Secretory Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Kluyveromyces lactis and Characterization of Its Biological Activity

Autor: Seçil Erden Tayhan, Hülya Kuduğ Ceylan, İsa Gökçe
Rok vydání: 2021
Předmět:
Zdroj: International Journal of Peptide Research and Therapeutics. 27:1989-2001
ISSN: 1573-3904
1573-3149
Popis: Vascular endothelial growth factor 165 (VEGF165) is the best characterized member and most potent endothelial cell mitogenic factor belonging to the VEGF family. Because VEGF165 is the major player in angiogenesis in vivo and has potential in therapeutic applications for accelerating wound repair, many expression systems have been developed to produce VEGF165. In this study, biological active recombinant human VEGF165 (rhVEGF165) was expressed for the first time in Kluyveromyces lactis GG799 cells. The gene encoding human VEGF165 was cloned in K. lactis genome by homologous recombination. The VEGF165 was secreted in culture medium at a level of 5.7 mg/L and expression was confirmed by SDS-PAGE and Western blotting. Downstream purification was performed using ammonium sulphate precipitation and affinity chromatography. The biological activity of rhVEGF165 was tested by the proliferation assay on the human umbilical vein-derived endothelial cells (HUVEC) in a dose and time-dependent manner. The K. lactis-derived rhVEGF165 exhibited proliferative activity with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF165 via a scratch assay. These findings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF165 for therapeutic use.
Databáze: OpenAIRE
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