Protein renaturation by the liquid organic salt ethylammonium nitrate

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Abstract
The room‐temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured‐reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one‐step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded‐active protein can be separated from the molten salt by simple desalting methods. Although the use of a low‐temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.