Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system
文献类型：期刊论文
| 作者 | Cheng Hao3; Fan Chen2; Zhang Siwei3; Wu Zhongshan3; Cui Zhicheng2; Melcher Karsten1; Zhang Chenghai3; Jiang Yi3 ; Cong Yao2; Eric Xu H3
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| 刊名 | 中国药理学报：英文版
 |
| 出版日期 | 2015 |
| 卷号 | 000期号:008页码:1013 |
| 关键词 | 烟碱型乙酰胆碱受体 哺乳动物细胞 层析纯化 表达系统 结晶 SDS-PAGE 热稳定性分析 麦芽糖结合蛋白 |
| ISSN号 | 1671-4083 |
| 英文摘要 | Aim： To report our methods for expression and purification of （α7 nicotinic acetylcholine receptor （α7-nAChR）, a ligand-gated pentameric ion channel and an important drug target. Methods： α7-nAChRs of lO different species were cloned into an inducible BacMam vector with aft N-terminal tag of a tandem maltose- binding protein （MBP） and a TEV cleavage site. This α7-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 α7-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 α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60℃, 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 α7-nAChR channel. Conclusion： We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. |
| 语种 | 英语 |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/294098]  |
| 专题 | 中国科学院上海药物研究所 |
| 作者单位 | 1.Laboratory of Structural Sciences, Van Andel Research Institute 2.中国科学院上海生命科学研究院 3.中国科学院上海药物研究所 |
| 推荐引用方式 GB/T 7714 | Cheng Hao,Fan Chen,Zhang Siwei,et al. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system[J]. 中国药理学报：英文版,2015,000(008):1013. |
| APA | Cheng Hao.,Fan Chen.,Zhang Siwei.,Wu Zhongshan.,Cui Zhicheng.,...&Eric Xu H.(2015).Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.中国药理学报：英文版,000(008),1013. |
| MLA | Cheng Hao,et al."Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system".中国药理学报：英文版 000.008(2015):1013. |
入库方式： OAI收割
来源：上海药物研究所
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