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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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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DOI: https://doi.org/10.1007/s12033-019-00211-4