Geertsma Eric R, Nik Mahmood N A B, Schuurman-Wolters Gea K, Poolman Bert
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Nat Protoc. 2008;3(2):256-66. doi: 10.1038/nprot.2007.519.
In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.
在本方案中，我们描述了一种将ATP结合盒（ABC）转运蛋白整合到大型单层囊泡（LUVs）中的方法，以及用于确定这些膜重组系统的配体结合和溶质转运的检测方法。所述的重组技术已针对ABC转运蛋白进行了优化，但可轻松适用于其他类型的转运系统。将纯化的转运蛋白插入去污剂破坏的预制脂质体中，随后通过吸附到聚苯乙烯珠上去除去污剂。接下来，通过一个或多个冻融循环将Mg-ATP或ATP再生系统掺入囊泡内腔，然后通过聚碳酸酯滤膜挤出以获得单层囊泡。使用同位素标记的配体和快速过滤来测量底物的结合和转运，以将蛋白脂质体与周围介质分离。可获得关于配体结合的解离常数（K(d)）、结合位点数量、转运亲和力（K(m)）、转运速率以及膜中方向相反的转运蛋白分子活性的定量信息。整个方案可在4-5天内完成。
