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 En, Russian]

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

  • Biological Assay*
  • Biotinylation
  • Cell Line, Tumor
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Gene Expression
  • Genetic Vectors
  • Humans
  • Immobilized Proteins / analysis
  • Immobilized Proteins / genetics
  • Immobilized Proteins / metabolism
  • Kinetics
  • Ligands
  • Luminescent Measurements
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sensitivity and Specificity
  • Vascular Endothelial Growth Factor A / analysis*
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism
  • Vascular Endothelial Growth Factor Receptor-1 / analysis*
  • Vascular Endothelial Growth Factor Receptor-1 / genetics
  • Vascular Endothelial Growth Factor Receptor-1 / metabolism

Substances

  • Immobilized Proteins
  • Ligands
  • Recombinant Proteins
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factor Receptor-1