| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 92 === This study mainly investigates the refolding efficiency of denatured protein using the method of direct dilution. The research is comprised of two major sections.The first section investigates the effect of denaturation period and different denatured agent on protein refolding efficiency. The second section primarily discusses the difference of protein recovery with different concentration of protein and dilution factor.
It is proven that the ratio [GSSG]/[GSH] increases with storage time [Liao,2003], which enhances the protein renaturation. For this reason, [GSSG]/[GSH] freshly prepared to model the previous work and the refolding efficiency obtained is found to be very similar. A ratio of absorbance, A280/A260 is used in this study to quantify the condition of refolding buffer. It is found that the A280/A260 and concentration of protein is a key factor that affects the refolding efficiency. As a result, we propose an empirical correlation to predict protein recovery efficiency:
Act(%) = 275N2.15 P-1.94N+1 I-0.14
From this correlation, we can approximate the recovey of enzyme activity if A280/A260(N), protein concentration(I) and dilution fold(P) are known.
Besides, we discover that the refolding efficiency of protein is different if EDTA is added or not in the refolding buffer. It is found that refolding buffer with EDTA will reduce the oxidation rate of GSH to GSSG and result in a serious protein aggregation. Therefore, it is recommended that EDTA addition is necessary for refolding buffer with a high A280/A260, and it is not necessary for refolding buffer with a low A280/A260.
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