Reteplase Fusion Protein: Expression, Puriflcaton And The Effect Of Chaperones On Its Renaturation

Objective To construct the prokaryotic expression system of reteplase fusion proteinand research the effect of chaperones on its renaturation. Methods Inserted thereteplase gene into the prokaryotie expression vector PET32a. Then the recombinantPET-32a-r-PA was transformed into competent cell of E.coli BL21afterPCR identification and EcoR I/BamH I digest identification. Expressed the positiverecombinant clones by the induction of IPTG and then investigated the expressionproductions by SDS-PAGE,Western blot analysis.The target protein inclusion bodiespurified by Ni2+metal-chelate affinity chromatograpy.37℃for engineered bacteriacontaining the recombinant plasmid of chaperones, then expressed by the induction ofIPTG. Researched the effect of chaperones on the renaturation of fusion protein byAdding different chaperones. The final step, measured the activity of fusion protein bythe method of agarose-fibrin plate. Results The result of PCR identification, EcoRI/BamH I digestion and DNA sequencing showed that the recombinant PET-32a-r-PAvector was successfully constructed; The analysis of SDS-PAGE and Western blotindicated that reteplase fusion protein was expressed correctly; The result of addingdifferent chaperones showed that Chaperones DsbA、pKJE7、pTf16had the conspicuouseffect on the renaturation of fusion protein and the rate of renaturation was about20%;The result of fibrinolytic activity detection indicated that the refolded reteplase fusionprotein had fibrinolytic activity and the activity by calculating was4.8x10~5IU/mg.Conclusion Chaperones can promote renaturation of reteplase fusion protein.