Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164).
Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation ( approximately 124mg/L) and purified by heparin affinity chromatography. SDS-PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences.
Geutjes
Paul J
PJ
Department of Biochemistry, Radboud University Nijmegen Medical Centre, HB Nijmegen, The Netherlands. p.geutjes@uro.umcn.nl
Nillesen
Suzan T M
ST
Lammers
Gerwen
G
Daamen
Willeke F
WF
van Kuppevelt
Toin H
TH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A