: 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.
Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays
Conti Nibali, Stefano;Turato, Cristian;Arrigoni, Cristina
;Lolicato, Marco
2024-01-01
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
