反相色谱法研究人粒细胞集落刺激因子的离子交换色谱复性和稀释复性

Study on Dilution and Ion Exchange Chromatographic Refolding of Recombinant Human Granulocyte Colony-stimulating Factor by Reversed Phase Chromatography

以反相色谱分析为主要手段,结合活性测定,研究了以包含体形式表达的重组人粒细胞集落刺激因子(rhG-CSF)的稀释复性和离子交换色谱复性. 建立了L-精氨酸离子交换复性蛋白质的方法,将变性、还原的rhG-CSF吸附到高浓度变性剂平衡的离子交换色谱柱上,用L-精氨酸作洗脱剂进行梯度洗脱并同时脱除变性剂,实现了rhG-CSF的复性. 与稀释复性相比,色谱复性的rhG-CSF处于一种中间状态,呈现快速而不同步的动力学特点,这与色谱复性的梯度洗脱及固定相的吸附有关. 同时发现了对复性峰进一步稀释并且温育则可以提高其活性的新现象.

A novel way to refold rhG-CSF by combining ion exchange chromatography (IEC) and L-arginine was developed: denatured rhG-CSF from inclusion body was attached to a DEAE column and eluted by a gradient from 6 mol/L urea to 0.5 mol/L L-arginine. Refolding of rhG-CSF was also done by dilution and by passing the rhG-CSF inclusion body through the DEAE column perfused with 0.5 mol/L arginine without adsorption. RPC analysis and bioactivity assay showed that rhG-CSF refolded by IEC had different pattern from those of the rhG-CSFs refolded by dilution and flowing-through, representing fast but asynchronous kinetics, due to the adsorption of the DEAE medium to the rhG-CSF and the gradient of denaturant. It was also found that subsequent dilution and incubation of the IEC-refolded rhG-CSF furthered its refolding and enhanced its bioactivity up to 10 folds.