Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori

Ping Zhao, Qingyou Xia, Juan Li, Hiroshi Fujii, Yutaka Banno, Zhonghuai Xiang

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100°C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor.

Original languageEnglish
Pages (from-to)349-357
Number of pages9
JournalProtein Journal
Volume26
Issue number5
DOIs
Publication statusPublished - Aug 1 2007

Fingerprint

Bombyx
Hemolymph
Cloning
Chymotrypsin
Purification
Amino acids
Organism Cloning
Hydrophobic chromatography
Gels
Molecular weight
Affinity chromatography
Polyacrylates
Electrophoresis
Websites
Silver
Electrophoresis, Gel, Two-Dimensional
Amino Acids
Polyacrylamide Gel Electrophoresis
Molecular Weight
Native Polyacrylamide Gel Electrophoresis

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry

Cite this

Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori. / Zhao, Ping; Xia, Qingyou; Li, Juan; Fujii, Hiroshi; Banno, Yutaka; Xiang, Zhonghuai.

In: Protein Journal, Vol. 26, No. 5, 01.08.2007, p. 349-357.

Research output: Contribution to journalArticle

Zhao, Ping ; Xia, Qingyou ; Li, Juan ; Fujii, Hiroshi ; Banno, Yutaka ; Xiang, Zhonghuai. / Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori. In: Protein Journal. 2007 ; Vol. 26, No. 5. pp. 349-357.
@article{a3dca72f15ef4cf9aeb22a96ce8c6d4b,
title = "Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori",
abstract = "Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100°C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor.",
author = "Ping Zhao and Qingyou Xia and Juan Li and Hiroshi Fujii and Yutaka Banno and Zhonghuai Xiang",
year = "2007",
month = "8",
day = "1",
doi = "10.1007/s10930-007-9077-0",
language = "English",
volume = "26",
pages = "349--357",
journal = "Protein Journal",
issn = "1572-3887",
publisher = "Springer New York",
number = "5",

}

TY - JOUR

T1 - Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori

AU - Zhao, Ping

AU - Xia, Qingyou

AU - Li, Juan

AU - Fujii, Hiroshi

AU - Banno, Yutaka

AU - Xiang, Zhonghuai

PY - 2007/8/1

Y1 - 2007/8/1

N2 - Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100°C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor.

AB - Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100°C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor.

UR - http://www.scopus.com/inward/record.url?scp=34250806530&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34250806530&partnerID=8YFLogxK

U2 - 10.1007/s10930-007-9077-0

DO - 10.1007/s10930-007-9077-0

M3 - Article

VL - 26

SP - 349

EP - 357

JO - Protein Journal

JF - Protein Journal

SN - 1572-3887

IS - 5

ER -