色谱

色谱

• 研究报告 •    下一篇

阴离子交换整体柱对蛋白质的分离与纯化

张轶华1,杨更亮1,2,张小乐1,3,赵菊敏1,蔡丽萍1,陈义2   

  1. 1. College of Pharmacy, Hebei University, Baoding 071002, China; 2. Molecular Science Center, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China; 3. College of Light Industry, Hebei Polytechnic University, Tangshan 063020, China
  • 收稿日期:2004-12-15 修回日期:1900-01-01 出版日期:2005-05-30 发布日期:1988-09-25
  • 通讯作者: 杨更亮
  • 基金资助:

Separation and Purification of Proteins on Monolithic Anion-Exchange Columns

ZHANG Yihua1, YANG Gengliang1,2, ZHANG Xiaole1,3, ZHAO Jumin1, CAI Liping1, CHEN Yi2   

  1. 1. College of Pharmacy, Hebei University, Baoding 071002, China; 2. Molecular Science Center, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China; 3. College of Light Industry, Hebei Polytechnic University, Tangshan 063020, China
  • Received:2004-12-15 Revised:1900-01-01 Online:2005-05-30 Published:1988-09-25

PDF

746

被引次数

16

摘要： 分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件，并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时，实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。

关键词: 纯化 , 蛋白质, 分离, 离子交换色谱, 纤维素降解酶, 修饰, 整体柱

Abstract: A monolithic anion-exchange column with glycidyl methacrylate as the functional monomer and ethylene dimethacrylate as the cross linker was prepared by a free radical polymerization. The epoxide groups of the column were modified respectively by triethylamine, diethylamine and ethylenediamine that afforded anionic functionalities required for the anion-exchange chromatographic mode. The properties of the monolithic columns were investigated and the columns were successfully used as stationary phases of high performance liquid chromatography for the separation of proteins. For chromatographic analysis the effects of mobile phase composition and pH on the separation were investigated. The optimum separation for bovine serum albumin, lysozyme and glutathione was achieved with a gradient elution of mobile phase A ( 0.01 mol/L Tris-HCl (pH 　7.0)) and mobile phase B (mobile phase A+1.0 mol/L NaCl) with a flow rate of 1.0 mL/min at 25 ℃. The optimum purification for cellulase enzyme was obtained with a gradient elution of mobile phase A (0.01 mol/L Tris-HCl (pH　7.1)) and mobile phase B (mobile phase A+1.0 mol/L KBr) with the same flow rate and temperature. The columns exhibited good stability, and cellulase enzyme could be separated and purified quickly on the monolithic anion-exchange column modified by diethylamine.

Key words: cellulase enzyme, ion-exchange chromatography, modification, proteins, purification , separation, monolithic column