阴离子交换整体柱对蛋白质的分离与纯化  

Separation and Purification of Proteins on Monolithic Anion-Exchange Columns

作　　者：张轶华[1] 杨更亮[1] 张小乐[1] 赵菊敏[1] 蔡丽萍[1] 陈义[2] 

机构地区：[1]河北大学药学院,河北保定071002 [2]中国科学院化学研究所分子科学中心,北京100080

出　　处：《色谱》2005年第3期219-222,共4页Chinese Journal of Chromatography

基　　金：国家自然科学基金资助项目(No.20375010);教育部高等学校博士学科点专项科研基金资助项目;教育部优秀青年教师资助计划和中国科学院"百人计划"项目.

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。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.

关 键 词：整体柱 修饰 离子交换色谱 纤维素降解酶 蛋白质 分离 纯化 

分 类 号：O658[理学—分析化学]