Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system
Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system
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使用 BacMam 表达系统从哺乳动物细胞中结晶规模纯化 α7 烟碱乙酰胆碱受体
DOI：
10.1038/aps.2015.34
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发表时间：
2015-08-01
影响因子：
8.2
通讯作者：
Xu, H. Eric
中科院分区：
文献类型：
--
作者：
Cheng, Hao;Fan, Chen;Xu, H. Eric
目的：报道配体门控五聚体离子通道和重要药物靶点α7烟碱乙酰胆碱受体(α7 nAChR)的表达和纯化方法：将10种不同物种的α7 nAChR克隆到带有串联麦芽糖结合蛋白(MBP)N端标签和TEV裂解位点的诱导型BacMam载体中。该α7-nAChR融合受体在哺乳动物HEK293F细胞中表达，并经Western印迹检测。这个表情被放大到了升。用直链淀粉树脂和体积排阻层析法对受体进行纯化。用SDS-PAGE凝胶、热稳定性分析和负染电子显微镜(EM)对纯化的受体进行了质量评价。通过末端截断和定点突变优化了表达结构。结果：表达筛选结果表明点滴带羽衣藻α7-nAChR的表达水平最高。融合受体主要在细胞表面表达，一步亲和层析法可有效纯化融合受体。从一升细胞培养中纯化了一到两毫克的优化的α7-nAChR表达载体。纯化的α7-nAChR样品表现出很高的热稳定性，其Tm为60℃，拮抗剂结合后热稳定性进一步增强，但在激动剂存在时热稳定性降低。结论：我们建立了α7-nAChR结晶度的表达和纯化方法，为利用X射线结晶学或单颗粒低温电子显微镜研究高分辨结构奠定了基础。
Aim: To report our methods for expression and purification of alpha 7 nicotinic acetylcholine receptor (alpha 7-nAChR), a ligand-gated pentameric ion channel and an important drug target.Methods: alpha 7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This alpha 7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.Results: Expression screening revealed that alpha 7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized alpha 7-nAChR expression construct were purified from one liter of cell culture. The purified alpha 7-nAChR samples displayed high thermal stability with a Tm of 60 degrees C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the alpha 7-nAChR channel.Conclusion: We have established methods for crystallization scale expression and purification of alpha 7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.