Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori.
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.
Zhao
Ping
P
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Chongqing, 400716, China.
Xia
Qingyou
Q
Li
Juan
J
Fujii
Hiroshi
H
Banno
Yutaka
Y
Xiang
Zhonghuai
Z
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006458:Hemolymph / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D019476:Insect Proteins; D015842:Serine Proteinase Inhibitors