Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF(165) in E-coli

Awatef Taktak-Benamar (1) , Maram Morjen (2) , Hazem Ben Mabrouk (2) , Rania Abdelmaksoud-Dammak (1) , Mohamed Guerfali (1) , Najla Fourati-Masmoudi (1) , Naziha Marrakchi (2) , Ali Gargouri (1)

1 CBS - Centre de Biotechnologie de Sfax
2 LR11IPT08 - Laboratoire des Venins et Biomolécules Thérapeutiques - Laboratory of Venoms and Therapeutic Biomolecules

Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF(165) in convenient yield. In this study, recombinant human VEGF(165) was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF(165) was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).

Mots clés: Cell migration and proliferation; Refolding; Purification; RT-PCR; Soluble VEGF(165) expression; Inclusion bodies; CAM assay; Soluble VEGF 165 expression

Domaines: Sciences du Vivant [q-bio]

Awatef Taktak-Benamar, Maram Morjen, Hazem Ben Mabrouk, Rania Abdelmaksoud-Dammak, Mohamed Guerfali, et al.. Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF(165) in E-coli. AMB Express, 2017, 7 (1), pp.33. ⟨10.1186/s13568-016-0300-2⟩. ⟨hal-01506947⟩