A strategy for the isolation and refolding of aggregation-prone proteins: application to two CRISPR-associated Cas12m effectors

Research Objective. To develop refolding conditions for recombinant proteins prone to aggregation and inclusion body formation during expression in Escherichia coli, using two homologs of the CRISPR effector Cas12m as an example. Materials and Methods. The Cas12m genes were amplified and cloned into expression vectors with an N-terminal His-tag. The resulting plasmids were transformed into E. coli for protein production. Protein expression was induced by IPTG, after which the proteins accumulated in inclusion bodies were solubilized in a chaotropic agent and purified by immobilized metal affinity chromatography (IMAC) on an Ni-TED column with simultaneous on-column refolding using the “dual-gradient” method. Results. A comprehensive screening of expression conditions was conducted; however, in all tested bacterial strains, the proteins formed inclusion bodies. Optimization of induction conditions and the use of various fusion tags did not yield significant improvements. To address the issue of low solubility, a refolding protocol was developed, which involved immobilizing the denatured protein on Ni-TED resin. This protocol proved to be the most effective compared to traditional methods. A key aspect was the application of the “dual-gradient” system — a simultaneous decrease in the denaturant concentration and an increase in the detergent concentration. As a result, the target proteins were successfully obtained in a soluble form. Despite protein loss due to partial aggregation and the presence of contaminant proteins typical for metal-chelate chromatography, the resulting preparations were of sufficient purity for subsequent studies of functional activity. Conclusion. The proposed method outperforms traditional refolding techniques, such as dialysis or dilution, in terms of reproducibility and the yield of soluble protein. This approach can be extended to a wide range of aggregation-prone proteins and is considered a viable strategy for obtaining difficult-to-solubilize proteins in a soluble form.