Functional Human ?7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

ABSTRACT: Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human ?7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified ?7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native ?7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to ?7nAChR. Moreover, the purified ?7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the ?7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human ?7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design.

SUBMITTER: Tillman TS 

PROVIDER: S-EPMC5000075 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature