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 language | English |
|---|---|
| Pages (from-to) | 349-357 |
| Number of pages | 9 |
| Journal | Protein Journal |
| Volume | 26 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Aug 1 2007 |
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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 journal › Article
}
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.
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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 -