Date Published: May 15, 2018
Author(s): 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.
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.
Encoded by almost one third of archaean, bacterial and eukaryote DNA , membrane proteins represent vital cellular components for all lifeforms. Given their essential roles towards sustaining life, it is unsurprising that membrane protein pathology accounts for a large number of debilitating conditions, such as Bartter syndrome, cardiac arrhythmia and hypertension, congenital deafness and myotonia, cystic fibrosis, epilepsy, osteoporosis and polycystic kidney disease [2,3]. Their significant therapeutic importance has led to many of today’s pharmaceuticals targeting membrane proteins [4,5], with the largest class being the G-protein coupled receptors (GPCRs). However, the discovery of novel membrane protein binders – including antibody-based medicines that have emerged throughout the last decade  – is not without issue. The high-throughput protocols employed by the drug discovery industry demand high levels of expression and purity from their designated screening targets, yet few membrane proteins can be expressed at high level within their native membranes. Moreover, the general study of membrane proteins is further complicated by the fact that advanced research techniques (e.g., kinetic and ligand-binding characterisation, nuclear magnetic resonance (NMR) or X-ray crystallography) cannot always be directly performed on crude cellular membranes and thus require generous amounts of recombinant protein of high purity and conformational stability, therefore becoming reliant on identifying optimised expression platforms, a suitable detergent for the solubilisation and, more often than not, demanding high-throughput methodologies [, , ].
A particular screening method that finds increasing use in both the pharmaceutical and biotechnological fields is that based on phage display. In principle, phage display screening can be performed using detergent-solubilised membrane protein targets. However, detergent-based screening methods come with their own drawbacks, including target denaturation over long periods of storage or the inability to solubilise certain membrane protein classes due to monomer packing defects resulting in their aggregation and, ultimately, inactivation following purification . A final problem with phage display screening against membrane protein targets is the immobilisation strategy. Globular proteins are typically adsorbed onto polymeric or streptavidin coated surfaces. However, detergent solubilisation of membrane proteins and the aforementioned problems with tagging can impede these strategies. Several alternative strategies have been described, such as whole cell panning  or embedding the proteins into nanodiscs . Whole cells provide a very complex environment for screening while nanodiscs still require the membrane proteins to be purified to a high yield and purity.
In conclusion, we have demonstrated that SSBLMs represent a promising platform for screening assays, where membrane protein targets are displayed embedded within a native-like lipid environment. We have also demonstrated that SSBLMs can be quickly and easily formed using purified proteins reconstituted into liposomes, as well as by directly employing crude membrane extracts. Here, the potential suitability of the SSBLM platform towards high-affinity antibody binding was established using ELISAs and cryo-EM imaging, where the former technique showed that non-specific binding can be minimised through suitable assay modifications. We are now investigating whether the SSBLM can be applied in phage display screening.