotides

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Cite this

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

In: Bioscience, Biotechnology and Biochemistry, Vol. 65, No. 4, 01.04.2001, p. 969-972.

Research output: Contribution to journalArticle

Kouzuma, Yoshiaki ; Tsukigata, Keiko ; Inanaga, Hideko ; Doi-Kawano, Keiko ; Yamasaki, Nobuyuki ; Kimura, Makoto. / Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds. In: Bioscience, Biotechnology and Biochemistry. 2001 ; Vol. 65, No. 4. pp. 969-972.
@article{7dde2dd370ad45008739a799a283e308,
title = "Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds",
abstract = "Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.",
author = "Yoshiaki Kouzuma and Keiko Tsukigata and Hideko Inanaga and Keiko Doi-Kawano and Nobuyuki Yamasaki and Makoto Kimura",
year = "2001",
month = "4",
day = "1",
doi = "10.1271/bbb.65.969",
language = "English",
volume = "65",
pages = "969--972",
journal = "Bioscience, Biotechnology and Biochemistry",
issn = "0916-8451",
publisher = "Japan Society for Bioscience Biotechnology and Agrochemistry",
number = "4",

}

TY - JOUR

T1 - Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds

AU - Kouzuma, Yoshiaki

AU - Tsukigata, Keiko

AU - Inanaga, Hideko

AU - Doi-Kawano, Keiko

AU - Yamasaki, Nobuyuki

AU - Kimura, Makoto

PY - 2001/4/1

Y1 - 2001/4/1

N2 - Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.

AB - Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.

UR - http://www.scopus.com/inward/record.url?scp=0035316454&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035316454&partnerID=8YFLogxK

U2 - 10.1271/bbb.65.969

DO - 10.1271/bbb.65.969

M3 - Article

C2 - 11388484

AN - SCOPUS:0035316454

VL - 65

SP - 969

EP - 972

JO - Bioscience, Biotechnology and Biochemistry

JF - Bioscience, Biotechnology and Biochemistry

SN - 0916-8451

IS - 4

ER -