Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor.
Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis. It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins. The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family. Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding. The ligand-binding site of the receptor is localized on extracellular Ig-like domains. The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore). Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods. Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6. Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e. sample volume. This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain.
von Tiedemann
Birgit
B
Division of Biochemical Engineering, German Research Center for Biotechnology Ltd. (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Bilitewski
Ursula
U
D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000032:analysis* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis* / Q000378:metabolism; D008222:Lymphokines / Q000032:analysis* / Q000378:metabolism; D055095:Optics and Photonics / Q000295:instrumentation; D010843:Pichia / Q000145:classification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D013045:Species Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000032:analysis* / Q000378:metabolism; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D040281:Vascular Endothelial Growth Factor Receptor-1