利用6× His/ Ni-NTA 表达纯化系统一步纯化乙型肝炎前S抗原  

One step purification of recombinant protein with 6×His and Ni NTA resin

作　　者：孟 莉[1] 韩保光[1] 陈 坤[1] 宋晓国[1] 张贺秋[1] 凌世淦[1] 马贤凯[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学科学院院刊》1999年第3期201-204,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：欧共体合作项目( C I1 C T940023)资助

摘　　要：目的：利用 ６× Ｈｉｓ／ Ｎｉ Ｎ Ｔ Ａ 表达纯化系统表达并纯化带有 ６ 个 Ｈｉｓ 纯化标签的乙型肝炎前 Ｓ抗原。方法：利用 Ｎｉ Ｎ Ｔ Ａ Ｓｕｐｅｒｆｌｏｗ 亲和层析，比较了 Ｐｒｅ Ｓ１／２ 与 Ｐｒｅ Ｓ２ 蛋白分别在非变性和变性条件下的各种纯化条件和产率，并用三种方式对 Ｐｒｅ Ｓ２ 蛋白进行了复性。结果： Ｐｒｅ Ｓ１／２ 与 Ｐｒｅ Ｓ２ 蛋白均存在于 ２５０ ｍ ｍ ｏｌ／ Ｌ 咪唑洗脱峰中。从 １ Ｌ 诱导菌体中可以分别纯化 Ｐｒｅ Ｓ１／２ 与 Ｐｒｅ Ｓ２ 蛋白 １０～１５ ｍ ｇ，纯度达到电泳纯。三种复性方式中，在 Ｎｉ Ｎ Ｔ Ａ 柱上复性 Ｐｒｅ Ｓ２ 蛋白的复性率达 ６０．５％ 。结论：６× Ｈｉｓ／ Ｎｉ Ｎ Ｔ Ａ 表达纯化系统不仅可以使外源蛋白在大肠杆菌中获得高效表达，而且可以通过简单的纯化步骤得到较高纯度的目的蛋白。该表达纯化系统为利用大肠杆菌制备重组蛋白提供了一种简便可行的方法。Objective: Using a 6×His/Ni NTA system to express two polypeptides of hepatitis B virus, PreS1/2 (PreS1 1-108 /PreS2 1-55 ) and PreS2 (PreS2 1-55 /S 1-9 ) with a six consecutive histidine residues (the 6×His tag),in E.coli and to purify them in a single step. Methods: The purification under native or denaturing conditions and the yield of the two proteins were studied using Ni NTA superflow affinity chromatography. The protein PreS2 was refolded with three methods. Results: Both protein PreS1/2 and protein PreS2 were eluted by 250 mmol/L imidazole. Ten to fifteen mg of each of the two proteins were purified from 1 L of induction bacteria with their purity attaining electrophoretically pure. The protein PreS2 can be efficiently refolded while immobilized on the Ni NTA column. Conclusion: The 6×His/Ni NTA system in combination with a high level bacterial expression system creates an elegant yet simple strategy for protein purification no matter whether the expressed protein is at low or high levels, native or denatured. It is a widely used method in the preparation of recombinant proteins from E.coli.

关 键 词：重组蛋白质 组氨酸标签 乙型肝炎 前S抗原 

分 类 号：Q51[生物学—生物化学] R373.21[医药卫生—病原生物学]