期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 69, 期 1, 页码 76-82出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2009.08.015
关键词
Growth factors; rrVEGF-164; Pichia pastoris; Angiogenesis
类别
资金
- Dutch Program for Tissue Engineering (DPTE) [6735]
- EMCM BV, Nijmegen, The Netherlands
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 A pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation (similar to 124 mg/L) and purified by heparin affinity chromatography. SDS-PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48 kDa 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 3 days, 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. (C) 2009 Elsevier Inc. All rights reserved.
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