Efficient lysozyme refolding at a high final concentration and a low dilution factor

摘要

Of the various protein refolding methods, direct dilution is one of the simplest and easiest for scaling up the refolding process. However, it requires a large amount of refolding buffer, often utilizes a number of chemicals, and results in a low final protein concentration. In this report, we demonstrate that reduced dithiothreitol (DTT~(red)), a carryover from denaturation, is a crucial and adverse factor in lysozyme refolding. Accordingly, we proposed a method of using high concentration of oxidized glutathione (GSSG) in the refolding buffer to eliminate excess DTT~(red) and aid in the refolding of lysozyme. The efficiency of this method is 84%, which resulted in a high final refolded protein concentration of 1.5g/l and required only a low dilution factor (4×). Furthermore, compared with the traditional 50x direct dilution (resulting in a similar yield of 74%), the low dilution factor required much less GSSG and other constituents.