壳聚糖固定化金属离子亲和层析法对重组乙醛脱氢酶的纯化

摘要

以壳聚糖为载体,甲醛为预交联剂,戊二醛为交联剂,用环氧氯丙烷法进行载体活化,乙二胺为螯合配基,制备了壳聚糖Zn2＋固定化亲和层析填料。利用该填料对重组ALDH粗酶液进行了纯化,经分段盐析和亲和层析,纯化倍数达到9.23,回收率达到29%。结果表明,制备的壳聚糖Zn2＋固定化亲和层析填料,能够用于带有组氨酸标签重组蛋白的快速分离与纯化。该方法对重组ALDH的纯化倍数和回收率均高于目前的其他分离纯化方法。%The immobilized metal affinity chromatography（IMAC） matrixes were successfully prepared with chitosan as support,formaldehyde as precrosslinking agent,glutaraldehyde as crosslinking agent,3-Chloro-1,2-epoxypropane as activated agent,ethylenediamine as chelating ligand and Zn2＋ as center ions.Then it was used to separate and purify aldehyde dehydrogenase（ALDH） tagged with 6×His.The yield of ALDH was 29.0 % and the purification fold reached 9.23 times by ammonium sulfate precipitation and immobilized metal affinity chromatography.It proved that the prepared high-efficiency immobilized metal affinity chromatography matrixes could be used to rapidly separate and purify the His-tagged recombinant protein and the purification fold and recovery rate purified by chitosan Zn2＋ immobilized affinity chromatography were higher than any other methods reported currently.