Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Jülich, Germany ; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
PLoS One. 2013 Sep 9;8(9):e72272. doi: 10.1371/journal.pone.0072272. eCollection 2013.
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.
在这项工作中，我们利用了一种方法，该方法使用多拓扑膜蛋白作为噬菌体展示选择的靶标。膜蛋白是最大的一类药物靶标，药物发现主要基于鉴定与靶分子结合的配体。针对膜蛋白的噬菌体展示文库筛选配体通常受到这些蛋白质对膜环境的需求的阻碍，这种需求对于保留正确的折叠和表位形成是必要的。特别是在具有多个跨膜结构域的蛋白质中，表位通常是非线性的，由蛋白质跨膜区之间的环的组合组成。在这里，我们使用菌紫质（bR）作为多拓扑膜蛋白的模型，组装成纳米尺度的磷脂双层，即所谓的纳米盘，以筛选针对潜在配体的噬菌体展示文库。纳米盘为膜蛋白提供了类似天然的环境，因此可以在接近生理状态下进行配体的选择。针对 bR 纳米盘筛选 12 肽噬菌体展示肽文库导致分离出特异性结合 bR 的噬菌体克隆。我们还能够鉴定选定噬菌体克隆的结合位点，证明这些克隆结合 bR 的膜外、非线性表位。因此，纳米盘提供了一种合适且通用的工具，允许在接近天然的环境下针对膜蛋白筛选噬菌体展示文库。
