体积排阻色谱法(SEC)对重组人粒细胞-巨噬细胞集落刺激因子的复性并同时纯化  

Renaturation and Simultaneous Purification of Recombinant Human Granulocyte Macrophage Colonly Stimulating Factor(rhGM-CSF) with Size Exclusive Chromatography

作　　者：刘建波[1] 王欢[1] 白泉[2] 耿信笃[2] 

机构地区：[1]咸阳师范学院化学系,陕西咸阳712000 [2]西北大学现代分离科学研究所,陕西西安710069

出　　处：《咸阳师范学院学报》2007年第6期43-46,共4页Journal of Xianyang Normal University

基　　金：国家自然科学基金项目(20175016)

摘　　要：采用体积色谱法对由E.coli表达的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)复性与同时纯化工艺进行了研究。分别讨论了不同浓度的脲、流动相pH以及在流动相中添加不同比例的GSSG/GSH对rhGM-CSF复性与同时纯化的影响。结果表明:三者对其复性效率和质量回收率均有较大的影响,GSSG/GSH可有效提高rhGM-CSF二硫键的正确对接和复性效率,而低浓度的脲则可有效抑制蛋白沉淀,可大大提高质量回收率。在最优化条件下,SEC可在30 min内使rhGM-CSF在分离纯化的同时进行复性,其比活为1.31×107 IU/mg,质量回收率为42.3%,纯度可达到86.4%。The renaturation and simultaneous purification of recombinant human granulocyte macrophage colonly stimulation factor （rhGM-CSF） expressed in E.coli with size exclusion chromatography （SEC） was studied. The effects of the different pH values, the ratios of concentrations of GSH/GSSG and urea concentrations in the mobile phase on the renaturation and simultaneous purification of rhGM-CSF with SEC were investigated respectively. The results show the three factors have the remarkable effects on the renaturation efficiency and mass recovery ofrhGM-CSF. GSH/GSSG can improve to form the correct disulfide bonds as a result that the renaturation efficiency ofrhGM-CSF can be increased. In addition, in order to enhance the mass recovery ofrhGM-CSF with SEC, the low concentration urea can be used in the mobile phase to prevent denatured protein aggregation. Under the optimal condition, rhGM-CSF can be renatured with simultaneous purification with SEC in 30 rain only by one step, and its specific activity, mass recovery and purity can be obtained to be 1.31× 10^7IU/mg,42.3% and 86.4%, respectively.

关 键 词：体积排阻色谱 蛋白复性 纯化 rhGM—CSF 

分 类 号：Q819[生物学—生物工程]