[Expression and function identification of extracellular domain 3 of human vascular endothelial growth factor receptor KDR].
Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessel. We prepared recombinant extracellular domain 3 (KDR3) of human vascular endothelial growth factor receptor-2 in Escherichia coli and studied its specific binding activity with its ligand. The target DNA was synthesized by overlapping PCR, ligated with expression vector p-ET32a and transformed into E. coli Rosetta (DE3). The soluble fusion protein Trx-KDR3 was expressed in cytoplasm, which was up to 20% of total soluble protein in cytoplasm after having been induced by 1 mmol/L IPTG for 5 h at 30 degrees C. It was characterized to be target protein by Western blotting. The product was purified by CM cation exchange resin and immobilized metal affinity chromatography(IMAC). Its VEGF-binding activity was determined by ELISA assay and its influence on the propagation of HUVEC induced by VEGF. The protein product showed high ligand binding activity in the ELISA and HUVEC propagation study compared to the control. Therefore, ligand binding active, soluble recombinant extracellular domain 3 of VEGFR-2 KDR was successfully expressed and purified in E. coli, which would be applied to anti-angiogenesis anti-tumor therapy and anti-KDR antibody development.
Zhang
Juan
J
Department of Molecular Biology, School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China.
Zhang
Yanli
Y
Li
Haixin
H
Chen
Wei
W
Zhang
Tao
T
Wang
Min
M
D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2