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Molecular Biotechnology

, Volume 61, Issue 11, pp 860–872 | Cite as

Biochemical and Conformational Characterization of Recombinant VEGFR2 Domain 7

  • Rossella Di StasiEmail author
  • Donatella Diana
  • Lucia De Rosa
  • Roberto Fattorusso
  • Luca D. D’AndreaEmail author
Original paper
  • 55 Downloads

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.

Keywords

Angiogenesis VEGF VEGFR Recombinant expression Allosteric binders 

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

Notes

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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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Istituto di Biostrutture e BioimmaginiCNRNaplesItaly
  2. 2.Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e FarmaceuticheUniversità della Campania “L. Vanvitelli”CasertaItaly
  3. 3.Istituto di Biostrutture e BioimmaginiCNRTurinItaly

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