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Biochemical and Conformational Characterization of Recombinant VEGFR2 Domain 7

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Abstract

Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains. VEGFR2 domain 7 has been shown to play a crucial role in receptor dimerization and regulation, therefore, representing a convenient target for the allosteric modulation of VEGFR2 activity. The ability to prepare a functional VEGFR2D7 domain represents the starting point to the development of novel VEGFR2 binders acting as allosteric inhibitors of receptor activity. Here, we describe a robust and efficient procedure for the preparation in E. coli of the VEGFR2 domain 7. The protein was obtained with a good yield and was properly folded. It was investigated in a biochemical and structural study, providing information on its conformational arrangement and in solution properties.

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Abbreviations

VEGFs:

Vascular endothelial growth factors

VEGFRs:

Vascular endothelial growth factor receptors

RTKs:

Receptor tyrosine kinases

TEMED:

Tetramethylethylenediamine

APS:

Ammonium persulfate

dNTPs:

Deoxynucleotide triphosphates

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

O.D.600 :

Optical density at 600 nm

Ni-NTA resin:

Nickel-charged nitrilotriacetic resin

TEV protease:

Tobacco etch virus protease

EDTA:

Ethylenediaminetetraacetic acid

DTT:

Dithiothreitol

RP-HPLC:

Reversed-phase high-performance liquid chromatography

TFA:

Trifluoroacetic acid

LC–MS:

Liquid chromatography–mass spectrometry

Trp:

Tryptophan

DSS:

4,4-Dimethyl-4-silapentane-1-sulfonic acid

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Acknowledgements

LDR is supported by Fondazione Umberto Veronesi-Post-Doctoral Fellowship 2019. We would like to thank Mr Leopoldo Zona for technical assistance and Dr Luigi Russo for skillful help with HYDROPRO.

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  1. Luca D. D’Andrea

    Present address: Istituto di Biostrutture e Bioimmagini, CNR, Via Nizza 52, 10126, Turin, Italy

Authors and Affiliations

  1. Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy

    Rossella Di Stasi, Donatella Diana, Lucia De Rosa & Luca D. D’Andrea

  2. Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università della Campania “L. Vanvitelli”, Via Vivaldi, 43 - 81100, Caserta, Italy

    Roberto Fattorusso

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  1. Rossella Di Stasi
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Di Stasi, R., Diana, D., De Rosa, L. et al. Biochemical and Conformational Characterization of Recombinant VEGFR2 Domain 7. Mol Biotechnol 61, 860–872 (2019). https://doi.org/10.1007/s12033-019-00211-4

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