The optimization of expression, refolding and purification of an Endostatin mutant

• 1、School of Pharmacy，Nanjing University of TCM
• 2、 School of Life Sciences, Nanjing University
• 3、 Research Institute of Phammaceutical Biotechnology, Jiangsu Industrial Technology Ｒesearch Institute
• 4、Nanjing Genrecom Laboratories, Ltd.

Abstract：Anti-angiogenesis has become an effective approach for cancer therapy recently. Endostatin can inhibit angiogenesis, and has been approved as an anti-tumor drug and has displayed good clinical therapeutic effect. Our lab constructed an endostatin mutant that combined with the penetrating peptide composing of nine polyarginine, and it is designed to promote the entry of endostatin into the cell membrane, in order to achieve better therapeutic effect. In the current study, a prokaryotic expression plasmid pBV-222 wasThe optimization of expression, refolding and purification of an Endostatin mutant used as a vector to construct a His-tagged endostatin mutant (9R-Endo-9R). The protein is expressed in the form of inclusion bodies. In order to further explore the biological properties of the endostatin mutant, this study explores the method of obtaining a soluble endostatin mutant by a refolding methodology and optimizing the purification to obtain a better recovery rate. The results of CCK-8 assay showed that the endostatin mutant had a proliferation inhibitory effect on vascular endothelial cells, indicating that the optimized refolding and purified endostatin mutant has good biological activity. This study lays a solid foundation for the study of further pharmaceutical properties and potential application of endostatin mutant.

Keywords： endostatin endostatin mutant inclusion body refolding expression and purification