Spherical-supported membranes as platforms for screening against membrane protein targets

V. Vasilca, A. Sadeghpour, S. Rawson, L.E. Hawke, S.A. Baldwin, T. Wilkinson, D. Bannister, V.L.G. Postis, M. Rappolt, S.P. Muench, L.J.C. Jeuken (Lead / Corresponding author)

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Abstract

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes (“SSBLM”). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.
Original languageUndefined/Unknown
Pages (from-to)58-65
Number of pages8
JournalAnalytical Biochemistry
Volume549
DOIs
Publication statusPublished - 15 May 2018

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