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.

Figure 1

Design of the fusion proteins between MalE and the extracellular domain of α7 nAChR subunit. The white box corresponds to the MalE protein sequence, black box to the linker segment, dark gray box to the mature rat α7 ECD sequence, and light gray boxes to the α7 transmembrane segments. Dashed lines correspond to the sites where the stop codon was introduced.

Figure 2

Coomassie-stained SDS/PAGE of the Mal-ent-α7 1–196 fusion protein. Lanes A and D, marker proteins; lane B, crude lysate; lane C, purified fraction of the fusion protein after affinity chromatography.

Figure 3

CD spectra of MalE (dashed line) and Mal-ent-α7 1–196 (solid line) in 25 mM K/Na-phosphate, pH 7.2.

Figure 4

Binding of ¹²⁵I-αBgtx on MalE-ent-α7 1–196 (first bar), and in the presence of various nicotinic competitors. Values represent the mean of three experiments performed in duplicate.

Figure 5

Typical Scatchard experiment for the binding of ¹²⁵I-αBgtx on Mal-ent-α7 1–196.