Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF(121) Expressed in E. coli Origami B (DE3) with Molecular Chaperones

0467201 - FGÚ 2017 RIV US eng J - Journal Article
Kaplan, Ondřej - Zárubová, Jana - Mikulová, Barbora - Filová, Elena - Bártová, J. - Bačáková, Lucie - Brynda, E.
Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF(121) Expressed in E. coli Origami B (DE3) with Molecular Chaperones.
PLoS ONE. Roč. 11, č. 10 (2016), č. článku e0163697. ISSN 1932-6203. E-ISSN 1932-6203
R&D Projects: GA MŠMT(CZ) LQ1604; GA MŠMT(CZ) ED1.1.00/02.0109; GA MZd NV15-29153A; GA TA ČR(CZ) TA04011345
Institutional support: RVO:67985823
Keywords : angiogenic gene-therapy * Escherichia coli * VEGF-A
Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery
Impact factor: 2.806, year: 2016

We describe the production of a highly-active mutant VEGF variant, alpha(2)-PI1-8-VEGF(121), which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The alpha(2)-PI1-8-VEGF(121) gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-alpha(2)-PI1-8-VEGF(121) fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7.The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified alpha(2)-PI1-8-VEGF(121) was 1.4 mg per liter of the cell culture. The alpha(2)-PI1-8-VEGF(121) expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF(121). This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, alpha(2)-PI1-8-VEGF(121) did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.
Permanent Link: http://hdl.handle.net/11104/0265311