Recombinant Expression And Activity Assay Of Fibrinolytic Enzyme Derived From Urechis Unicinchis

Thrombosis is a serious disease in the field of medicine due to its high disability rate, high fatality rate, and its serious threat to people’s health and life. Currently, the thrombolytic drugs still have some drawbacks, although they have been developed to the third generation, which is mainly dominated by recombinant plasminogen activator, the study of the natural thrombolytic provides more possibilities for the development of novel thrombolytic drug.After years of efforts, a series of serine protease with fibrinolytic activity was extracted form marine invertebrate Urechis uniconctus, and they were named Urechis unicinctus fibrinolytic enzyme I, II, III, IV (UFEI, UFEII, UFEIII, UFEIV). The naturally extracted UFEs possess good anticoagulant activity, good fibrinolytic activity and higher biosafety. However, there are still some problems, including complicated extraction procedure, low yield and high cost. Here, to address these issues, the cloning and over-expression of UFEs from heterogenous expression system were carried out. The study is described as following:1. pINA-1317-ufeII and pINA-1317-ufeⅢ expression plasmid were constructed and transformed into Y. lipolytica Polh. After extensive amout of stain screenings, no high expression stain was obtained. We then constructed expression vector pPICZaA-ufeⅡ and transformed into X-33, and unfortunately no high expression stain was observed after screening. These results indicated that the expressions of UFEII and UFEIII in the yeast expression system were difficult.2. pET32a-ufeⅡ expression system was constructed. After induced by IPTG, inclusion body protein was expressed in the OrigamiB(DE3) high expression stain. After refolding from inclusion body protein, UFEII with fibrinolytic activity was obtained. Owing to the instable of expression of inclusion body protein in pET32a-ufeⅡ expression system, the expression and cloning strategy of toxic protein was optimized (the concentration of IPTG, induction time and the temperature, etc). But the effort was not obvious.3. pMSCG9-ufeⅡ expression system was constructed, and UFEII with fibrinolytic activity was obtained after the separation and purification of the fusion protein.4. pET32a-ufeⅢ expression system was constructed, relatively pure protein UFEIII was obtained after the refolding and purification of the inclusion body protein. Unfortunately the resulting protein was no detectable fibrinolytic activity.5. Peptide mass fingerprinting method was used to identify the purified UFEII, and the result confirmed that the protein with fibrinolytic activity is UFEII.6. Fibrin plate method, fibrinolytic protein degradation experiment and amide degradation assay were carried out to characterize the fibrinolytic activity of UFEII. The result suggests that UFEII can not only degrade fibrin, but also can activate the plasminogen. The degrade sequence to fibrinogen is a chia、βchain、 γchain. Using polypeptide as substrate, the reaction constant of the enzyme kinetics of UFEII is Vmax=3.74μM/min, Km=6.02 mM。Taken together, the cloning and over-expression of the UFEs in different expression systems were conducted. UFEII with fibrinolytic activity was obtained in the pMSCG9-ufeⅡ and pET32a-ufeⅡ expression system. The enzymatic activity and cleavage specificity was determined for the first time. Our study will facilitate the in-depth research of this enzyme.