RRC ID 11235
Author Zhao P, Xia Q, Li J, Fujii H, Banno Y, Xiang Z.
Title Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori.
Journal Protein J
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 degrees 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.
Volume 26(5)
Pages 349-57
Published 2007-8-1
DOI 10.1007/s10930-007-9077-0
PMID 17503165
MeSH Animals Base Sequence Bombyx / enzymology Bombyx / genetics* Cloning, Molecular DNA, Complementary Hemolymph / enzymology Hot Temperature Hydrogen-Ion Concentration Insect Proteins / chemistry Insect Proteins / genetics* Insect Proteins / isolation & purification Molecular Sequence Data Protein Structure, Tertiary Sequence Analysis, Protein Sequence Homology, Amino Acid Serine Proteinase Inhibitors / chemistry Serine Proteinase Inhibitors / genetics* Serine Proteinase Inhibitors / isolation & purification
IF 1.317
Times Cited 6
Silkworms b40