Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. Its biological activity is mediated by the binding to the extracellular domain of two tyrosine kinase transmembrane receptors: VEGFR1 and VEGFR2. Deletion studies showed that VEGF binding site resides in the first three domains of VEGFR1 and in domains 2 and 3 of VEGFR2. In particular, the second extracellular domain of VEGFR1 (VEGFR1D2) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1D2. The protein was expressed in E. coli and refolded from inclusion bodies. The recombinant protein assumes the correct fold as assessed by a combination of biochemical and functional assays as well as by NMR characterization. Furthermore, the recombinant VEGFR1D2 was analyzed by circular dichroism and fluorescence spectroscopy. The protein obtained by this procedure is suitable for the structural characterization of the complexes with receptor binders and to be used in interaction/screening studies.

VEGFR1D2 in drug discovery: Expression and molecular characterization

Diana, D;ROMANELLI, ALESSANDRA;
2010

Abstract

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. Its biological activity is mediated by the binding to the extracellular domain of two tyrosine kinase transmembrane receptors: VEGFR1 and VEGFR2. Deletion studies showed that VEGF binding site resides in the first three domains of VEGFR1 and in domains 2 and 3 of VEGFR2. In particular, the second extracellular domain of VEGFR1 (VEGFR1D2) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1D2. The protein was expressed in E. coli and refolded from inclusion bodies. The recombinant protein assumes the correct fold as assessed by a combination of biochemical and functional assays as well as by NMR characterization. Furthermore, the recombinant VEGFR1D2 was analyzed by circular dichroism and fluorescence spectroscopy. The protein obtained by this procedure is suitable for the structural characterization of the complexes with receptor binders and to be used in interaction/screening studies.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/375471
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