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


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.


Angiogenesis VEGF VEGFR Recombinant expression Allosteric binders 



Vascular endothelial growth factors


Vascular endothelial growth factor receptors


Receptor tyrosine kinases




Ammonium persulfate


Deoxynucleotide triphosphates


Isopropyl β-d-1-thiogalactopyranoside


Optical density at 600 nm

Ni-NTA resin

Nickel-charged nitrilotriacetic resin

TEV protease

Tobacco etch virus protease


Ethylenediaminetetraacetic acid




Reversed-phase high-performance liquid chromatography


Trifluoroacetic acid


Liquid chromatography–mass spectrometry




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



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.


  1. 1.
    Patel-Hett, S., & D’Amore, P. A. (2011). Signal transduction in vasculogenesis and developmental angiogenesis. The International Journal of Developmental Biology, 55, 353–363.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ribatti, D. (2008). Judah Folkman, a pioneer in the study of angiogenesis. Angiogenesis, 11, 3–10.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Carmeliet, P. (2003). Angiogenesis in health and disease. Nature Medicine, 9, 653–660.CrossRefPubMedGoogle Scholar
  4. 4.
    Shibuya, M. (2014). VEGF-VEGFR signals in health and disease. Biomolecules & Therapeutics, 22, 1–9.CrossRefGoogle Scholar
  5. 5.
    Olsson, A. K., Dimberg, A., Kreuger, J., & Claesson-Welsh, L. (2006). VEGF receptor signalling-in control of vascular function. Nature Reviews Molecular Cell Biology, 7, 359–371.CrossRefPubMedGoogle Scholar
  6. 6.
    Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A., Abramsson, A., et al. (2003). VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. The Journal of Cell Biology, 161, 1163–1177.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Takahashi, H., & Shibuya, M. (2005). The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clinical Science, 109, 227–241.CrossRefPubMedGoogle Scholar
  8. 8.
    Lania, G., Ferrentino, R., & Baldini, A. (2015). TBX1 represses Vegfr2 gene expression and enhances the cardiac fate of VEGFR2+ cells. PLoS ONE, 10, e0138525.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Smith, N. R., Baker, D., James, N. H., Ratcliffe, K., Jenkins, M., Ashton, S. E., et al. (2010). Vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3 are localized primarily to the vasculature in human primary solid cancers. Clinical Cancer Research : An Official Journal of the American Association for Cancer Research, 16, 3548–3561.CrossRefGoogle Scholar
  10. 10.
    Yamagishi, N., Teshima-Kondo, S., Masuda, K., Nishida, K., Kuwano, Y., Dang, D. T., et al. (2013). Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells. BMC Cancer, 13, 229.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chatterjee, S., Heukamp, L. C., Siobal, M., Schottle, J., Wieczorek, C., Peifer, M., et al. (2013). Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. The Journal of Clinical Investigation, 123, 1732–1740.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ruch, C., Skiniotis, G., Steinmetz, M. O., Walz, T., & Ballmer-Hofer, K. (2007). Structure of a VEGF-VEGF receptor complex determined by electron microscopy. Nature Structural & Molecular Biology, 14, 249–250.CrossRefGoogle Scholar
  13. 13.
    Kisko, K., Brozzo, M. S., Missimer, J., Schleier, T., Menzel, A., Leppanen, V. M., et al. (2011). Structural analysis of vascular endothelial growth factor receptor-2/ligand complexes by small-angle X-ray solution scattering. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25, 2980–2986.CrossRefGoogle Scholar
  14. 14.
    Shinkai, A., Ito, M., Anazawa, H., Yamaguchi, S., Shitara, K., & Shibuya, M. (1998). Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. The Journal of Biological Chemistry, 273, 31283–31288.CrossRefPubMedGoogle Scholar
  15. 15.
    Dosch, D. D., & Ballmer-Hofer, K. (2010). Transmembrane domain-mediated orientation of receptor monomers in active VEGFR-2 dimers. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 24, 32–38.CrossRefGoogle Scholar
  16. 16.
    Brozzo, M. S., Bjelic, S., Kisko, K., Schleier, T., Leppanen, V. M., Alitalo, K., et al. (2012). Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization. Blood, 119, 1781–1788.CrossRefPubMedGoogle Scholar
  17. 17.
