Protocol for the production and reconstitution of VDAC1 for functional assays

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 • Expression of VDAC1 in E. coli and purification in urea • Detailed description of protein folding in LDAO • Step-by-step details of VDAC1 cross-linking and reconstitution into liposomes • Fluorescence-based assay of VDAC channel and scramblase activity Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.