The Investigation of Redox Pairs in the Lysozyme refolding Process
Date Issued
2010
Date
2010
Author(s)
Lin, Wei-Tai
Abstract
Currently, protein refolding process is the crucial issue in the bio-industry. In this research, denatured hen egg-white lysozyme was refolded by the direct dilution and the dialysis methods, while the denaturing condition was in 8M urea and reduced dithiothreitol (DTTred) for simulating the solubilized inclusion body. The goal of this work was to investigate the role of redox pairs in the refolding process.
There were two redox pairs existing in the refolding system, DTTred -GSSG and GSH-GSSG, shuffling between thiol group and new-forming disulfide bonds to regain the activity. In this research, DTTred -GSSG has better refolding efficiency than GSH-GSSG on lysozyme renaturation. Moreover, in the refolding condition without DTTred, the ratio of GSH to GSSG had little influence on the activity yield. One of the reasons was that the degree of reaction of GSH upon disulfide bonds would depend on the urea concentration. On the other hand, regarding to the redox pair DTTred -GSSG, as the ratios of GSSG to DTTred are higher than 1 inside the dialysis tube and higher than 2.5 in refolding buffer of direct dilution within our experimental conditions, the activity yield would be enhanced significantly. However, while the ratio was higher than the critical value for both methods, little change was obtained in the activity yield.
At the beginning of the dialysis refolding operation, high concentration GSSG was added into dialysis tube, which could react with residual DTTred and reduce the concentration of DTTred. The above protein refolding strategy was called fed-batch dialysis refolding method in this work. Fed-batch dialysis was an effective way to refold protein, for example, 80% of refolding yield could be obtained for 1g/l of lysozyme by fed-batch dialysis refolding method, 20% higher than batch operation.Besides, This fed-batch could have better performance than batch dialysis and continuous dialysis in chemicals consumption. Furthermore, via the fed-batch dialysis conbines gradually step-changing concentration of urea, the activity yield of lysozyme with high concentration which are 4g/l、6g/l and 8g/l could reach 70% after 30 hr-refolding process.
In addition, in a direct dilution by only 4 times, 70% and 90 % of activity yield could be obtained with an appropriate ratio of GSSG to DTTred for lysozyme concentration of 1.25g/l and 0.75g/l, respectively.
Subjects
Lysozyme
Oxidized glutathione
Reduced dithiothreitol
Refolding
Direct dilution
Dialysis
Type
thesis
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