title: Purification and functional characterization of a truncated human α4β2 nicotinic acetylcholine receptor.
abstract: Nicotinic acetylcholine receptors (nAChR) are abundant in the brain and are essential in cognitive function, learning and memory. Previous efforts on α4β2 nAChR had been focused on functional and pharmacological characterization, where high expression yield is not essential. For structural studies though, large amounts of pure protein is important but heterologous overexpression of membrane proteins can be a burdensome task, especially if high amounts are required. In the current study, a truncated mutant of the human α4β2 nAChR was designed in order to improve expression and solubility and to obtain material suitable for high resolution structural studies. We showed that the wild type α4β2 nAChR presented low expression and solubilization yield both of which were improved with the truncated construct. The truncated nAChR showed similar binding profile to the wild type, was purified by a two-step chromatography and isolated in high purity and adequate quantity.
authors.affiliation: Department of Neurobiology, Hellenic Pasteur Institute, 127 Vass. Sofias Avenue, GR11521 Athens, Greece.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Kouvatsos
authors.affiliation: Department of Pharmacy, University of Patras, GR26500 Patras, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Niarchos
authors.affiliation: Department of Neurobiology, Hellenic Pasteur Institute, 127 Vass. Sofias Avenue, GR11521 Athens, Greece.
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Zisimopoulou
authors.affiliation: Department of Agricultural Biotechnology, Agricultural University of Athens, Holy Street 75, GR11855 Athens, Greece.
authors.forename: Elias
authors.initials: E
authors.lastname: Eliopoulos
authors.affiliation: Department of Pharmacy, University of Patras, GR26500 Patras, Greece. Electronic address: kpoulas@upatras.gr.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Poulas
authors.affiliation: Department of Neurobiology, Hellenic Pasteur Institute, 127 Vass. Sofias Avenue, GR11521 Athens, Greece; Department of Pharmacy, University of Patras, GR26500 Patras, Greece. Electronic address: tzartos@pasteur.gr.
authors.forename: Socrates
authors.initials: S
authors.lastname: Tzartos
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D008024:Ligands; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C082748:epibatidine
keywords: Baculovirus expression system; Ion channel receptor; Membrane protein; Protein engineering; α4β2 nAChR