Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165in E. coli.
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 VEGF165in convenient yield. In this study, recombinant human VEGF165was 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 VEGF165was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).
Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.
Awatef
A
Taktak-BenAmar
Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis, Tunisia.
Maram
M
Morjen
Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis, Tunisia.
Hazem
H
Ben Mabrouk
Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.
Rania
R
Abdelmaksoud-Dammak
Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.
Mohamed
M
Guerfali
Service Analyse, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.
Najla
N
Fourati-Masmoudi
Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis, Tunisia.
Naziha
N
Marrakchi
Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia. faouzi.gargouri@cbs.rnrt.tn.
Ali
http://orcid.org/0000-0001-5009-9000
A
Gargouri
CAM assay; Cell migration and proliferation; Inclusion bodies; Purification; RT-PCR; Refolding; Soluble VEGF165 expression