金属螯合亲和层析介质用于六聚组氨酸融合蛋白的纯化研究

Preparation of Metal Chelate Affinity Chromatographic Medium and Its Application in the Purification of 6×Histidine-tagged Protein

摘要:

以Sepharose CL-6B为载体，环氧氯丙烷为活化剂，羧甲基天冬氨酸（CM-Asp）为螯合配基制备载有Co2+的金属螯合亲和层析介质Co-CM-Asp-Sepharose，并将其用于六聚组氨酸融合蛋白的纯化研究。对纯化200 μL细胞裂解液中靶蛋白所需Co-CM-Asp-Sepharose介质用量，Co-CM-Asp-Sepharose与细胞裂解液的孵育时间，介质清洗条件及靶蛋白洗脱时所需咪唑浓度等进行了优化。比较了Co-CM-Asp-Sepharose与Ni-NTA-Agarose(Qiagen公司)两种螯合介质对融合蛋白的纯化效果，开展了从5mL细胞裂解液中放大规模纯化融合蛋白的研究，并通过Bradford法测定了Co-CM-Asp-Sepharose对CD155D1蛋白的纯化量。结果表明：对200μL细胞裂解液纯化体系， Co-CM-Asp-Sepharose（50%悬浮液）的优选体积为60μL，最佳孵育时间为30min，洗脱液最佳咪唑浓度为200mmol/L，纯化得到融合蛋白的量约为200μg。介质用量放大为1.5mL（50%悬浮液）对CD155D1蛋白的纯化量可达4.6mg。与商品化Ni-NTA-Agarose相比，本介质具有选择性好，清洗条件简单，得到的靶蛋白纯度高等优点。

Abstract:

Using Sepharose CL-6B as support, 3-Chloro-1,2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co²⁺, named Co-CM-Asp-Sepharose, was prepared and used to purify 6×His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200μL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60μL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200μL of lysate, the optimal incubation time of medium and lysate was 30min, the optimal imidazole concentration in the eluting buffer was 200mmol/L, and 200μg of fusion protein was obtained. In a big scale experiment, 4.6mg of fusion protein was obtained from 5 mL of lysate using 1.5mL of Co-CM-Asp-Sepharose(50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.