Purification and characterization of chymotrypsin inhibitor CI-3 from hemolymph of silkworm, Bombyx mori

The chymotrypsin inhibitor 3 (CI-3) whose expression is controlled by Ict-E gene on the 22nd linkage group was purified from the larval hemolymph of silkworm Bombyx mori by combination of a series of column chromatography and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The molecular weight of CI-3 was 40 kDa and its isoelectric point was 5.5. The results of analysis on the protein properties revealed that the inhibitory activity of CI-3 against alpha-chymotrypsin was remarkably stable at pH 6.9-10.6, but lost 30% and 70% of the inhibitory activity at pH 5.9 and pH 11.9, respectively. CI-3 was quite stable at the temperature between 0degreesC to 50degreesC and lost inhibitory activity completely at the temperatures higher than 60degreesC. CI-3 showed strong inhibitory activity toward a-chymotrypsin and silkworm digestive juice (DJ) protease.