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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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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Academic Journal of Second Military Medical University Year: 2010 Type: Article