Expression of biologically active human vascular endothelial growth factor in yeast.
Vascular endothelial growth factor (VEGF) is a glycoprotein consisting of two identical polypeptide chains linked by a disulfide bond. The unique biological activities of VEGF include its potent mitogenic and permeability inducing properties specific for the vascular endothelium. VEGF is implicated in tumor angiogenesis, wound healing, and the stimulation of collateral vessel formation at the site of arterial occlusion. Therefore, in order to produce large quantities of biologically active VEGF, a splice variant (VEGF165) was cloned and expressed in a yeast expression system. The coding region of VEGF165 was isolated from U937 cells by RT-PCR, sequenced and then cloned into the yeast expression vector pHILS1. VEGF165 was secreted into the medium as a dimer. Recombinant VEGF reacted to antibodies raised against the N-terminal and C-terminal synthetic polypeptides of human VEGF. As much as 35-40 mg/L of purified VEGF could be obtained from the yeast expression system. The recombinant protein was biologically active in inducing vascular endothelial cell proliferation in vitro and permeability changes in vivo.
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
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Mohanraj
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Olson
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Ramakrishnan
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005070:Evans Blue / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C037405:heparin-sepharose; D005070:Evans Blue; D012685:Sepharose