Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor with a target-cell specificity highly restricted to vascular endothelial cells. Recombinant baculovirus were constructed for the production of two different forms of the human VEGF protein in insect cells. VEGF165 and VEGF121 proteins produced by Sf158 cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. Only one of these forms, VEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogenicity by a two-step heparin-affinity chromatography. The biological activity of the purified 43-kDa homodimer was demonstrated by high-affinity binding to VEGF receptors, and by the induction of DNA synthesis in vascular endothelial cells. A positive angiogenic activity in vivo was demonstrated by the day-13 chorioallantoic-membrane assay. The mitogenic potency of VEGF121 for human umbilical vein endothelial cells was very similar compared to VEGF165. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 5 micrograms/ml) in insects cells.
Fiebich
B L
BL
Institute of Molecular Cell Biology, University of Freiburg, Federal Republic of Germany.
Jäger
B
B
Schöllmann
C
C
Weindel
K
K
Wilting
J
J
Kochs
G
G
Marmé
D
D
Hug
H
H
Weich
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D066298:In Vitro Techniques; D007915:Lepidoptera; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D006023:Glycoproteins; D008222:Lymphokines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D006493:Heparin