Denaturation, rapid dilution refolding, and single-step purification of the core histones using desalting...

Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and interpreted by readers to control gene expression, chromatin structure, and essential cellular processes such as differentiation and development. Accurate Post Translational modifications analysis requires high-purity histones due to the sensitivity of epigenetic assays. Recombinant histones are expressed in E. coli as inclusion bodies, requiring denaturation, refolding, and purification. These traditional purification methods involve complicated and lengthy protocols taking days and potentially exposing the histones to oxidation and proteolytic degradation. We developed a rapid method for refolding histones from inclusion bodies in a one-step purification using a desalting column achieving > 90 % purity. This method is compared to our previous High Performance Liquid Chromatography (HPLC)-based protocol. Our single-step desalting purification reduces purification time from multiple days to one day, lowers operational cost, and eliminates the need for reverse-phase HPLC, making high-purity histone production accessible to laboratories without specialized chromatography infrastructure.