Vasilca, V, Sadeghpour, A, Rawson, S orcid.org/0000-0002-2973-2630 et al. (8 more authors) (2018) Spherical-supported membranes as platforms for screening against membrane protein targets. Analytical Biochemistry, 549. pp. 58-65. ISSN 0003-2697
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 13 Mar 2018 15:40 |
Last Modified: | 25 Jun 2023 21:16 |
Status: | Published |
Publisher: | Elsevier Masson |
Identification Number: | 10.1016/j.ab.2018.03.006 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:128471 |