Prediction And Verification Of Cocoonase's Catalytic Triad From Antheraea Pernyi

Thrombosis is a leading cause of morbidity and mortality throughout the world. Existing thrombolytic agents are effective at dissolving the thrombi, but at the same time, it will also result in high risk of hemorrhagic complications. Cocoonase is an enzyme which come from tussah pupa's secretion in the period of eclosion. Cocoonae can soft the cocoons to permit exit of the adult moth. We have validated cocoonase can be used for thrombo lysis with low bleeding risk by experiment. This provides a new way for the development of thrombo lysis drug.In order to study the relationship between cocoonase's structure and thrombo lysis, homology modeling was adopted to build cocoonase's 3D structure by using the cocoonase's sequence as template. The substrates pocket and active center of cocooonase was searched by calculation. Molecular docking with substrate BAEE (N alpha Benzoyl-L-arginine baton rouge ester hydrochloride) was also carried out to predict the possible active sites. By homology modeling and molecular docking,44-bit histidine,87-bit aspartic acid,183-bit serine were proved to be the key amino acids of cocoonase. alanine GCG was obtained by site-directed mutagenesis on 44 amino acids CAT password (coding histidine H44),87 amino acids GAT password (D87 coding aspartic acid) and 183 amino acids of GCG password (coding serine S183) using the method of overlap extension PCR. Then the cocoonase gene and mutated cocoonase gene were expressed by eukaryotic expression, recombinant expression vector pFastBac-EGFP-GST-Cocoonase and mutation expression vector pFastBac-EGFP-GST-Cocoonase-(H44A/D87A/S183A) were also constructed. We use Bac to Bac insect baculovirus expression system to express cocoonase and its mutants.The result of SDS-PAGE and Western Blot proved the coconase's gene and mutated genes were expressed successfully. The fusion protein was purified and then activity assay was carried out. In the fibrinogen degradation experiments, the mutaion weaked cocoonase's degradation ability on fibrinogen, all the three mutants lose their degaation ablility on fibrinogen.All the experiment results mentioned above proved bioinformatics methods was efficient on the prediction of cocoonase's key amino acid, this will lay the foundation of further study on thrombolysis mechanisms of cocoonase.