Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment
Bull Exp Biol Med. 2012 Apr;152(6):707-11.
doi: 10.1007/s10517-012-1612-0.
[Article in
English,
Russian]
Affiliation
- 1 Department and Division of Medical Nanobiotechnologies, Medico-Biological Faculty, N. I. Pirogov Russian State Medical University, Russia. ahnavleopold@yandex.ru
Abstract
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.
MeSH terms
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Biological Assay*
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Biotinylation
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Cell Line, Tumor
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Cloning, Molecular
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Escherichia coli / genetics
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Gene Expression
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Genetic Vectors
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Humans
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Immobilized Proteins / analysis
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Immobilized Proteins / genetics
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Immobilized Proteins / metabolism
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Kinetics
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Ligands
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Luminescent Measurements
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Protein Binding
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Protein Structure, Tertiary
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Recombinant Proteins / analysis
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sensitivity and Specificity
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Vascular Endothelial Growth Factor A / analysis*
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Vascular Endothelial Growth Factor A / genetics
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Vascular Endothelial Growth Factor A / metabolism
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Vascular Endothelial Growth Factor Receptor-1 / analysis*
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Vascular Endothelial Growth Factor Receptor-1 / genetics
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Vascular Endothelial Growth Factor Receptor-1 / metabolism
Substances
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Immobilized Proteins
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Ligands
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Recombinant Proteins
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VEGFA protein, human
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factor Receptor-1