A universal method for the purification of C2H2 zinc finger arrays

Abstract

Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and for the targeted delivery of effector domains for purposes such as gene regulation and editing. Despite recent advances in both the design and application of ZF-containing proteins, there is still a lack of a general method for their expression and purification. Here we describe a simple method, involving two chromatographic steps, for the production of homogeneous, functional ZF proteins in high yield (one milligram per liter of bacterial culture), and we demonstrate the generality of this method by applying it to a diverse set of eight C2H2-type ZF proteins. By incorporating a surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, we confirm the suitability of these probes for in situ labeling of specific DNA sequences in human cells.