Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer

Earlier, we formally established an effective refolding procedure for a protein by gradient removal of a solubilizer such as urea [Maeda et al. (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 8, 201-205]. However, this procedure was less effective for unstable proteins. We developed here an excellent method to add protein stabilizer so as to get reasonable amounts of folded protein under the concentration of solubilizer where the unstable protein does not form aggregate. We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins.