Enzyme Activation Mechanism of Cocoonase

Abstract

Cocoonase, a protein produced by the silkworm (Bombyx mori), consists of a pro-peptide and a mature region with three disulfide bonds. Cocoonase is known to specifically digest the sericin protein which is a coating protein of the fibroin of silkworm cocoons. However, the specificity of the protease activity and the mechanism responsible for its activation related to folding have not yet been determined in detail although its primary structure shows a high homology to that of trypsin. Therefore, to further study the activation mechanism and biological activity of cocoonase, we prepared recombinant cocoonase using an E. coli expression system, and examined its folding and enzymatic activity.For this purpose, a recombinant pro-cocoonase (pro-CCN), cocoonase (CCN), and its mutant proteins were expressed using the T7 expression system in E. coli cells. The recombinant proteins were expressed in the forms of inclusion bodies. Therefore, the refolding reaction was carried out in the presence of 2 mM GSH/1 mM GSSG under several different conditions after purification. In general, pro-CCN was treated with trypsin to eliminate the pro-peptide region for its enzyme activation. To avoid contamination by the trypsin activity in the final product, the refolded pro-CCN was further processed to the mature form, CCN, by the autolysis of the pro-peptide region in the absence of trypsin at several temperatures. The enzyme activity of the recombinant CCN derived from pro-CCN was then estimated, either by a casein assay or the hydrolysis of Bz-Arg-OEt as a substrate. In addition, to eliminate the non-specific degradation of CCN during the refolding reaction, a protease-resistant mutant was also prepared and its folding examined.