title: Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.
abstract: We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 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-α2-PI1-8-VEGF121 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 α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. 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, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.
authors.affiliation: Institute of Macromolecular Chemistry, Czech Academy of Sciences, CZ-162 06, Prague, Czech Republic.|Institute of Physiology, Czech Academy of Sciences, CZ-142 20, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Kaplan
authors.affiliation: Institute of Physiology, Czech Academy of Sciences, CZ-142 20, Prague, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Zárubová
authors.affiliation: Institute of Physiology, Czech Academy of Sciences, CZ-142 20, Prague, Czech Republic.|Faculty of Science, Charles University in Prague, CZ-128 40, Prague, Czech Republic.
authors.forename: Barbora
authors.initials: B
authors.lastname: Mikulová
authors.affiliation: Institute of Physiology, Czech Academy of Sciences, CZ-142 20, Prague, Czech Republic.
authors.forename: Elena
authors.initials: E
authors.lastname: Filová
authors.affiliation: School of Dental Medicine, General University Hospital in Prague, CZ-128 08, Prague, Czech Republic.
authors.forename: Jiřina
authors.initials: J
authors.lastname: Bártová
authors.affiliation: Institute of Physiology, Czech Academy of Sciences, CZ-142 20, Prague, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bačáková
authors.affiliation: Institute of Macromolecular Chemistry, Czech Academy of Sciences, CZ-162 06, Prague, Czech Republic.
authors.forename: Eduard
authors.initials: E
authors.lastname: Brynda
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D013879:Thioredoxins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013879:Thioredoxins; D005337:Fibrin