title: High yield and efficient expression and purification of the human 5-HT3A receptor.
abstract: OBJECTIVE
  To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies.
  
  
  METHODS
  Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM).
  
  
  RESULTS
  The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure.
  
  
  CONCLUSIONS
  With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor.
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China [2] 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: 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: 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: 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: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors.forename: Karsten
authors.initials: K
authors.lastname: Melcher
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Jiang
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: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Hua-Liang
authors.initials: HL
authors.lastname: Jiang
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: Yao
authors.initials: Y
authors.lastname: Cong
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, 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; D016367:Baculoviridae / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D018076:DNA, Complementary; C509409:HTR3A protein, human; D044406:Receptors, Serotonin, 5-HT3