内皮抑素变体蛋白的表达及复性与纯化工艺的优化

The Optimization of Expression,Refolding and Purification of an Endostatin Mutant

目前抗血管新生已经成为一种有效的癌症治疗手段。内皮抑素(Endostatin)可以抑制血管新生和形成,已经作为抑癌药物上市并作为辅助用药已起到较好的治疗效果。课题组构建了将内皮抑素与穿膜肽九聚精氨酸融合的内皮抑素变体,拟促进内皮抑素进入细胞膜以期达到更好的治疗效果。该研究以原核表达质粒p BV222为载体,构建了带有His标签的内皮抑素变体(9R-Endo-9R),内皮抑素变体蛋白的相对分子质量约21 k。在1 L的发酵体系下,蛋白以包涵体形式表达,为了深入探究该内皮抑素变体的生物学性能,该研究探索以变复性的方法获得可溶性内皮抑素变体,并对纯化方法进行优化以获得较高回收率。CCK-8实验结果表明该内皮抑素变体对血管内皮细胞具有增殖抑制作用,显示优化后的复性和纯化的内皮抑素变体具有较好的生物学活性。该研究为内皮抑素变体的进一步药学性质的研究奠定了基础。

Anti-angiogenesis has become an effective approach for cancer therapy recently. Endostatin can inhibit angiogenesis and vessel development,and has been approved as an anti-tumor drug combined with chemotherapy and has displayed good clinical therapeutic effect. In previous work,the authors’ research team 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 a better therapeutic effect. In this paper,a prokaryotic expression plasmid p BV222 was used as a vector to construct a His-tagged endostatin variant( 9R-Endo-9R),of which the relative molecular mass was about 21 k. In 1 L fermentation system,the prokaryotic vector carried a temperature-controlled element that activated the downstream expression site at high temperature of 42 ℃. The protein was expressed in the form of inclusion bodies. In order to further explore the biological properties of the endostatin variant,this paper explored the method of obtaining a soluble endostatin variant by a renaturation method and optimizing the purification method to obtain a higher recovery rate. Firstly,after the protein was expressed by E. coli,the cells were lysed by high-pressure crushing,and the target protein in the obtained lysate was formed into a large amount of inclusion bodies. After the inclusion body was washed several times with the washing liquid,the inclusion body protein was denatured and dissolved by using a high concentration salt solution,and the target protein with higher purity could be obtained at this time. The dissolved protein was further purified by Ni-NTA affinity chromatography to obtain a high purity target protein. The concentration of the target protein was removed by a concentration gradient dialysis method,and the salt in the solution was removed to obtain a soluble protein. The results of CCK-8 experiments showed that the endostatin variant had a proliferation inhibitory effect on vascular endothelial cells,indicating that the optimized renaturation and purified endostatin variants had good biological activity. This study lays a solid foundation for the study of further pharmaceutical properties and potential application of endostatin mutant.