Department of Cellular and Molecular Physiology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Biochemistry. 2014 Jan 14;53(1):4-6. doi: 10.1021/bi401412e. Epub 2013 Dec 30.
The scintillation proximity assay is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously expressed proteins, aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes, and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.
闪烁接近分析是一种强大的技术，可用于测量放射性配体与膜转运蛋白的结合，已成为高通量药物发现筛选工作的重要组成部分。在这里，我们将该方法改编为用于纯化的 LeuT，即一种原核二级转运蛋白，重新构建到磷脂双层纳米盘中。该应用克服了内源性表达蛋白背景干扰、从天然细胞膜中去污剂溶解常伴随的聚集和结合活性丧失以及大小和转运蛋白方向异质性的潜在挑战，其中至少一些配体结合位点不可用，与脂质体重建相关。
