Production of Homogeneous, Functional Zinc-Finger Arrays in High Yield With Two Chromatographic Steps

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 <i>E. coli</i>, 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.