Protein Science, Vol 9, Issue 10 2001-2008, Copyright © 2000 by The Protein Society

Protein renaturation by the liquid organic salt ethylammonium nitrate [In Process Citation]

CA Summers and RA Flowers 2nd
Department of Chemistry, The University of Toledo, Ohio 43606 USA.

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.
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