层析辅助体外复性含有多对二硫键的融合蛋白质

Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds

摘要:

为建立一种基于阴离子交换介质辅助的含多对二硫键的抗凝溶栓双功能水蛭素12肽-瑞替普酶融合蛋白质 (HV12p-rPA) 的复性方法，采用Q Sepharose XL作为层析复性介质，通过正交实验考察蛋白质上样量、流速、脲梯度、洗脱液中精氨酸浓度、脲浓度、pH、还原型及氧化型谷胱甘肽等因素对复性过程的影响，探索最佳层析复性条件。结果表明：脲梯度、上样量及精氨酸浓度是影响复性的3个主要因素。脲梯度是复性成功的关键，上样量增大时复性蛋白质比活降低，精氨酸辅助HV12p-rPA复性的最佳浓度为1 mol/L。创建了脲、pH双梯度下的阴离子交换层析辅助HV12p-rPA的复性方法，复性后蛋白质的溶栓比活达到46 520 IU/mg，抗凝比活达到9 980 ATU/mg，与稀释复性方法相比，该方法能使复性蛋白质的溶栓比活提高14~15倍，抗凝比活提高7~8倍。

Abstract:

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.