Stefano Conti Nibali, Andrea Magrì, Angela Messina, Armin Wagner, Ramona Duman, Vito De Pinto, Cristian Turato, Cristina Arrigoni, Marco Lolicato
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引用次数: 0
Abstract
Voltage-dependent anion channel 1 (VDAC1) is a key protein in cellular metabolism and apoptosis. Here, we present a protocol to express and purify milligram amounts of recombinant VDAC1 in Escherichia coli. We detail steps for a fluorescence polarization-based high-throughput screening assay using NADH displacement, along with procedures for thermostability, fluorescence polarization, and X-ray crystallography. In this context, we demonstrate how 2-methyl-2,4-pentanediol (MPD), a crystallization reagent, interferes with VDAC1 small-molecule binding, hindering the detection of these ligands in the crystal. For complete details on the use and execution of this protocol, please refer to Conti Nibali et al.1.
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