Membrane reconstitution of ABC transporters and assays of translocator function
Membrane reconstitution of ABC transporters and assays of translocator function
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DOI：
10.1038/nprot.2007.519
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Poolman, Bert
中科院分区：
文献类型：
--
作者：
Geertsma, Eric R.;Mahmood, N. A. B. Nik;Poolman, Bert
在本方案中，我们描述了将ATP结合盒（ABC）转运蛋白整合到大单层囊泡（LUV）中的程序，并通过测定这些膜重建系统的配体结合和溶质转运进行测定。所描述的重构技术已经针对ABC转运蛋白进行了优化，但是可以容易地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去稳定的预形成脂质体中，随后通过吸附到聚苯乙烯珠上除去去污剂。接下来，通过一个或多个冻融循环将Mg-ATP或ATP再生系统掺入囊泡腔中，然后通过聚碳酸酯过滤器挤出以获得单层囊泡。使用同位素标记的配体和快速过滤以将脂蛋白体与周围介质分离来测量底物的结合和易位。获得有关配体结合的解离常数（Kd）、结合位点的数目、运输亲和力（Km）、运输速率和在膜中具有相反取向的运输分子的活性的定量信息。整个方案可在4-5 d内完成。
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.