Ni2+-induced selective precipitation of His-tagged recombinant proteins shortens purification time while maintaining high yield.

Nickel-NTA affinity chromatography is the current standard method for purifying His-tagged recombinant proteins. However, this process involves repetitive tasks, can be time-consuming, and reduces protein yield. Here, we present a simple, fast, and handy method for purifying His-tagged proteins using free Ni²⁺. This approach allows the fractional precipitation of His-tagged proteins directly from E. coli cell lysates. We successfully applied this Ni²⁺-based method to purify three His₆-tagged recombinant proteins overexpressed in E. coli. We found that Ni²⁺ at a final concentration of as low as 1 mM precipitates the His-tagged proteins with near-complete specificity as confirmed by SDS-PAGE analysis. The Ni²⁺-precipitated proteins were dissolved by adding 10 % acetic acid and further purified by reverse-phase HPLC. The final yields were between 3.5 and 8.0 mg per 200 mL culture, similar to or even higher than purification using conventional Ni-NTA chromatography. The purified proteins exhibited natively folded characteristics, as assessed by CD, SLS, and DLS, and binding activity, as assessed by ELISA and BLI, demonstrating the method's potential in both small and large-scale settings.