    Hyde, C. A., Giese, A., Stuttfeld, E., Abram, Saliba J., Villemagne, D., Schleier, T., et al. (2012). Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites. Molecular and Cellular Biology, 32, 3802–3813.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yang, Y., Xie, P., Opatowsky, Y., & Schlessinger, J. (2010). Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling. Proceedings of the National academy of Sciences of the United States of America, 107, 1906–1911.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sarabipour, S., Ballmer-Hofer, K., & Hristova, K. (2016). VEGFR-2 conformational switch in response to ligand binding. eLife, 5, e13876.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Thieltges, K. M., Avramovic, D., Piscitelli, C. L., Markovic-Mueller, S., Binz, H. K., & Ballmer-Hofer, K. (2018). Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling. Angiogenesis, 21, 533–543.CrossRefPubMedGoogle Scholar
  21. 21.
    Ellis L. M. (2005). Bevacizumab. Nature Reviews Drug Discovery, Suppl S8–9.CrossRefGoogle Scholar
  22. 22.
    Krupitskaya, Y., & Wakelee, H. A. (2009). Ramucirumab a fully human mAb to the transmembrane signaling tyrosine kinase VEGFR-2 for the potential treatment of cancer. Current Opinion in Investigational Drugs, 10, 597–605.PubMedGoogle Scholar
  23. 23.
    D’Andrea, L. D., Del Gatto, A., De Rosa, L., Romanelli, A., & Pedone, C. (2009). Peptides targeting angiogenesis related growth factor receptors. Current Pharmaceutical Design, 15, 2414–2429.CrossRefPubMedGoogle Scholar
  24. 24.
    Feng, S., Zou, L., Ni, Q., Zhang, X., Li, Q., Zheng, L., et al. (2014). Modulation, bioinformatic screening, and assessment of small molecular peptides targeting the vascular endothelial growth factor receptor. Cell Biochemistry and Biophysics, 70, 1913–1921.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Di Stasi, R., De Rosa, L., Romanelli, A., & D’Andrea, L. D. (2016). Peptides interacting with growth factor receptors regulating angiogenesis. Frontiers in Medicinal Chemistry, 9, 103–160.CrossRefGoogle Scholar
  26. 26.
    De Rosa, L., Di Stasi, R., & D’Andrea, L. D. (2018). Pro-angiogenic peptides in biomedicine. Archives of Biochemistry and Biophysics, 660, 72–86.CrossRefPubMedGoogle Scholar
  27. 27.
    D’Andrea, L. D., Romanelli, A., Di Stasi, R., & Pedone, C. (2010). Bioinorganic aspects of angiogenesis. Dalton Transactions, 39, 7625–7636.CrossRefPubMedGoogle Scholar
  28. 28.
    D’Andrea, L. D., De Rosa, L., Vigliotti, C., & Cataldi, M. (2017). VEGF mimic peptides: Potential applications in central nervous system therapeutics. New Horizons in Translational Medicine, 3, 233–251.Google Scholar
  29. 29.
    Mendel, D. B., Laird, A. D., Xin, X., Louie, S. G., Christensen, J. G., Li, G., et al. (2003). In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: Determination of a pharmacokinetic/pharmacodynamic relationship. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 9, 327–337.Google Scholar
  30. 30.
    Kendrew, J., Eberlein, C., Hedberg, B., McDaid, K., Smith, N. R., Weir, H. M., et al. (2011). An antibody targeted to VEGFR-2 Ig domains 4–7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding. Molecular Cancer Therapeutics, 10, 770–783.CrossRefPubMedGoogle Scholar
  31. 31.
    Di Stasi, R., De Rosa, L., Diana, D., Fattorusso, R., & D’Andrea, L. D. (2019). Human recombinant VEGFR2D4 biochemical characterization to investigate novel anti-VEGFR2D4 antibodies for allosteric targeting of VEGFR2. Molecular Biotechnology, 61, 513–520.CrossRefPubMedGoogle Scholar
  32. 32.
    Keunen, O., Johansson, M., Oudin, A., Sanzey, M., Rahim, S. A., Fack, F., et al. (2011). Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proceedings of the National academy of Sciences of the United States of America, 108, 3749–3754.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Chung, A. S., Kowanetz, M., Wu, X., Zhuang, G., Ngu, H., Finkle, D., et al. (2012). Differential drug class-specific metastatic effects following treatment with a panel of angiogenesis inhibitors. The Journal of Pathology, 227, 404–416.CrossRefPubMedGoogle Scholar
  34. 34.
    Di Stasi, R., Diana, D., Capasso, D., Palumbo, R., Romanelli, A., Pedone, C., et al. (2010). VEGFR1(D2) in drug discovery: Expression and molecular characterization. Biopolymers, 94, 800–809.CrossRefPubMedGoogle Scholar
  35. 35.
    Provencher, S. W., & Glockner, J. (1981). Estimation of globular protein secondary structure from circular dichroism. Biochemistry, 20, 33–37.CrossRefPubMedGoogle Scholar
  36. 36.
