人TRAIL分子胞外区的纯化及活性检测

Purification of human TRAIL extracellular region and its activity

目的 :利用大肠杆菌表达hTRAIL4 1～ 2 81 蛋白 ,并纯化复性成生物活性形式。方法 :以IPTG诱导表达 ,使用Ni NTA层析柱分离纯化蛋白 ,透析法复性 ,SDS PAGE和Westernblot法进行鉴定 ,DNA断裂实验检测hTRAIL4 1～ 2 81 蛋白的凋亡诱导活性。结果 :表达的蛋白以包涵体的形式存在 ,纯化后得到分子量为 30 5kD、纯度在 90 %以上的蛋白 ,Westernblot证实为hTRAIL4 1～ 2 81 分子。经复性 ,DNA断裂实验检测出该蛋白有较好的诱导肿瘤细胞凋亡活性。结论 :成功地利用大肠杆菌表达、Ni NTA层析柱分离纯化、透析法复性hTRAIL4 1～ 2 81 蛋白 ,并证实其具有诱导肿瘤细胞凋亡的生物学活性。

Objective:To express hTRAIL_ 41～281 protein using E.coli and refold it into a functional form.Methods:The expression of protein was induced by IPTG,protein was purified by Ni-NTA chromatography column,and refolded by dialysis ,protein purified was determined by SDS-PAGE and Western blot.Antitumor activity of hTRAIL_ 41～281 protein was measured by DNA fragmentation electropherogram.Results:Results of SDS-PAGE and Western blot proved that the 30.5 kD protein purified were hTRAIL_ 41～281 protein,the purity of protein was more than 90%.After refolding,DNA fragmentation electropherogram showed that hTRAIL_ 41～281 protein had good antitumor activity.Conclusion:hTRAIL_ 41～281 protein with antitumor activity was successfully expressed with E.coli and purified by Ni-NTA chromatography column,refolded by dialysis.