title: Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1] 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.
authors.forename: Hao
authors.initials: H
authors.lastname: Cheng
authors.affiliation: 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.
authors.forename: Chen
authors.initials: C
authors.lastname: Fan
authors.affiliation: 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.
authors.forename: Si-wei
authors.initials: SW
authors.lastname: Zhang
authors.affiliation: 1] 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] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Zhong-shan
authors.initials: ZS
authors.lastname: Wu
authors.affiliation: 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.
authors.forename: Zhi-cheng
authors.initials: ZC
authors.lastname: Cui
authors.affiliation: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors.forename: Karsten
authors.initials: K
authors.lastname: Melcher
authors.affiliation: 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.
authors.forename: Cheng-hai
authors.initials: CH
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Yi
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: 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.
authors.forename: Yao
authors.initials: Y
authors.lastname: Cong
authors.affiliation: 1] 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] Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors.forename: H Eric
authors.initials: HE
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D016415:Sequence Alignment; D013696:Temperature; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
chemical_list: D064569:alpha7 Nicotinic Acetylcholine Receptor