Expression of soluble VEGF receptor 2 and characterization of its binding by surface plasmon resonance.
Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.
Huang
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X
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, 75235, USA. HuangX@mail.mmc.org
Gottstein
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Brekken
R A
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Thorpe
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PE
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008222:Lymphokines / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D017978:Receptors, Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D011975:Receptors, Mitogen / Q000096:biosynthesis* / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008024:Ligands; D008222:Lymphokines; D017978:Receptors, Growth Factor; D011975:Receptors, Mitogen; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor