Effect of Additives on Refolding of a Denatured Protein

Denatured lysozyme was refolded by a dilution method. The refolding yield depended greatly on the lysozyme concentration in the refolding mixture. When the concentration of denatured lysozyme was 0.02 g/L, the refolding yield was as high as 60%. However, when the concentration of denatured lysozyme was 0.2 g/L, the refolding yield was as low as 10% due to the formation of aggregates. To prevent the formation of aggregates and to increase the refolding yield at a low cost, inexpensive additives were screened. The addition of acetone, acetoamide, or urea derivatives was very effective for improving the refolding yield. To clarify why the addition of acetoamide in the refolding mixture improved the refolding yield at the high lysozyme concentration, the time courses of the concentration and the average diameter of the aggregates in the refolding mixture were monitored by the dynamic light scattering method. The experimental results showed that acetoamide played a role in preventing the formation and growth of aggregates and secondary aggregation between the lysozyme aggregates.