Preparation of extracellular domain 3 of human VEGF receptor-2 and the monitoring of its real-time binding to VEGF by biosensors.
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 vessels, which is involved in tumor angiogenesis, a process that is essential for tumor growth and metastasis. In this study, we describe a method for high yield of recombinant extracellular domain 3 (KDR3) of human VEGFR-2 in an Escherichia coli system with further purification by cation exchange chromatography and immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR3 was performed by sequestering VEGF in HUVEC proliferation assay. The real-time binding of human VEGF to immobilized KDR3 was monitored by a label-free biosensor, Optical waveguide lightmode spectroscopy (OWLS). Under the given experimental conditions, the association rate constant k(a) was 4.2 x 10(3) M(-1) s(-1) and the dissociation rate k(d) was 5.1 x 10(-3) s(-1). The dissociation constant K(D) was then calculated to be 1.2 x 10(-6) M. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptors and the evaluation of developed anti-KDR antibodies. In addition, such a scenario established by the use of OWLS will potentiate the kinetic study of ligand/receptor and antigen/antibody. The receptor discussed here, which block VEGF binding to cell membrane KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.
Zhang
Juan
J
School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China.
Li
Haixin
H
Chen
Wei
W
Cao
Peng
P
Wang
Min
M
D015374:Biosensing Techniques / Q000379:methods*; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013057:Spectrum Analysis; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2