【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200μL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。更多还原

【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 Co 2+,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.更多还原