Context: The kringle 2 plus serine protease domains (K2S) of human tissue plasminogen activator (tPA) is an efficacious thrombolytic drug, which has been used to treat heart attacks and strokes by breaking up the clots that cause them. It has nine disulfide bridges, which are needed for proper folding and be the bottleneck in improving the production in the Escherichia coli system. So far, few reports have described the production of soluble active K2S from E. coli.
Objective: To achieve high-level expression of active K2S in the E. coli system.
Materials and methods: The DNA fragment coding for K2S was fused with the E. coli disulfide isomerase DsbC. The constructed fusion protein was expressed in E. coli, and then purified with the Ni(2+)-chelating affinity chromatography. K2S was released by cleavage with Factor Xa protease, and the thrombolytic activity was determined using the fibrin plate assay.
Results: The fusion protein DsbC-K2S was found in the culture supernatant of recombinant E. coli as a soluble form of ~40%. The result of fibrinolysis fibrin plate assay showed that the purified recombinant K2S exhibited significant fibrinolysis activity in vitro.
Discussion and conclusion: These works provided a novel approach for the production of active K2S in E. coli without the requirements of in vitro refolding process, and might establish a significant foundation for the following production of K2S.