ANKIB1的克隆、原核表达和纯化  

Cloning , Prokaryotic Expression and Purification of ANKIB1

作　　者：谢云飞[1] 杨子善[1] 马振玲[1] 解博红[2] 刘世饶[1] 

机构地区：[1]新乡医学院生命科学技术学院河南省遗传性疾病与分子靶向药物高校重点实验室培育基地,新乡453003 [2]新乡医学院基础医学院,新乡453003

出　　处：《生物技术通报》2013年第7期189-194,共6页Biotechnology Bulletin

基　　金：新乡医学院博士科研启动基金项目(100792)

摘　　要：含锚蛋白重复序列和IBR结构域蛋白1(ANKIB1)含有RING-IBR-RING结构域、ANK模体和泛素结合模体,可能具有E3泛素连接酶活性和特殊的催化机制,但其功能目前所知甚少。采用巢式PCR技术分段扩增了人ANKIB1基因的cDNA,并通过基因重组构建了全长ANKIB1基因的完整编码序列。将ANKIB1连接到原核表达载体pGEX-5X-3中,用于表达GST-ANKIB1融合蛋白。在含有RING型蛋白稳定剂ZnCl2的培养基中,转化pGEX-5X-ANKIB1的大肠杆菌BL21(DE3)经过0.1 mmol/L IPTG在15℃低温条件下诱导10 h,成功表达了分子量高达148.5 kD的可溶性GST-ANKIB1蛋白。利用谷胱甘肽琼脂糖凝胶亲和层析,纯化了GST-ANKIB1融合蛋白。Ankyrin repeat and IBR domain-containing protein 1 （ ANKIB1 ） contains RING-IBR-RING domain, ankyrin repeat （ ANK ） motif and ubiquitin-interacting motif （ UIM ） . It is likely that ANKIB1 has E3 ubiquitin ligase activity and specific catalytic mechanism. However, the function of ANKIB1 remains unclear. For the first time we cloned human ANKIB1 eDNA using nestled PCR and segmental amplication. Full length of ANKIB1 coding DNA sequence was constructed by gene recombination technology. The cDNA was ligated into prokaryotic expression vector pGEX-5X-3, and the recombinant plasmid pGEX-5X-ANKIB1 was transformed into E.coli BL21 （ DE3 ） . Using culture medium cotaining 200 mmol/L ZnC12, the stabilizer for RING-type protein, expression of soluble GST-ANKIB1 fusion protein with a molecular weight of 148.5 kD was successfully induced by 0.1 mmol/L IPTG at 15℃ for 10 h. The recombinant protein was purified by affinity chromatography using Glutathione Sepharose 4B.

关 键 词：ANKIB1基因 克隆 表达 纯化 

分 类 号：Q78[生物学—分子生物学]