| 초록 |
The process monitoring method for the traditional solution-phase refolding could not directly applied to the solid-phase refolding of immobilized proteins. In this study, we estimated the structural change of the immobilized proteins on the gold surface of an SPR sensor chip by detecting the reflective index change. After demonstrating the positive relationship between the reflective index value and the protein's degree of folding, we tested the effect of denaturant concentration such as urea and guanidine-HCl on the protein structure. To investigate the effect of disulfide bonds on the refolded structure, 2 mM dithiothreitol was added during the uvitro refolding experiment of urokinase (12 disulfides) and disulfide-free myoglobin. Unlike the myoglobin, the RU value of the urokinase was not fully recovered to the original value, which suggested that the unrecovered RU value represented the mis-shuffled structure. We proposed the unique method to monitor the unfolding/refolding of proteins by using the simple SPR sensor. This method can be applied to identify the optimal condition for the solid-phase refolding process of immobilized proteins.
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