高等学校化学学报

高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (02): 303.doi: 10.3969/j.issn.0251-0790.2012.02.016

• 生物化学 • 上一篇    下一篇

基于Fe3O4@SiO2/Ni-NTA磁性微球的His-tag融合蛋白纯化体系的建立

王文加1, 郭晓林2, 韦安慧1, 付常皓1, 韩振国3   

  1. 1. 吉林大学药学院, 长春 130021;
    2. 吉林大学第一医院, 长春 130021;
    3. 吉林大学中日联谊医院, 长春 130031
  • 收稿日期:2011-03-14 出版日期:2012-02-10 发布日期:2012-01-13
  • 通讯作者: 韩振国, 男, 教授, 主要从事肿瘤与细胞因子相关性研究. E-mail: zhenguohan@163.com E-mail:zhenguohan@163.com

Purification of Histidine-tagged Fusion Proteins Based on Fe3O4@SiO2/Ni-NTA Magnetic Spheres

WANG Wen-Jia1, GUO Xiao-Lin2, WEI An-Hui1, FU Chang-Hao1, HAN Zhen-Guo3   

  1. 1. School of Pharmacy, Jilin University. Changchun 130021, China;
    2. First Hospital, Jilin University, Changchun 130021, China;
    3. China-Japan Union Hospital, Jilin University, Changchun 130031, China
  • Received:2011-03-14 Online:2012-02-10 Published:2012-01-13
  • Contact: WenJia wang E-mail:zhenguohan@163.com

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摘要： 合成了表面共价结合Ni-氨基三乙酸(Ni-NTA)基团的Fe3O4@SiO2微球, 这种磁性微球可用于分离含有His-tag标签的融合蛋白. 微球中心由尺寸约402 nm的Fe3O4微粒组成, 赋予了微球极好的磁性分离和离心分离的特性. 应用Fe3O4@SiO2/Ni-NTA磁性微球对含有6×His-tag(6聚组氨酸)标签的蛋白进行了分离纯化, 结果表明, 10 mg Fe3O4@SiO2/Ni-NTA微球能够从10 mL重组蛋白裂解液中纯化出约1 mg带有6×His-tag标签的融合蛋白. 微球的高效分离效果使其能够用于含量较低的带有6×His-tag标签蛋白的分离纯化.

关键词: 磁性微球, 6×His-tag蛋白, 亲和纯化, Ni-氨基三乙酸

Abstract: The development of reliable and efficient methods to separate proteins of interest from a biological source is still a challenging work. Currently, using the His-tagged fusion proteins to separate and purifiy proteins has been the most widely used technology. This study deals with the synthesis of Ni-nitrilotriacetic acid(Ni-NTA) modified Fe3O4@SiO2 magnetic spheres, which can act as a general agent to separate and purify Histidine-tagged fusion proteins. The spheres have a core composed of carbon particles and a shell composed of Fe3O4 having a uniform size of ca.402 nm, which provides the spheres with excellent magnetic responsivity and dispersity. The recombinant proteins that had been engineered to have six consecutive histidine residues(6×His) were separated by means of Fe3O4@SiO2/Ni-NTA magnetic spheres. The results show that 10 mg of Fe3O4@SiO2/Ni-NTA spheres are able to purify about 1 mg of 6×His-tagged proteins from 10 mL crude E. coli. lysate. Owing to the high separation capacity, Fe3O4@SiO2/Ni-NTA spheres can be used to separate 6×His-tagged proteins with low-concentrations.

Key words: Magnetic sphere, 6×His-tagged protein, Affinity Purification, Ni-nitrilotriacetic acid

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