Liang Jingchang, Azubel Maia, Wang Guanqiao, Nie Yan, Kornberg Roger D, Beel Andrew J, Matteï Pierre-Jean
Department of Structural Biology, Stanford University, Stanford, CA, USA.
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
Bio Protoc. 2025 Aug 20;15(16):e5420. doi: 10.21769/BioProtoc.5420.
Zinc-finger (ZF) arrays are compact, sequence-specific polynucleotide-binding domains, which have been used to target the delivery of diverse effector domains, enabling applications such as gene identification, localization, regulation, and editing. To facilitate in vitro applications of ZF arrays, we have developed a general method for their expression and purification. Here, we describe a protocol involving two chromatographic steps that yields homogeneous and functional ZF arrays in milligram quantities. Key features • A general method for expressing and purifying C2H2 ZF arrays in , compatible with both natural and artificial ZFs. • The His-SUMO tag improves ZF-array solubility, eliminating the need for denaturation and refolding steps. • Simple, two-step purification yields milligram-scale ZF arrays suitable for downstream applications.
锌指（ZF）阵列是紧凑的、序列特异性的多核苷酸结合结构域，已被用于靶向递送多种效应结构域，从而实现基因识别、定位、调控和编辑等应用。为了促进锌指阵列的体外应用，我们开发了一种表达和纯化锌指阵列的通用方法。在此，我们描述了一种包含两个色谱步骤的方案，该方案可产生毫克级的均一且具有功能的锌指阵列。关键特性• 一种在大肠杆菌中表达和纯化C2H2锌指阵列的通用方法，适用于天然和人工锌指。• His-SUMO标签提高了锌指阵列的溶解度，无需变性和复性步骤。• 简单的两步纯化可产生适合下游应用的毫克级锌指阵列。
