Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli.
Identifieur interne : 000001 ( Main/Exploration ); précédent : 000000; suivant : 000002Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli.
Auteurs : Awatef Taktak-Benamar [Tunisie] ; Maram Morjen [Tunisie] ; Hazem Ben Mabrouk [Tunisie] ; Rania Abdelmaksoud-Dammak [Tunisie] ; Mohamed Guerfali [Tunisie] ; Najla Fourati-Masmoudi [Tunisie] ; Naziha Marrakchi [Tunisie] ; Ali Gargouri [Tunisie]Source :
- AMB Express ; 2017.
Abstract
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 VEGF165 in convenient yield. In this study, recombinant human VEGF165 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 VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).
DOI: 10.1186/s13568-016-0300-2
PubMed: 28168572
Affiliations:
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El Manar</orgName></affiliation></author><author><name sortKey="Ben Mabrouk, Hazem" sort="Ben Mabrouk, Hazem" uniqKey="Ben Mabrouk H" first="Hazem" last="Ben Mabrouk">Hazem Ben Mabrouk</name><affiliation wicri:level="4"><nlm:affiliation>Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis, Tunisia.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis</wicri:regionArea><placeName><settlement type="city">Tunis</settlement><region nuts="2">Gouvernorat de Tunis</region></placeName><orgName type="university">Université de Tunis - El Manar</orgName></affiliation></author><author><name sortKey="Abdelmaksoud Dammak, Rania" sort="Abdelmaksoud Dammak, Rania" uniqKey="Abdelmaksoud Dammak R" first="Rania" last="Abdelmaksoud-Dammak">Rania Abdelmaksoud-Dammak</name><affiliation wicri:level="1"><nlm:affiliation>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax</wicri:regionArea><wicri:noRegion>Sfax</wicri:noRegion></affiliation></author><author><name sortKey="Guerfali, Mohamed" sort="Guerfali, Mohamed" uniqKey="Guerfali M" first="Mohamed" last="Guerfali">Mohamed Guerfali</name><affiliation wicri:level="1"><nlm:affiliation>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax</wicri:regionArea><wicri:noRegion>Sfax</wicri:noRegion></affiliation></author><author><name sortKey="Fourati Masmoudi, Najla" sort="Fourati Masmoudi, Najla" uniqKey="Fourati Masmoudi N" first="Najla" last="Fourati-Masmoudi">Najla Fourati-Masmoudi</name><affiliation wicri:level="1"><nlm:affiliation>Service Analyse, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Service Analyse, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax</wicri:regionArea><wicri:noRegion>Sfax</wicri:noRegion></affiliation></author><author><name sortKey="Marrakchi, Naziha" sort="Marrakchi, Naziha" uniqKey="Marrakchi N" first="Naziha" last="Marrakchi">Naziha Marrakchi</name><affiliation wicri:level="4"><nlm:affiliation>Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis, Tunisia.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire des Venins et Biomolécules Thérapeutiques, LR11IPT08, Institut Pasteur de Tunis, Université de Tunis el Manar, 13, Place Pasteur, 1002, Tunis</wicri:regionArea><placeName><settlement type="city">Tunis</settlement><region nuts="2">Gouvernorat de Tunis</region></placeName><orgName type="university">Université de Tunis - El Manar</orgName></affiliation></author><author><name sortKey="Gargouri, Ali" sort="Gargouri, Ali" uniqKey="Gargouri A" first="Ali" last="Gargouri">Ali Gargouri</name><affiliation wicri:level="1"><nlm:affiliation>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax, Tunisia. faouzi.gargouri@cbs.rnrt.tn.</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018, Sfax</wicri:regionArea><wicri:noRegion>Sfax</wicri:noRegion></affiliation></author></analytic><series><title level="j">AMB Express</title><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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 VEGF165 in convenient yield. In this study, recombinant human VEGF165 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 VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).</div></front></TEI><affiliations><list><country><li>Tunisie</li></country><region><li>Gouvernorat de Tunis</li></region><settlement><li>Tunis</li></settlement><orgName><li>Université de Tunis - El Manar</li></orgName></list><tree><country name="Tunisie"><noRegion><name sortKey="Taktak Benamar, Awatef" sort="Taktak Benamar, Awatef" uniqKey="Taktak Benamar A" first="Awatef" last="Taktak-Benamar">Awatef Taktak-Benamar</name></noRegion><name sortKey="Abdelmaksoud Dammak, Rania" sort="Abdelmaksoud Dammak, Rania" uniqKey="Abdelmaksoud Dammak R" first="Rania" last="Abdelmaksoud-Dammak">Rania Abdelmaksoud-Dammak</name><name sortKey="Ben Mabrouk, Hazem" sort="Ben Mabrouk, Hazem" uniqKey="Ben Mabrouk H" first="Hazem" last="Ben Mabrouk">Hazem Ben Mabrouk</name><name sortKey="Fourati Masmoudi, Najla" sort="Fourati Masmoudi, Najla" uniqKey="Fourati Masmoudi N" first="Najla" last="Fourati-Masmoudi">Najla Fourati-Masmoudi</name><name sortKey="Gargouri, Ali" sort="Gargouri, Ali" uniqKey="Gargouri A" first="Ali" last="Gargouri">Ali Gargouri</name><name sortKey="Guerfali, Mohamed" sort="Guerfali, Mohamed" uniqKey="Guerfali M" first="Mohamed" last="Guerfali">Mohamed Guerfali</name><name sortKey="Marrakchi, Naziha" sort="Marrakchi, Naziha" uniqKey="Marrakchi N" first="Naziha" last="Marrakchi">Naziha Marrakchi</name><name sortKey="Morjen, Maram" sort="Morjen, Maram" uniqKey="Morjen M" first="Maram" last="Morjen">Maram Morjen</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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