Low Dilution Refolding for Lysozyme
Date Issued
2012
Date
2012
Author(s)
Sheim, Yu-An
Abstract
Protein refolding is mainly carried out by eliminating denaturant through various methods. Direct dilution method is a traditional one by diluting the denaturant with a large amount of refolding buffer. However, this research added GSSG to react with carry-over DTTRed which was a crucial hinderence to successful protein refolding. The experimental results indicated that low dilution refolding method was as good as, if not better than, that of high dilution refolding method with this strategy. Therefore, this research further discussed the impacts of refolding environmental factors including GSSG and carry-over DTTRed.
According to the experiments, there was a relationship between refolding environments and lysozyme activity recovery depending on its concentrations. Namely, it was classified into three regions, including redox control region, transition region, and aggregation control region. For example, in redox control region, the activity of lysozyme up to final concentration of 1.5g/L would regain 80% in 2M urea condition with appropriate GSSG refolding buffer.
In addition, lysozyme of higher concentration in aggregation control region usually needed the help of urea to minimize aggregation, and thus obtain better activity recovery. For instance, the activity recovery of the 3g/L final lysozyme concentration by 4-times dilution in 2M urea condition was merely 60%; nevertheless,
the activity recovery could be enhanced to 70% in 3M urea.
Subjects
lysozyme
protein refolding
direct dilution
DTT
Type
thesis
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