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Updated: Apr 10, 2026
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Published on: April 9, 2018
Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam
Hao Cheng1, Chen Fan2, Si-wei Zhang3
11] VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China.
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.
