镍离子亲和层析介质的制备及其用于组氨酸标记蛋白质的纯化  

Preparation of Ni Affinity Chromatographic Adsorbent and Its Application on Purification of His-Tagged Protein

作　　者：夏海锋[1] 张显[1] 金雄华[1] 刘婷婷[1] 郑志永[1] 饶志明[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《江南大学学报（自然科学版）》2010年第6期685-689,共5页Joural of Jiangnan University (Natural Science Edition)　

基　　金：国家自然科学基金项目(30970056);国家863计划项目(2007AA02Z207);江苏省自然科学基金项目(BK2009516);江南大学自主科研基金项目(JUSRP11012)

摘　　要：以交联琼脂糖微球为基质,通过环氧氯丙烷活化,偶联亚胺二乙酸并螯合N i2+,制备得到一种镍离子亲和层析介质。结果发现,在强碱条件下环氧活化率达到了38.0μmol/mL,最终N i2+配基密度达到了30.9μmol/mL,偶联效率为81.3%。利用得到的镍离子亲和介质对基因工程大肠杆菌表达的组氨酸标记3-羟基丁酮还原酶进行了一步层析纯化,酶活回收率达到了58.8%,纯化倍数为2.1,蛋白纯度在85%左右,纯化效果与常用商品介质基本相当。Ni affinity adsorbent was prepared by epichlorohydrin activation,iminodiacetic acid（IDA） coupling and then chelated with Ni2＋ based on the crosslinked agrose particles.As a result,the epoxy group on the matrix reached 38.0 μmol/mL,the Ni2＋ ligands density reached 30.9 μmol/mL,and the coupling efficiency was 81.3%.The adsorbents were used to purify the his-tagged 2,3-butanediol dehydrogenase which is expressed by E.coli.It was found that the enzyme recovery reached 58.8%,the purification factor reached 2.1 and the purity of protein was about 85%.The performance of prepared adsorbents was almost equal to that of Ni Sepharose 6FF.

关 键 词：琼脂糖微球 镍离子亲和层析 组氨酸标记 克隆表达 纯化 

分 类 号：O647.316.2[理学—物理化学]