Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.
cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.
Department and Division of Medical Nanobiotechnologies, Medico-Biological Faculty, N. I. Pirogov Russian State Medical University, Russia. ahnavleopold@yandex.ru
A V
AV
Leopol'd
V P
VP
Baklaushev
A A
AA
Korchagina
S A
SA
Shein
N F
NF
Grinenko
K A
KA
Pavlov
I A
IA
Ryabukhin
V P
VP
Chekhonin
D001681:Biological Assay; D019921:Biotinylation; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D055767:Immobilized Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008163:Luminescent Measurements; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D055767:Immobilized Proteins; D008024:Ligands; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1