One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography
One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography
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DOI：
10.1016/s1046-5928(02)00614-9
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发表时间：
2003-02-01
影响因子：
1.6
通讯作者：
Christopoulos, TK
中科院分区：
文献类型：
--
作者：
Glynou, K;Ioannou, PC;Christopoulos, TK
Apoaequorin的N-末端融合了一个六组氨酸标签。构建了编码融合蛋白的载体，并将其转化到大肠杆菌JM109细胞中。在tac启动子的控制下，Apoaequorin基因过表达。然而，研究发现，大部分蛋白质以包涵体的形式存在。包涵体用尿素溶解，(His)(6)-Apoaquorin用Ni2+-氮三乙酸琼脂糖亲和层析纯化和复性。经SDS-PAGE分析，纯度大于80%。从50ml的细菌培养中得到0.7-1 mg的麦冬苦素。纯化后的水飞蓟素在加入钙离子后的发光动力学符合典型的商品水杨酸发光动力学。纯化后的水飞蓟素的发光与其浓度呈线性关系，其变化范围超过六个数量级。低至0.5阿托摩尔的精制木犀草素的信噪比为1.8。(C)2002年埃尔塞维尔科学公司(美国)。版权所有。
A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni2+-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca2+ was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8. (C) 2002 Elsevier Science (USA). All rights reserved.