用于高通量生物物理分析的电压依赖性阴离子通道1的高产细菌表达和纯化方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-01-11 DOI:10.1016/j.xpro.2024.103557

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

摘要

电压依赖性阴离子通道1 （VDAC1）是细胞代谢和凋亡的关键蛋白。在这里，我们提出了一种在大肠杆菌中表达和纯化毫克量的重组VDAC1的方案。我们详细介绍了使用NADH置换的基于荧光偏振的高通量筛选分析的步骤，以及热稳定性，荧光偏振和x射线晶体学的程序。在这种情况下，我们证明了2-甲基-2,4-戊二醇（MPD），一种结晶试剂，如何干扰VDAC1小分子结合，阻碍这些配体在晶体中的检测。有关本协议使用和执行的完整细节，请参考Conti Nibali等人1。

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

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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