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

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

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-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/L GSH和0.3mmol/L GSSG。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1.58×107U/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。

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.