Gene Expression and Activity Analysis of a Novel Fusion Protein (RGD)3/tTF

A novel fusion protein (RGD)3/tTF was developed for the therapy of selective thrombosis of tumor blood vessels. The fused gene (RGD)3/tTF was reconstructed using polymerase chain reaction, cloned into vector pET22b(+), and expressed in Escherichia coli BL21(DE3). The fusion protein was purified using nickel-affinity chromatography column. The tTF activity of the fusion protein was detected using clotting assay and FX activation assay. The specific binding of the (RGD)3/tTF to αvβ3 was analyzed using indirect enzyme linked immunosorbent assay. The recombinant plasmid pET22b(+)/(RGD)3/tTF was obtained and expressed in E. coli BL21(DE3). The purified fusion protein could induce blood coagulation and activate FX. The ability of the (RGD)3/tTF to specifically bind to αvβ3 was increased by 32%, compared with the RGD/tTF. The novel fusion protein (RGD)3/tTF was successfully expressed in E. coli BL21(DE3). The expressed proteins retained the tTF activity and showed a higher binding to αvβ3 than that of the RGD/tTF.