高等学校化学学报

高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (05): 925.doi: 10.3969/j.issn.0251-0790.2012.05.011

• 分析化学 • 上一篇    下一篇

谷胱甘肽键合柱对变性核糖核酸酶的复性研究

柯从玉, 孟祖超   

  1. 西安石油大学化学化工学院, 西安 710065
  • 收稿日期:2011-06-03 出版日期:2012-05-10 发布日期:2012-05-10
  • 作者简介:柯从玉, 男, 博士, 讲师, 主要从事生物大分子高效液相色谱研究. E-mail: kcy@xsyu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号: 39880003)和西安石油大学博士科研启动基金(批准号: YS29030804)资助.

Denatured Ribonuclease Refolding by Glutathione Bonding Column

KE Cong-Yu, MENG Zu-Chao   

  1. College of Chemistry & Chemical Engineering, Xi'an Shiyou University, Xi'an 710065, China
  • Received:2011-06-03 Online:2012-05-10 Published:2012-05-10

PDF (PC)

被引次数

6

摘要： 将氧化还原型谷胱甘肽(GSH/GSSG)共价键合到色谱固定相上, 实现了对变性核糖核酸酶(RNase)的复性. 实验发现, 谷胱甘肽键合柱具有典型的弱阳离子交换性质, 在离子交换(IEC)模式下能够对4种标准蛋白进行基线分离, 且具有较高的柱效. 当蛋白浓度为5 mg/mL, 流速为0.2 mL/min时, 在流动相中不加GSH/GSSG的条件下, GSH/GSSG柱对变性核糖核酸酶的活性回收率可达(39.5±3.8)%, 而普通IEC柱对变性核糖核酸酶的活性回收率几乎为0, 说明其对变性蛋白二硫键的正确对接具有明显的促进作用; 在收集液中加入GSH/GSSG后, 其活性回收率可达到(81.5±4.3)%. 本文结果对蛋白折叠液相色谱法的发展及降低蛋白复性成本具有一定的应用价值.

关键词: 蛋白折叠, 核糖核酸酶, 离子交换色谱, 谷胱甘肽键合柱

Abstract: The renaturation of denatured ribonuclease was studied by covalent bonding the glutathione/glutathione disulfide(GSH/GSSG) to the surface of stationary phase. The results showed that the glutathione bonding column had a typical characters of weak-cation exchange, under the mode of ion-exchange chromatography(IEC), the column had a better column efficiency and four kinds of standard proteins could be baseline separated. With the protein concentration of 5 mg/mL, flow rate of 0.2 mL/min and no GSH/GSSG in mobile phase, the bioactivity recovery of denatured ribonuclease could reach (39.5?3.8)% refolded by glutathione bonding column, which was almost zero by common IEC column. The glutathione bonding column had an evident promoter action to the right refolding of denatured proteins disulfide bonds. Moreover, the bioactivity recovery could reach (81.5?4.3)% by adding GSH/GSSG to sample collections directly. These results may be useful for the development of protein folding liquid chromatography(PFLC) and in reducing the cost of proteins folding.

Key words: Protein folding, Ribonuclease, Ion-exchange chromatography, Glutathione bonding column

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