@article{APS9374,
author = {Hao Cheng and Chen Fan and Si-wei Zhang and Zhong-shan Wu and Zhi-cheng Cui and Karsten Melcher and Cheng-hai Zhang and Yi Jiang and Yao Cong and H Eric Xu},
title = {Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system},
journal = {Acta Pharmacologica Sinica},
volume = {36},
number = {8},
year = {2017},
keywords = {},
abstract = {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 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 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 °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 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.},
issn = {1745-7254}, url = {http://www.chinaphar.com/article/view/9374}
}