离子交换色谱法去除rhOP-1蛋白溶液中内毒素的方法

Removal of Endotoxin from rhOP-1 Solution Using Negative Chromatographic Column

目的建立一种去除重组人成骨蛋白(recombination human osteogenic protein-1,rhOP-1)溶液中内毒素的方法。方法提取包涵体蛋白,经纯化、复性得rhOP-1溶液。此溶液经DEAE离子交换色谱柱,通过线性增加盐浓度方式先将蛋白洗脱,内毒素则由较强盐浓度和NaOH洗脱。收集洗脱峰,用鲎试剂检测其内毒素含量。结果凝胶法检测rhOP-1溶液在DEAE-琼脂糖色谱柱上样前内毒素含量在限值以上,DEAE-琼脂糖色谱柱洗脱峰的蛋白内毒素含量降到限值以下,而经0.22μm滤膜除菌的蛋白溶液中内毒素含量在限值以上。结论rhOP-1蛋白溶液经DEAE-琼脂糖色谱柱洗脱,可得低于内毒素限值的蛋白溶液,因此这种方法可作为一种去除rhOP-1缓冲液中内毒素的方法。

Objective To establish a method of removing bacterial endotoxin from protein solution. Methods Inclusion body was extracted from engineered bacteria. Then the active rhOP-1 was collected by purification and renaturation. When the rhOP-1 solution was obtained it was subjected to flow negative chromatographyi column, thereby binding the rhOP-1 and endotoxin with the media. Endotoxin carried strong negative charges, and it bound more tightly with media than with protein. Protein could be eluted by using increasing salt gradients and endotoxin could be desorbed by high concentration salt and NaOH solution. The elution peak was collected, and its endotoxin was detected by tachypleus amebocyte lysate. Results The content of endotoxin was above the limit before dosing on chromatographic column, and after the elution, endotoxin was below the limit. After the membrane filtration for bacterial removal, the endotoxin was above the limit. Conclusion Elution by DEAE-S chromatographic column can serve to remove the endoxin in RhOP-1 solution and can be used as an alternative for the removal of endotoxin in rhOP-1 solution.