title: Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor.
abstract: Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA 98, 3567-3570] and glutathione S-transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem. 276, 15810-15815]. However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm). Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. vits@ibch.ru
authors.forename: Victor I
authors.initials: VI
authors.lastname: Tsetlin
authors.forename: Natalia I
authors.initials: NI
authors.lastname: Dergousova
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Azeeva
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kryukova
authors.forename: Irina A
authors.initials: IA
authors.lastname: Kudelina
authors.forename: Elena D
authors.initials: ED
authors.lastname: Shibanova
authors.forename: Igor E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: Christoph
authors.initials: C
authors.lastname: Methfessel
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D003039:Cobra Neurotoxin Proteins / Q000378:metabolism; D009154:Mutation; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C579165:Chrna7 protein, rat; D003039:Cobra Neurotoxin Proteins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C025154:alpha-cobratoxin