Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays
Summary: 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 assa...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724007226 |
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| author | Stefano Conti Nibali Andrea Magrì Angela Messina Armin Wagner Ramona Duman Vito De Pinto Cristian Turato Cristina Arrigoni Marco Lolicato |
| author_facet | Stefano Conti Nibali Andrea Magrì Angela Messina Armin Wagner Ramona Duman Vito De Pinto Cristian Turato Cristina Arrigoni Marco Lolicato |
| author_sort | Stefano Conti Nibali |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-a5914788faa643ef982d41154b7a6ac3 |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-a5914788faa643ef982d41154b7a6ac32025-01-13T04:19:11ZengElsevierSTAR Protocols2666-16672025-03-0161103557Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assaysStefano Conti Nibali0Andrea Magrì1Angela Messina2Armin Wagner3Ramona Duman4Vito De Pinto5Cristian Turato6Cristina Arrigoni7Marco Lolicato8Department of Molecular Medicine, University of Pavia, Pavia, ItalyDepartment of Biological, Geological and Environmental Sciences, University of Catania, Catania, ItalyDepartment of Biological, Geological and Environmental Sciences, University of Catania, Catania, ItalyDiamond Light Source, Harwell Science and Innovation Campus, OX11 0DE Didcot, UKDiamond Light Source, Harwell Science and Innovation Campus, OX11 0DE Didcot, UKDepartment of Biomedical and Biotechnological Sciences, Section of Biology & Genetics, University of Catania, Catania, ItalyDepartment of Molecular Medicine, University of Pavia, Pavia, ItalyDepartment of Molecular Medicine, University of Pavia, Pavia, Italy; Corresponding authorDepartment of Molecular Medicine, University of Pavia, Pavia, Italy; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724007226BiophysicsProtein BiochemistryProtein expression and purificationX-ray Crystallography |
| spellingShingle | Stefano Conti Nibali Andrea Magrì Angela Messina Armin Wagner Ramona Duman Vito De Pinto Cristian Turato Cristina Arrigoni Marco Lolicato Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays STAR Protocols Biophysics Protein Biochemistry Protein expression and purification X-ray Crystallography |
| title | Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays |
| title_full | Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays |
| title_fullStr | Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays |
| title_full_unstemmed | Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays |
| title_short | Protocol for high-yield bacterial expression and purification of the voltage-dependent anion channel 1 for high-throughput biophysical assays |
| title_sort | protocol for high yield bacterial expression and purification of the voltage dependent anion channel 1 for high throughput biophysical assays |
| topic | Biophysics Protein Biochemistry Protein expression and purification X-ray Crystallography |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724007226 |
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