    Manavalan, P., & Johnson, W. C., Jr. (1987). Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Analytical Biochemistry, 167, 76–85.CrossRefPubMedGoogle Scholar
  37. 37.
    Sreerama, N., & Woody, R. W. (2000). Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Analytical Biochemistry, 287, 252–260.CrossRefPubMedGoogle Scholar
  38. 38.
    Hwang, T. L., & Shaka, A. J. (1995). Water suppression that works. Excitation sculpting using arbitrary wave-forms and pulsed-field gradients. Journal of Magnetic Resonance Series A, 112, 275–279.CrossRefGoogle Scholar
  39. 39.
    Dalvit, C. (1998). Efficient multiple-solvent suppression for the study of the interactions of organic solvents with biomolecules. Journal of Biomolecular NMR, 11, 437–444.CrossRefGoogle Scholar
  40. 40.
    Goddard, T. D., & Kneller, D. G. (2004). SPARKY 3. California: University of San Francisco.Google Scholar
  41. 41.
    Keller, R. L. J. (2004). The computer aided resonance assignement tutorial. Newport Beach: CANTINA.Google Scholar
  42. 42.
    Morris, K. F., & Jr, Johnson C. S. (1992). Diffusion-ordered two-dimensional nuclear magnetic resonance spectroscopy. Journal of the American Chemical Society, 114, 3139–3141.CrossRefGoogle Scholar
  43. 43.
    Price, W. S., Nara, M., & Arata, Y. (1997). A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin. Biophysical Chemistry, 65, 179–187.CrossRefPubMedGoogle Scholar
  44. 44.
    Garcia de la Torre, J., Huertas, M. L., & Carrasco, B. (2000). HYDRONMR: Prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations. Journal of Magnetic Resonance, 147, 138–146.CrossRefPubMedGoogle Scholar
  45. 45.
    Chemes, L. B., Alonso, L. G., Noval, M. G., & de Prat-Gay, G. (2012). Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains. Methods in Molecular Biology, 895, 387–404.CrossRefPubMedGoogle Scholar
  46. 46.
    Greenfield, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols, 1, 2876–2890.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Chen, Y., & Barkley, M. D. (1998). Toward understanding tryptophan fluorescence in proteins. Biochemistry, 37, 9976–9982.CrossRefPubMedGoogle Scholar
  48. 48.
    Yang, Y., Zhang, Y., Cao, Z., Ji, H., Yang, X., Iwamoto, H., et al. (2013). Anti-VEGF- and anti-VEGF receptor-induced vascular alteration in mouse healthy tissues. Proceedings of the National academy of Sciences of the United States of America, 110, 12018–12023.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Gautier, B., Goncalves, V., Diana, D., Di Stasi, R., Teillet, F., Lenoir, C., et al. (2010). Biochemical and structural analysis of the binding determinants of a vascular endothelial growth factor receptor peptidic antagonist. Journal of Medicinal Chemistry, 53, 4428–4440.CrossRefPubMedGoogle Scholar
  50. 50.
    Basile, A., Del Gatto, A., Diana, D., Di Stasi, R., Falco, A., Festa, M., et al. (2011). Characterization of a designed vascular endothelial growth factor receptor antagonist helical peptide with antiangiogenic activity in vivo. Journal of Medicinal Chemistry, 54, 1391–1400.CrossRefPubMedGoogle Scholar
  51. 51.
    De Rosa, L., Diana, D., Basile, A., Russomanno, A., Isernia, C., Turco, M. C., et al. (2014). Design, structural and biological characterization of a VEGF inhibitor beta-hairpin-constrained peptide. European Journal of Medicinal Chemistry, 73, 210–216.CrossRefPubMedGoogle Scholar
  52. 52.
    Wang, L., Coric, P., Broussy, S., Di Stasi, R., Zhou, L., D’Andrea, L. D., et al. (2019). Structural studies of the binding of an antagonistic cyclic peptide to the VEGFR1 domain 2. European Journal of Medicinal Chemistry, 169, 65–75.CrossRefPubMedGoogle Scholar
  53. 53.
    Ruegg, C., Hasmim, M., Lejeune, F. J., & Alghisi, G. C. (2006). Antiangiogenic peptides and proteins: From experimental tools to clinical drugs. Biochimica et Biophysica Acta, 1765, 155–177.PubMedGoogle Scholar
  54. 54.
    Sulochana, K. N., & Ge, R. (2007). Developing antiangiogenic peptide drugs for angiogenesis-related diseases. Current Pharmaceutical Design, 13, 2074–2086.CrossRefPubMedGoogle Scholar
  55. 55.
    Wilkins, D. K., Grimshaw, S. B., Receveur, V., Dobson, C. M., Jones, J. A., & Smith, L. J. (1999). Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry, 38, 16424–16431.CrossRefPubMedGoogle Scholar
  56. 56.
    Clarke, J., Cota, E., Fowler, S. B., & Hamill, S. J. (1999). Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway. Structure, 7, 1145–1153.CrossRefPubMedGoogle Scholar

Copyright information

© 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

Personalised recommendations