Protein Refolding by Size Exclusion Chromatography
Date Issued
2007
Date
2007
Author(s)
Chang, Yi-Tin
DOI
zh-TW
Abstract
In recent decades, protein refolding by size exclusion chromatography (SEC) has been proven feasible in terms of its good mass recovery and activity recovery. One of the advantages of SEC is that there is no chemical interaction between protein and the column matrix. In addition, the porous property enables SEC to separate the protein with various conformations. Protein refolding by SEC can minimize the formation of aggregates with careful consideration of the following two important aspects: (1) the formation of aggregates in the inlet tubing prior to column, and (2) the formation of correct disulfide bond in refolding protein. The former is related to protein mass recovery, while the latter is crucial to activity recovery. From the experimental results, it can be concluded that the urea concentration in the refolding buffer has a significant effect on mass recovery as demonstrated by the higher urea concentration yielding higher mass recovery (up to 94 % when urea concentration is higher than 6 M). The pH value of the refolding buffer also affects the recovery of refolded protein significantly with the maximum activity of recovery of 70 % at pH 10.5.
This research work mainly focuses on the effects of operating variables of SEC on protein refolding, and can be divided into three parts: (1) the effect of denaturation process on SEC protein refolding; (2) the effect of renaturation process on SEC protein refolding; (3) the simulation of the elution profile of native protein in SEC. In the first part, the influences of the duration of denaturation and the concentration of denatured protein on SEC protein refolding process are discussed. Our experimental results indicated that the activity recovery was decreased with increasing denaturation time. When protein concentrations were within 1 to 10 g/L, the mass recovery ranged from 70% to 75 % and the activity of recovery roughly remained constant (30 %). However, when the concentration of denatured protein reached 20 g/L, the mass recovery and the activity recovery of refolded protein were dropped to 61 % and 24%, respectively. In the second part, low mobile phase velocity led to the better activity recovery, while high velocity resulted in higher mass recovery. It was found that elevating urea concentration in refolding buffer suppress formation of aggregates, and appropriate choice of pH, for example, pH 10.5, enhanced the activity recovery. Finally, a program using the Matlab software was devised to describe the chromatogram of the native protein in the SEC column. Results demonstrated that, via the program we developed, the elution profiles of the native protein under different mobile velocities could be simulated in a reasonable manner.
Subjects
溶菌(酉每)
大小排阻層析
蛋白質復性
Matlab 程式
lysozyme
size exclusion chromatography
protein refolding
Matlab program
Type
thesis
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