用疏水色谱法复性并同时纯化重组人粒细胞-巨噬细胞集落刺激因子

目的：建立一种简单、快速复性并同时纯化大肠杆菌表达的重组人粒细胞一巨噬细胞集落刺激因子（rhGM-CSF）的方法。方法：研究rhGM-CSF在疏水色谱（HIC）上的复性和纯化机理，并对固定相和流动相进行选择和优化，包括固定相配基、流动相中盐的种类、流动相pH值、流动相中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的比例，以及流动相中尿素的浓度。结果：优化后的固定相为PEG600，流动相中的盐为（NH4）2SO4，流动相pH值为7．0，流动相中添加2．0mol／L尿素、1．8mmol／LGSH和0-3mmol／LGSSG。在优化条件下，HIC可使rhGM-CSF在分离纯化的同时得到复性，比活达1．58×10^7U／mg，纯度为95．7％，质量回收率为56．8％。结论：建立的疏水色谱复性和纯化工艺可简化操作步骤，缩短生产周期。

Refoding and Purification of Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor with Hydrophobic Interaction Chromatography

Objective： To establish a simple method to refold and purify the recombinant human granulocyte-macrophage colony stimulating factor（rhGM-CSF） expressed in Escherichia coli rapidly. Methods： Based on the refolding and purification mechanisms of rhGM-CSF in hydrophobic interaction chromatography （HIC）, we investigated the stationary phase type and the mobile phase factors including salts, pH values, ratios of GSH/GSSG, and urea concentrations. Results： We chose PEG600 as the stationary phase and （NH4）2SO4 as the salt in mobile phase. The optimized conditions are pH7.0, addition of 2.0 mol/L urea, 1,8 mmol/L GSH and 0.3 mmoL/L GSSG into mobile phase. Under the optimal conditions, rhGM-CSF was renatured and purified simultaneously on HIC column. Its specific bioactivity, purity, and mass recovery reached 1.58× 107 U/mg, 95.7% and 56.8% respectively. Conclusion： Using this craft, we got the purified rhGM-CSF with high specific bioactivity in one step. This craft has the advantages of simplicity and time-saving.