Cloning, Expression And Function Of Human Endothelial Growth Factor Receptor Ⅱ Extracelluar Domain 3(KDR3)

Proliferation and metastasis of tumor cells are angiogenesis dependent. Antiangiogenic therapy is a new developing strategy of anti-tumor biotherapy. Vascular endothelial growth factor (VEGF) is the most important regulative factor. The interaction between VEGF and its receptor can stimulate the proliferation of vascular endothelial cells, then induce the proliferation and metastasis of tumor cells.Because human VEGFR â…¡ ( KDR) encoding 1354 amino acids , it is difficult to express or research the function of integral domain of KDR .Therefore , the main binding domain was selected for search . The extracellular region of KDR consists of seven immunoglobulin-like domains,and the experiments have shown that the domain 3 is necessary for high affinity binding to VEGF.Based on the reference and the results of CAD molecular modeling and analysis system, three KDR3 mutants: M1, M2 and M3 were designed aiming at the critical amino acid that played important role in KDR3 binding to VEGF. A cDNA fragment encoding mature KDR3 was amplified by RT-PCR using a template of mRNA extracted from the umbelical venous endothelial cells. Then, the DNA fragments encoding the KDR3 mutants were obtained using overlap extension polymerase chain reaction, and then inserted into the expression vector pET-His respectively. The recombinant expression plasmids pET-KDR3 and pET-KDR3M were then introduced into the competent cells of E.coli BL21, respectively. The recombinant proteins exist as inclusion bodies(IBs). After washing the IBs, the proteins were purified by chromatography on Sephacyl-200. Then renaturation was carried out in the presence of reduced and oxidized glutathione system. Then, the purity of recombinant proteins of KDR3 and KDR3M reached over 90%.