VEGFR1(D2) in drug discovery: Expression and molecular characterization.
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.
Di Stasi
Rossella
R
Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.
Diana
Donatella
D
Capasso
Domenica
D
Palumbo
Rosanna
R
Romanelli
Alessandra
A
Pedone
Carlo
C
Fattorusso
Roberto
R
D'Andrea
Luca D
LD
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
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