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 α7nAChR 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 α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR 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 α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design.
Highlights
Often has multiple subtypes that form numerous functional distinct receptors
After testing multiple E. coli strains, we found Rosetta(DE3)pLysS to be most suitable for ␣7nAChR expression
For the choice of detergents, we found that n-dodecylphosphocholine (DPC) was most effective for extracting ␣7nAChR from cell membranes
Summary
Often has multiple subtypes that form numerous functional distinct receptors. Among the human Cys-loop receptors, highresolution structures have been obtained for only the 3 GABAA and ␣3 glycine receptors [6, 7]. Available structures for Cys-loop receptors were obtained from proteins expressed in mammalian [6, 8] and insect (7, 9 –11, 22) cell lines. The native pathway for ␣7nAChR expression in mammalian cells involves subcellular trafficking through multiple subcellular compartments, where specific chaperone proteins and post-translational modifications aid folding and assembly.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
