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ANKIB1的克隆、原核表达和纯化

, PP. 189-194

Keywords: ANKIB1基因,克隆,表达,纯化

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Abstract:

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0.1mmol/LIPTG在15℃低温条件下诱导10h,成功表达了分子量高达148.5kD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。

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