Abstract：
The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.
摘要：
电压依赖性阴离子通道(VDAC)是一种丰富而多功能的线粒体外膜蛋白,在神经变性中起关键作用，凋亡，和线粒体膜生物发生。这里,我们提出了生产和重建高产量的洗涤剂溶解的VDAC的协议，在大肠杆菌中表达为包涵体。我们描述了通过亲和色谱纯化和在月桂基二甲胺-N-氧化物（LDAO）中重新折叠的步骤。然后，我们详细介绍了将VDAC重构为膜囊泡以通过基于荧光的测定法测定其通道和磷脂加扰酶活性的程序。有关此协议的使用和执行的完整详细信息，请参考Bergdoll等人。,1Queralt-Martín等人。,2和Jahn等3.
