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.
Highlights
Encoded by almost one third of archaean, bacterial and eukaryote DNA [1], membrane proteins represent vital cellular components for all lifeforms
Comparing individual experiments shows that this is due to varying amounts of target proteins incorporated from the crude membrane extract into the spherical-supported bilayer lipid membranes (SSBLM), as errors are similar to those in Fig. 4 when Enzyme-linked immunosorbent assay (ELISA) are performed on the same SSBLM batch. We propose that this is due to the need to mix crude membrane extracts with POPC liposomes, which might result in slight variations in incorporation of membrane proteins into the SSBLMs, even when fixed ratios of POPC versus crude membrane extracts are used
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
Summary
Encoded by almost one third of archaean, bacterial and eukaryote DNA [1], 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]. SAXS patterns were analysed using an extended core shell model [30] This model provides the scattering from a spherical core (silica) and six concentric shell structures: five used to build up the POPC bilayer and one for the space between the silica sphere and the lipid bilayer (i.e. the intermediate water layer). In order to get statistically reliable data and increased signal to noise ratio, we acquired 12 scattering frames each with 30 min exposure time and computed the average curve for the further analysis
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