Prokaryotic Expression And Purification Of Vascular Endothelial Growth Factor C (VEGF-C) Fusion Protein

As a potential specific endothelial cel(lEC)mitogen,Vascular Endothelial Growth Factor(VEGF) promotes the division, proliferation andmigration of Ecs, and also plays an important role in the maturing and remodeling of blood vessels. VEGF-C is a peculiar regulator of vascular and lymphatic endothelial cells.It mainly increases vascular endothelial cells permeation and induces chemotaxis of lymphatic endothelial cells,cellular proliferation and hyperplasia of lymphagion. Many researches have testified that lymphatic metastasis of malignant tumors is closely associated with the expression of VEGF-C. So VEGF-C provided a new path for anti-lymphatic metastasis of malignant tumors. This study was to construct the recombinant expression plasmid pGEX-VEGF-C, and abtain eficient expressed VEGF-C protein in E.Coli. So as to study its biological function, and get depth understanding of the pathogenesis of tumor, and promote anti-cancer drugs market.The main research contents and results are as follows:1 The VEGF-C was synthesized by chemical methods, and constructed it with pGEX-6p-1 expression vector. The pGEX-VEGF-C plasmid was transformed into E.coli BL21 and got efficient screening transformants. shake-flask culture for high-level expression of proteins induced by IPTG. The expressed fusion protein was purified by using Glutamine-Sepharose 4B affinity chromatography and isoelectric focusintg. And determines the protein density with the Lowry method .2 Prokaryotic expression plasmid of recombinant pGEX/VEGF-C was constructed successfully. SDS-PAGE indicated that the E.coli containing the recombinant expression plasmid pGEX/VEGF-C expressed a 61kd fusion protein after induced with IPTG. And after optimizing induction conditions, the expression of fusion protein reached peak level at 16℃after 16 hours induced with 0.4 mM IPTG .3 VEGF-C-GST fusion protein with high purity could be obtained after it was eficiently expressed in E.coli and purified with Glutamine-Sepharose 4B affinity chromatography.And also got a high purity enzyme digestion of VEGF-C protein. The Protein content is 20mg/ml. This will facilitate the crystallization of recombinant, Preparation of monoclonal antibodies, also facilitate our study of the structure and function of VEGF-C .