Document Detail


Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor.
MedLine Citation:
PMID:  12392947     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Birgit von Tiedemann; Ursula Bilitewski
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Publication Detail:
Type:  Comparative Study; Evaluation Studies; Journal Article; Validation Studies    
Journal Detail:
Title:  Biosensors & bioelectronics     Volume:  17     ISSN:  0956-5663     ISO Abbreviation:  Biosens Bioelectron     Publication Date:  2002 Dec 
Date Detail:
Created Date:  2002-10-23     Completed Date:  2003-06-18     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  9001289     Medline TA:  Biosens Bioelectron     Country:  England    
Other Details:
Languages:  eng     Pagination:  983-91     Citation Subset:  IM    
Affiliation:
Division of Biochemical Engineering, German Research Center for Biotechnology Ltd. (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
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MeSH Terms
Descriptor/Qualifier:
Binding Sites
Biosensing Techniques / instrumentation,  methods*
Cells, Cultured
Endothelial Growth Factors / analysis*,  metabolism
Intercellular Signaling Peptides and Proteins / analysis*,  metabolism
Lymphokines / analysis*,  metabolism
Optics and Photonics / instrumentation
Pichia / classification,  metabolism*
Protein Binding
Recombinant Proteins / analysis,  metabolism
Sensitivity and Specificity
Species Specificity
Surface Plasmon Resonance / methods*
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1 / analysis*,  metabolism
Vascular Endothelial Growth Factors
Chemical
Reg. No./Substance:
0/Endothelial Growth Factors; 0/Intercellular Signaling Peptides and Proteins; 0/Lymphokines; 0/Recombinant Proteins; 0/Vascular Endothelial Growth Factor A; 0/Vascular Endothelial Growth Factors; EC 2.7.10.1/Vascular Endothelial Growth Factor Receptor-1

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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