The Recombinant Expression Of Component 7～# Of Earthworm Fibrinolyntic Enzyme And Renaturation Of Inclusion Body Product

Earthworm fibrinolytic enzymes(EFE) is a group of fibrinolytic enzyme derived from earthworm. The enzymes exhibited strong dissolving activity to fibrin and thrombus, meanwhile it can activate the endogenous fibrinolysis system. So EFE is a ideal medicine for treatment of thrombosis. But EFE is a multicomponent enzymes and not only froward in quality but also complicated in pharmacology. So the best medicine is a single component of EFE, which is high effective and low poisonous. But there were many problems in separating single component of EFE from earthworm, such as the difficulty of purifying process, the complicated product technique, the high cost, etc. So our early research had cloned and expressed the gene of component T of EFE in engineering bacteria which has the highest fibrinolytic activity. We hope the product can be developed to a new medicine for treatment of thrombosis. But it is necessary to select the best one from many expression vectors and systems, because it is a puzzle to find which expression system can express a gene of protein best.In this research, the works had been done are as follows:The vector pBV221-EFE constructed in Escherichia coli expressed a non-fusion protein. We optimized the inducing conditions including OD₆₀₀ value and time and increased the product, but which is inclusion body. We attempted to renatured the inclusion body by dilution and fed-batch operation, etc.The gene was also expressed as a fusion protein in E. coli using vector pTYB11-EFE. We optimized the concentration of IPTG and induction temperature to express the gene. The most of the product became soluble from inclusion body by lowering induction temperature. At the same time the gene was cloned into vector pMAL-p2X, which was translated in E. coli TB1 and expressed a fusion protein. We optimized the induction time and achieved high expression. But there was no obvious product secreted in the periplasm extract. Almost all fusion protein is expressed soluble in cytoplasm.On the other hand, the gene of EFE-7^# was expressed secretly in Pichia pastoris using vector pPIC9K. We induced recombinants in different culture mediums and at different temperature, but the quantity of expression was still lower than 2.5mg/L.We did not achieve the primary target, that the recombinant EFE has fibrinolitic activity although sequence analysis showed the ORF is correct. We presume the 11 cysteines make the product not soluble or the product lock the glycan which is need for maintaining conformation and activity.