title: Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.
abstract: Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.
authors.affiliation: Pfizer Inc., Pfizer Global Research and Development, Groton, CT 06340, United States. shenping.liu@pfizer.com
authors.forename: Shenping
authors.initials: S
authors.lastname: Liu
authors.forename: Merrill S
authors.initials: MS
authors.lastname: Babcock
authors.forename: Jacob
authors.initials: J
authors.lastname: Bode
authors.forename: Jeanne S
authors.initials: JS
authors.lastname: Chang
authors.forename: H David
authors.initials: HD
authors.lastname: Fischer
authors.forename: Robert L
authors.initials: RL
authors.lastname: Garlick
authors.forename: Gurnam S
authors.initials: GS
authors.lastname: Gill
authors.forename: Eric T
authors.initials: ET
authors.lastname: Lund
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Margolis
authors.forename: W Rodney
authors.initials: WR
authors.lastname: Mathews
authors.forename: Bruce N
authors.initials: BN
authors.lastname: Rogers
authors.forename: Mark
authors.initials: M
authors.lastname: Wolfe
authors.forename: Vince
authors.initials: V
authors.lastname: Groppi
authors.forename: Eric T
authors.initials: ET
authors.lastname: Baldwin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000378:metabolism; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C579167:Chrna7 protein, human; C579166:Chrna7 protein, mouse; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor