On-Column Dual-Gradient Refolding for Efficient Recovery of Insoluble Affinity-Tagged Recombinant Proteins

This article presents an efficient protocol for refolding recombinant proteins that are prone to aggregation and form inclusion bodies during expression in Escherichia coli. As a model system, the homolog of CRISPR-associated effector protein CasV-M was investigated. The key element of the developed approach is refolding directly on a metal-affinity Ni-TED (N,N,N´-tris(carboxymethyl)ethylendiamine) resin using a dual-gradient system: a stepwise reduction in the concentration of the chaotropic agent combined with a simultaneous increase in the concentration of a mild nonionic detergent. This combination ensures spatial separation of protein molecules, minimizes aggregation, and promotes the recovery of the native conformation. The resulting method appears to be an alternative to conventional refolding strategies, with potential improvements in the reproducibility and yield of soluble protein compared to dialysis or dilution. The proposed approach can be extended to a broad range of aggregation-prone proteins and is considered a promising strategy for obtaining otherwise insoluble recombinant proteins. Key features • This protocol requires optimizing E. coli protein expression and an FPLC system, being particularly suitable for insoluble proteins that form inclusion bodies. • The method utilizes a dual-gradient refolding strategy on a Ni-TED column, integrating solubilization, refolding, and initial purification into a single workflow. • Effectively rescues challenging proteins from inclusion bodies, demonstrated with aggregation-prone CRISPR-associated effector homologs (Cas12m) refractory to traditional refolding methods. • Utilizes Ni-TED resin for its compatibility with high β-mercaptoethanol concentrations and moderate binding affinity, reducing nonspecific binding of host cell proteins.