Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays

Stefano Conti Nibali; Andrea Magrì; Angela Messina; Armin Wagner; Ramona Duman; Vito De Pinto; Cristian Turato; Cristina Arrigoni; Marco Lolicato

doi:10.1016/j.xpro.2024.103557

Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays

STAR Protoc. 2025 Jan 11;6(1):103557. doi: 10.1016/j.xpro.2024.103557. Online ahead of print.

Authors

Stefano Conti Nibali¹, Andrea Magrì², Angela Messina², Armin Wagner³, Ramona Duman³, Vito De Pinto⁴, Cristian Turato¹, Cristina Arrigoni⁵, Marco Lolicato⁶

Affiliations

¹ Department of Molecular Medicine, University of Pavia, Pavia, Italy.
² Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy.
³ Diamond Light Source, Harwell Science and Innovation Campus, OX11 0DE Didcot, UK.
⁴ Department of Biomedical and Biotechnological Sciences, Section of Biology & Genetics, University of Catania, Catania, Italy.
⁵ Department of Molecular Medicine, University of Pavia, Pavia, Italy. Electronic address: cristina.arrigoni01@unipv.it.
⁶ Department of Molecular Medicine, University of Pavia, Pavia, Italy. Electronic address: marco.lolicato@unipv.it.

PMID: 39799574
DOI: 10.1016/j.xpro.2024.103557

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.

Keywords: Biophysics; Protein Biochemistry; Protein expression and purification; X-ray Crystallography.