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Crystallization scale purification of alpha 7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

文献类型：期刊论文

作者Cheng, Hao1; Fan, Chen4; Zhang, Si-wei1; Wu, Zhong-shan1,3; Cui, Zhi-cheng4; Melcher, Karsten2; Zhang, Cheng-hai1; Jiang, Yi1; Cong, Yao4; Xu, H. Eric1,2
刊名ACTA PHARMACOLOGICA SINICA
出版日期2015-08
卷号36期号:8页码:1013-1023
关键词alpha 7-nAChR membrane protein receptor expression receptor purification BacMam HEK293F cells
ISSN号1671-4083
DOI10.1038/aps.2015.34
文献子类Article
英文摘要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.
WOS关键词GATED ION-CHANNEL ; X-RAY-STRUCTURE ; MEMBRANE-PROTEINS ; 5-HT3 RECEPTOR ; LIGAND-BINDING ; RESOLUTION ; BRAIN ; LEVEL ; LOOP
资助项目Jay and Betty Van Andel Foundation[00000000] ; Ministry of Science and Technology (China)[2012ZX09301001] ; Ministry of Science and Technology (China)[2012CB910403] ; Ministry of Science and Technology (China)[2013CB910600] ; Ministry of Science and Technology (China)[XDB08020303] ; Ministry of Science and Technology (China)[2013ZX09507001] ; Ministry of Science and Technology (China)[XDB08030201] ; Ministry of Science and Technology (China)[13JC1406300] ; Ministry of Science and Technology (China)[31222016] ; Ministry of Science and Technology (China)[31270771] ; Shanghai Science and Technology Committee[13JC1406300] ; Shanghai Science and Technology Committee[13ZR1447600]
WOS研究方向Chemistry ; Pharmacology & Pharmacy
语种英语
WOS记录号WOS:000358934000012
出版者ACTA PHARMACOLOGICA SINICA
源URL[http://119.78.100.183/handle/2S10ELR8/276453]  
专题药物靶标结构与功能中心
通讯作者Xu, H. Eric
作者单位1.Chinese Acad Sci, Shanghai Inst Mat Med, CAS Key Lab Receptor Res, Ctr Struct & Funct Drug Targets,VARI SIMM Ctr, Shanghai 201203, Peoples R China;
2.Van Andel Res Inst, Lab Struct Sci, Grand Rapids, MI 49503 USA
3.Huazhong Univ Sci & Technol, Wuhan Natl Lab Optoelect, Britton Chance Ctr Biomed Photon, Wuhan 430074, Peoples R China;
4.Chinese Acad Sci, Natl Ctr Prot Sci Shanghai, State Key Lab Mol Biol, Inst Biochem & Cell Biol,Shanghai Inst Biol Sci, Shanghai 201210, Peoples R China;
推荐引用方式
GB/T 7714		 Cheng, Hao,Fan, Chen,Zhang, Si-wei,et al. Crystallization scale purification of alpha 7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system[J]. ACTA PHARMACOLOGICA SINICA,2015,36(8):1013-1023.
APA Cheng, Hao.,Fan, Chen.,Zhang, Si-wei.,Wu, Zhong-shan.,Cui, Zhi-cheng.,...&Xu, H. Eric.(2015).Crystallization scale purification of alpha 7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.ACTA PHARMACOLOGICA SINICA,36(8),1013-1023.
MLA Cheng, Hao,et al."Crystallization scale purification of alpha 7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system".ACTA PHARMACOLOGICA SINICA 36.8(2015):1013-1023.

入库方式： OAI收割

来源：上海药物研究所

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