重组毒素DT389-IL13的复性与纯化工艺

为了获得有较高生物活性的靶向重组毒素DT389-IL13,在大肠杆菌中高效表达DT389-IL13,并采用凝胶过滤的方法对DT389-IL13进行柱上复性及初步纯化,然后使用离子交换色谱方法进一步纯化,最后应用CCK-8细胞活力检测试剂盒检测DT389-IL13对肿瘤细胞的抑制作用.实验结果显示,经柱上复性和纯化后,DT389-IL13的纯度可达90%以上,且对脑胶质瘤细胞U251有明显的抑制作用,存在明确的剂量依赖关系,48h的半数抑制浓度为8.87×10-10mol/L.表明联合应用凝胶过滤和离子交换两种色谱方法可获得高纯度的复性蛋白,是可行的复性及纯化方案.

Refolding and Purification of Recombinant Toxin DT389-IL13

In order to obtain the targeted recombinant toxin DT389-IL13 with a high biological activity,the DT389-IL13 gene was overexpressed in E.coli.The recombinant protein was refolded and initially purified using a size exclusive chromatograph column,then the resulting protein was further purified by ion exchange chromatography.In the end,we performed cytotoxicity assay to verify the inhibitive effective of DT389-IL13 on tumor cells.The results showed that the purity of the refolded and purified DT389-IL13 was higher than 90%.DT389-IL13 could significantly suppress the U251 cells in a dose dependent manner and the IC50 at 48 hours was 8.87×10-10 mol/L.Therefore,the combination of size exclusive chromatography and ion exchange chromatography were the feasible program to refold and purify recombinat toxin DT389-IL13.