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Cloning, Large-Scale Production, and Purification of Active Dimeric Rat Vascular Endothelial Growth Factor (rrVEGF-164)

Cloning, Large-Scale Production, and Purification of Active Dimeric Rat Vascular Endothelial Growth Factor (rrVEGF-164)

Paul J Geutjes, Suzan T M Nillesen, Gerwen Lammers, Willeke F Daamen, Toin H van Kuppevelt

Protein Expr Purif. 2010 Jan;69(1):76-82.

PMID: 19733244

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4248694	Vascular Endothelial Growth Factor 164 from rat			Price

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