A novel method to purify recombinant vascular endothelial growth factor (VEGF121) expressed in yeast.
VEGF is a potent mitogen for vascular endothelial cells in vitro and acts as an angiogenic factor in vivo. VEGF121 differs from the other isoforms in that it lacks the heparin-binding domain. To study the potential differences in biological functions of the VEGF isoforms, we cloned and expressed VEGF121 in a yeast expression system. VEGF121 was secreted from the yeast cells as a homodimer with a molecular weight of 34-36 kDa. By taking advantage of the consecutive histidine residues present at position 11 and 12 in VEGF, a novel method of purification using Nickel affinity chromatography was developed. Since all the isoforms of VEGF have an identical amino terminal end, this method can be used to purify not only VEGF121 but also the other forms of VEGF. The level of expression achieved using this system was as high as 40 mg/L. The recombinant protein was biologically active in stimulating the in vitro proliferation of vascular endothelial cells and positively reacted to an antiserum made against recombinant VEGF165.
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009532:Nickel; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D014162:Transfection; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009532:Nickel