Department of Chemistry, Lehigh University, 6 E. Packer Ave. Bethlehem, Pennsylvania, 18015, USA.
Department of Chemical Engineering, The Pennsylvania State University, 121 Chemical and Biomedical Engineering Building, University Park, PA, 16802, USA.
Arch Biochem Biophys. 2021 Nov 15;712:109051. doi: 10.1016/j.abb.2021.109051. Epub 2021 Oct 2.
Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample. A panoply of biophysical techniques including analytical ultracentrifugation, dynamic light scattering, gel filtration chromatography, circular dichroism spectroscopy, and cryogenic electron microscopy were employed to unequivocally confirm that aggregates formed by this method are indeed nanodiscs. We believe that this method will be attractive for time-sensitive and high-throughput experiments.
纳米盘是一种碟状实体，其中包含一个由两亲性螺旋构成的环形中央脂双层。它已经成为一种领先的类似天然膜的模拟物。目前形成纳米盘的方法涉及创建一种包含膜支架蛋白、脂质和去污剂的混合胶束溶液，然后进行耗时的透析和/或与吸附珠孵育过程，以从样品中去除去污剂分子。相比之下，本文所述的方法提供了一种简便、快速的方法，可在几分钟内（<15 分钟）使用 Sephadex® G-25 树脂从样品中去除去污剂来制备纳米盘。一系列生物物理技术，包括分析超速离心、动态光散射、凝胶过滤色谱、圆二色性光谱和低温电子显微镜，被用来明确证实通过这种方法形成的聚集体确实是纳米盘。我们相信这种方法将对时间敏感和高通量实验具有吸引力。
