tailieunhanh - Báo cáo Y học: Refolding of the Escherichia coli expressed extracellular domain of a7 nicotinic acetylcholine receptor Cys116 mutation diminishes aggregation and stabilizes the b structure

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 a7 subunit of the homo-oligomeric a7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat a7 ECDs (amino-acid residues 1–210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein. | Eur. J. Biochem. 269 2801-2809 2002 FEBS 2002 doi Refolding of the Escherichia coli expressed extracellular domain of a7 nicotinic acetylcholine receptor Cys116 mutation diminishes aggregation and stabilizes the b structure Victor I. Tsetlin1 Natalia I. Dergousova1 Ekaterina A. Azeeva1 Elena V. Kryukova1 Irina A. Kudelina1 Elena D. Shibanova1 Igor E. Kasheverov1 and Christoph Methfessel2 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences Moscow Russia 2Central Research Biophysics Bayer Leverkusen Germany 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 a7 subunit of the homo-oligomeric a7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat a7 ECDs amino-acid residues w 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-trans-ferase 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-a7- 1-208 protein in the presence of SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the a7 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 a-bungarotoxin from Kd w 300 to 150 dm . Gel-permeation HPLC was used to isolate the monomeric form of the GST-a7- 1-208 protein and its .