VEGFR1 D2 in Drug Discovery: Expression and Molecular Characterization Rossella Di Stasi, 1 Donatella Diana, 1 Domenica Capasso, 2 Rosanna Palumbo, 1 Alessandra Romanelli, 2 Carlo Pedone, 2 Roberto Fattorusso, 1,3 Luca D. D’Andrea 1 1 Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy 2 Dipartimento delle Scienze Biologiche, Universita ` di Napoli ‘‘Federico II,’’ Napoli, Italy 3 Dipartimento di Scienze Ambientali, Seconda Universita ` di Napoli, Caserta, Italy Received 28 December 2009; revised 9 March 2010; accepted 18 March 2010 Published online 4 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21448 This article was originally published online as an accepted preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION V ascular endothelial growth factor (VEGF) is a cyto- kine playing a major role in regulating angiogenesis and vasculogenesis, inducing the development of the vascular system and the differentiation of endothe- lial cells. 1 Moreover, deregulation of VEGF synthesis is related to angiogenesis-based diseases. 2 VEGF binds to two tyrosine kinase receptors, VEGF receptor 1 (VEGFR1/Flt-1) and receptor 2 (VEGFR2/KDR), which are localized on the cell surface of various cell types. 3 The receptors are organized in two intracellular kinase domains, a short transmembrane region and an extracellular portion constituted of seven im- munoglobulin-like domains. The binding of VEGF to the extracellular domain induces receptor dimerization, trans- phosphorylation of the kinase domains, and activation of the intracellular signaling. Deletion studies have shown that the ligand binding function resides within the first three domains of VEGFR1 4 and in domains 2 and 3 of VEGFR2. 5 Deletion of the second extracellular domain of VEGFR1 (VEGFR1 D2 ) completely abolishes the binding to VEGF, whereas VEGFR1 D2 binds VEGF with an affinity that is only 60 times lower than the entire extracellular portion of the receptor. 6 VEGFR1 D2 in Drug Discovery: Expression and Molecular Characterization Correspondence to: Luca D. D’Andrea, Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134 Napoli, Italy; e-mail: ldandrea@unina.it 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. # 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 800– 809, 2010. Keywords: VEGF receptors; angiogenesis; circular dichroism; protein refolding; E. coli; fluorescence spectroscopy Contract grant sponsor: Centro Regionale di Competenza in Diagnostica e Farma- ceutica Molecolari of Regione Campania Contract grant numbers: PRIN2008 N. 200875WHMR; FIRB RBRN07BMCT V V C 2010 Wiley Periodicals, Inc. 800 PeptideScience Volume 94 / Number 6