Expression and purification of high purity soluble chimeric protein VEGI+ / 第二军医大学学报
Academic Journal of Second Military Medical University
;
(12): 1324-1328, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-840987
ABSTRACT
Objective:
To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI+, so as to lay a basis for studying its biological activity.Methods:
Chimeric molecule VEGI+ was constructed by grafting oligopeptide CTTH-WGFTLC to extracellular region of VEGI (VEGI23-174). Before ligation into pET30a(+) expression vector, PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing, then pET30a-VEGI was used to transfect BL21 (modified E. coli strain). The chimeric protein was purified by metal affinity chromatography. Western blotting and coomassie blue staining were used for protein identification.Results:
The chimeric molecule VEGI+ was confirmed by restriction enzyme digestion and DNA sequencing. The constructed pET30a-VEGI was confirmed by enzymatic digestion. The expression was mainly in the form of inclusion body. SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23 000, with a purity of about 90%.Conclusion:
We have successfully constructed the recombinant plasmid pET30a-VEGI+ and expressed it in E. coli. And we have obtained high purity of soluble chimeric protein VEGI+ through affinity chromatography.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Academic Journal of Second Military Medical University
Year:
2010
Type:
Article
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