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

Jingchang Liang; Maia Azubel; Guanqiao Wang; Yan Nie; Roger D Kornberg; Andrew J Beel; Pierre-Jean Matteï

doi:10.21769/BioProtoc.5420

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

Bio Protoc. 2025 Aug 20;15(16):e5420. doi: 10.21769/BioProtoc.5420.

Authors

Jingchang Liang^{1

2

3}, Maia Azubel¹, Guanqiao Wang², Yan Nie², Roger D Kornberg¹, Andrew J Beel¹, Pierre-Jean Matteï¹

Affiliations

¹ Department of Structural Biology, Stanford University, Stanford, CA, USA.
² Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
³ WLA Laboratories, Shanghai, China.

Abstract

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 E. coli, 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.

Keywords: Cation exchange chromatography; His-SUMO tag; Ni-NTA chromatography; On-column cleavage; Protein purification; Zinc fingers.