Gene Expression And Optimization Of Fibrinolytic Enzyme In Phasolosma Esculenta

Thrombotic disease is a serious hazard to human health with an increasing influence, It is urgent to develop new effective thrombolysis medicine. The fibrinolytic enzymes are one of the main sources of thrombolytic drugs. There are four generations of thrombolysis drug research and development so far, Each has own advantages and disadvantages. Marine fibrinolytic enzymes, including from animals, plants, microorganisms, are of different fibrinolytic effects.Our lab previously discovered a fibrinolytic activity enzyme from Marine esculenta’s gut(Phascolosoma esculenta) with strong fibrinolytic activity, named PFE-1 and PFE-2 respectively. The aim of this study is to expression these two fibrinolytic enzymes with genetic engineering technologies respectively in order to development a novel fibrinolytic drug.First of all, Prokaryotic expression vector of p ET-22b(+)-PFE–2 was constructed successfully, the recombinant protein was well expression after vector was under lactose induce, however the recombinant protein was not of fibrinolytic activity by enzyme assay;Then the expression vector with histidine label p ET-22b(+)-PFE-2(his) and molecular chaperone expression vector were also constructed. Although expression of soluble recombinant proteins increased significantly but no enzyme activity was detected.Prokaryotic expression vector of p Pic9-PFE-2 and p Pic9-PFE-2(his) constructed respectively and then vectors were shocked into pichia pastoris GS115 competent cells successfully, the two engineering bacterium p Pic9-PFE-2-GS115 and p Pic9-PFE-2(his)-GS115 were selected. Protein expression was induced with methanol. Fiber plate assay showed that appeared to circle dissolved appeared on plate, indicating that the recombinant protein was of well fibrinolytic activity and the eukaryotic expression system was superior to prokaryotic expression system for p ET-22b(+)-PFE-2(his) expression.In summary, the results of this study demonstrated that using eukaryoticexpression system could be used to express recombinant fibrinolytic enzyme with high activity. Thus it is feasible to use the heterogenous expression system to expression PFE-2 in order to widen marine fibrinolytic enzyme resources. Provide an novel approach for the research and development of new thrombolytic drugs.