Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor
Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor
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DOI：
10.1016/j.pep.2006.12.003
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发表时间：
2007-05-01
影响因子：
1.6
通讯作者：
Gawrisch, Klaus
中科院分区：
文献类型：
--
作者：
Yeliseev, Alexel;Zoubak, Lioudmila;Gawrisch, Klaus
人外周大麻素受体 (CB2) 表达为与麦芽糖结合蛋白（位于 N 末端）、硫氧还蛋白 A（位于 C 末端）和两个小亲和标签（链球菌标签和多组氨酸标签）的融合体。重组受体在大肠杆菌 BL21(DE3) 细胞中的表达水平取决于表达构建体中标签的位置和类型，并且高达每升细菌培养物 1-2 mg。重组受体具有配体结合能力，并在体外偶联测定中激活同源 G 蛋白。通过 Ni-NTA 树脂上的固定化金属亲和层析纯化融合 CB2-125 蛋白。通过烟草蚀纹病毒 (Tev) 蛋白酶处理，从融合物中去除麦芽糖结合蛋白、硫氧还蛋白和十组氨酸标签。通过在 StrepTactin 树脂上进行亲和层析，将所得含有 N 末端 Strep 标签的 CB2 纯化至同质性超过 90%。圆二色光谱表明纯化的重组蛋白的α螺旋含量接近54%。表达和纯化方案允许生产大量（毫克）功能性外周大麻素受体，适合后续结构表征。重构实验的初步结果表明CB2保留了其配体结合特性。 (c) 2006 Elsevier Inc. 保留所有权利。
The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2 mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an alpha-helical content of the purified recombinant protein of similar to 54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties. (c) 2006 Elsevier Inc. All rights reserved.