纯化组氨酸标签蛋白金属螯合亲和色谱填料的制备与性能

Preparation of metal chelate affinity chromatographic material and its application in purification of histidine-tagged protein

以琼脂糖(agarose)凝胶的珠状产品SepharoseCl-6B为基质,以环氧氯丙烷为活化剂,将天冬氨酸固定在琼脂糖凝胶微球上,在经与溴乙酸修饰后,最终得到含有羧甲基天冬氨酸结构的琼脂糖凝胶微球.以凝胶微球负载的多羧基为配体分别与Co2+及Ni2+配位分别得到两种金属螯合亲和层析介质.依照上述方法制备了具有不同负载羧基含量的色谱介质.以六聚组氨酸融合蛋白为分离样品,研究了所制备的色谱分离介质的分离纯化性能,并与商品化色谱分离介质Agarose-NTA-Ni进行了比较.结果表明,目标介质蛋白结合容量大,与蛋白结合快、易洗脱、选择性高,金属离子不易脱落.

The metal chelate affinity chromatographic materials were successfully prepared with Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, carboxyrnethylated aspartate （CM-Asp） as chelating ligand and Co^2＋ and Ni^2＋ as center ions. The metal chelate affinity chromatographic materials were characterized using acid-base titration, IR, AAS, SEM and EDX, respectirely. The results indicate that metal ions are chelated to the carboxyl groups immobilized onto agarose to form multidentate complexes. The results from purification of 6× His recombination protein release that the efficiency in purification of tagged protein derived from the affinity chromatographic material containing Co^2＋ is higher than that containing Ni^2＋. Additionally, the prepared affinity chromatographic material containing Co^2 ＋ is more efficient in purification of 6 ×His recombination protein than a commercially available affinity chromatographic material, Agarose-NTA-Ni.