A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain - Institut Pasteur Access content directly
Journal Articles Proceedings of the National Academy of Sciences of the United States of America Year : 2001

A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain

Abstract

We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the alpha7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the alpha7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist alpha-bungarotoxin with 2.5 microM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.

Domains

Neurobiology

Dates and versions

pasteur-01659650 , version 1 (08-12-2017)

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Markus Fischer, Pierre-Jean Corringer, Karin Schott, Adelbert Bacher, Jean-Pierre Changeux. A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98 (6), pp.3567 - 3570. ⟨10.1073/pnas.041594798⟩. ⟨pasteur-01659650⟩
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