title: VEGFR1(D2) in drug discovery: Expression and molecular characterization.
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 (VEGFR1(D2)) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1(D2). 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 VEGFR1(D2) 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.
authors.affiliation: Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Di Stasi
authors.forename: Donatella
authors.initials: D
authors.lastname: Diana
authors.forename: Domenica
authors.initials: D
authors.lastname: Capasso
authors.forename: Rosanna
authors.initials: R
authors.lastname: Palumbo
authors.forename: Alessandra
authors.initials: A
authors.lastname: Romanelli
authors.forename: Carlo
authors.initials: C
authors.lastname: Pedone
authors.forename: Roberto
authors.initials: R
authors.lastname: Fattorusso
authors.forename: Luca D
authors.initials: LD
authors.lastname: D'Andrea
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D055808:Drug Discovery; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2