一种减少包涵体变复性过程中产生的蛋白多聚体的方法

A method for reducing protein polymers formed during denaturation and renaturation of inclusion bodies

目的优化蛋白制备工艺,减少原核表达包涵体变复性产生的多聚体。方法根据原始工艺进行菌体培养、破壁以及包涵体收集和变复性。然后在包涵体复性液中直接添加硫酸铵固体(优化工艺)。离心去沉淀,收集上清液制备蛋白粗制品。对原始工艺和优化工艺制备的蛋白粗制品进行凝胶蛋白结合量、蛋白回收率、纯化蛋白量以及细胞杀伤活性比较。结果与原始工艺相比,增加30%饱和度硫酸铵沉淀步骤的优化工艺可使1 ml层析凝胶的蛋白结合量由1 mg提高至20 mg,单位体积发酵液蛋白回收率由10%增加至90%,400 ml发酵液所得的纯化蛋白量由3 mg提升至15 mg。蛋白粗制品的平均半数抑制浓度从0.077 99 μg/ml降至0.045 69 μg/ml。结论在包涵体复性液中添加硫酸铵可有效减少多聚体。

Objective To optimize protein preparation process and reduce polymers formed during denaturation and renaturation of inclusion bodies.Methods The inclusion bodies were harvested, denatured and renatured after bacteria cultivation and wall-broken according to original process. Then, solid ammonium sulfate was added directly to renaturation solution of inclusion bodies (optimized process). The precipitate was removed by centrifugation. Supernatant was collected for preparing crude protein product. The crude products prepared by the original and optimized processes were compared in terms of protein-binding capacity of chromatographic gel, protein recovery rate, purified protein amount and cytotoxic activity.Results By adding a step of 30% saturated ammonium sulfate precipitation, the optimized process increased protein-binding capacity of 1 ml chromatographic gel from 1 mg to 20 mg, the protein recovery rate of unit volume fermentation fluid from 10% to 90%, and the amount of protein purified out of 400 ml fermentation fluid from 3 mg to 15 mg. The mean 50% inhibitory concentration of crude products decreased from 0.077 99 μg/ml to 0.045 69 μg/ml.Conclusion Adding ammonium sulfate in renaturation solution can effectively reduce polymers.