Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.
Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. 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 20mg/L) in Sf21 cells.
Lee
Geum Young
GY
MyGene Bioscience Institute, 202-16, Nonhyun-Dong, Kangnam-Gu, Seoul 135-010, Republic of Korea.
Jung
Woon Won
WW
Kang
Chang Soo
CS
Bang
In Seok
IS
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D046188:Protein Modification, Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D004220:Disulfides; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A