Nanodiscs Allow Phage Display Selection for Ligands to Non-Linear Epitopes on Membrane Proteins

Abstract

In this work, we exploited a method that uses polytopic membrane proteins as targets for phage display selections. Membrane proteins represent the largest class of drug targets and drug discovery is mostly based on the identification of ligands binding to target molecules. The screening of a phage display library for ligands against membrane proteins is typically hindered by the requirement of these proteins for a membrane environment, which is necessary to retain correct folding and epitope formation. Especially in proteins with multiple transmembrane domains, epitopes often are non-linear and consist of a combination of loops between transmembrane stretches of the proteins. Here, we have used bacteriorhodopsin (bR) as a model of polytopic membrane protein, assembled into nanoscale phospholipid bilayers, so called nanodiscs, to screen a phage display library for potential ligands. Nanodiscs provide a native-like environment to membrane proteins and thus selection of ligands can take place in a near physiological state. Screening a 12-mer phage display peptide library against bR nanodiscs led to the isolation of phage clones binding specifically to bR. We were further able to identify the binding site of selected phage clones proving that the clones bind to extramembranous, non-linear epitopes of bR. Thus, nanodiscs provide a suitable and general tool that allows screening of a phage display library against membrane proteins in a near native environment.

Nanodiscs in Membrane Biochemistry and Biophysics

“…477 bR was expressed in cell-free system and incorporated in Nanodiscs assembled with DMPC lipids. The samples as well as a control of empty Nanodiscs were immobilized in 96-well microtiter plates for screening with the library of random peptide 12-mers.…”

Formation of stable nanodiscs by bihelical apolipoprotein A‐I mimetic peptide

“…Nanodiscs consisting of discoidal phospholipid bilayer surrounded by a scaffold protein are used as membrane platforms to solubilize a variety of membrane proteins into their phospholipid bilayer environments . Scaffold protein has amphipathic α ‐helices to cover hydrocarbon chains of phospholipids with a double belt structure .…”

Applications and Advances in Bioelectronic Noses for Odour Sensing

“…Given that only the wet protein samples are functionally active, it is challenging to keep the ORs constitutively active under dry conditions. As a promising candidate to figure out this problem, nanodiscs can be regarded because they possess the ability to provide a native-like environment to membrane proteins and thus binding of odorants onto ORs can occur in a near physiological state [ 19 , 111 ]. Along with advances in nanotechnology, the integration of ORs with nanostructured devices enables the bioelectronic nose to have high sensitivity and specificity.…”