A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain.
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.
Fischer
M
M
Lehrstuhl fur Organische Chemie und Biochemie der Technischen Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Corringer
P J
PJ
Schott
K
K
Bacher
A
A
Changeux
J P
JP
D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D018733:Nicotinic Antagonists / Q000378:metabolism; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D064569:alpha7 Nicotinic Acetylcholine Receptor
D018528:ATP-Binding Cassette Transporters; D002038:Bungarotoxins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C461599:maltose transport system, E coli