The Effect of Dithiothreitol Carry-over and Urea on Renaturation Procedure of Lysozyme
Date Issued
2009
Date
2009
Author(s)
Lin, You-Chen
Abstract
Before the renaturation process, proteins need to be denatured and dissolved. Thus, when entering renaturation process, the denaturing chemicals are carried over into the refolding system and affected the performance of renaturation process. In this investigation, we used the direct dilution method to refold lysozyme, and measured the activity recovery by an efficient 96 well microplate method (0.8g/L Micrococcus lysodeikticus, 10s/90s measuring time, substrate and lysozyme volume ratio of 200μl and 10μl, for the lysozyme concentration range 0.01g/L~0.1g/L.). We examined the effect of the carried-over DTTRed from the denaturation process on the refolding performance. The effect of urea on the refolding of lysozyme was also explored by varying the concentration of urea in the refolding condition.ize exclusion chromatography (SEC) was applied to determine the carried-over DTTRed after lysozyme denaturation. With the initial concentration of DTTRed and lysozyme（Lyi）, we could calculate the concentration of DTTOxi by the equation , and accordingly the concentration of the carried-over DTTRed. From the results of direct dilution method, the relationship among lysozyme activity recovery, lysozyme concentration, and carried-over DTTRed concentration could be divided into three regions including the redox control region, aggregate control region, and DTTRed control region. An improvement in the activity recovery could be achieved through the proper regulation of the contributing factors in each region. Generally speaking, the activity recovery was inversely proportional to the lysozyme concentration and the addition of urea could reduce the aggregation. 1M of urea helps to recover the activity of lysozyme up to final concentration of 0.16g/L with more than 80% yield. 2M of urea could recover the enzyme activity up to 0.5g/L with the yield more than 75%. Although 3M of urea resulted in stronger hydrophobic interaction, it could still recover the activity of lysozyme of final concentration more than 1g/L efficiently. According to our experimental results, the rapid formation of aggregates would occur as soon as the denaturated lysozyme was in contact with the refolding buffer. With 1M of final concentration of urea and 10g/L of initial concentration of lysozyme, the amount of initial aggregates would lead to a reduction in activity recovery. However, a better inhibition of initial aggregate formation was observed when the final concentration of urea was 2M and initial concentration of urea was above 1.76M. We believe our work may contribute to a better design of protein refolding processes.
Subjects
dithiothreitol(DTT)
lysozyme
urea
denaturation
refolding
direct dilution method
Type
thesis
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