A Study on the Protein Refolding Using Size Exclusion Chromatography
Date Issued
2005
Date
2005
Author(s)
Chang, Tse-Kuei
DOI
zh-TW
Abstract
The formation of incorrectly folded protein, in particular aggregates, was recognized as the hindrance of good yield in refolding processes. Results from our study have shown that the formation of aggregates between the injection valve and column inlet was found to strongly hamper (adversely affect) the efficiency of lysozyme refolding in size-exclusion chromatography (SEC). To enhance the performance of protein refolding, our studies were directed toward seeking out methods or means to reduce the aggregate production during the SEC refolding. Furthermore, we compared the refolding performances between the direct dilution and SEC refolding methods under different constituents of refolding buffer or dilution factors, and then examined how the protein refolding was assisted via porous matrix in SEC column.
In this study, chaperone solvent plug strategy and step change of mobile phase flowrate strategy were developed to overcome the aggregate formation between the injector and column inlet. For the former one, the denatured protein was escorted from the injector into the column by a solvent plug that could inhibit aggregate formation or stabilize the denatured protein. As to the latter one, a higher flowrate of mobile phase should be applied to reduce the traveling time of denatured protein from injector to column inlet. After the denatured protein penetrated into the column, a lower flowrate should be used to allow enough time for protein to refold. Enough residence time inside the column was identified to be the key for proper protein refolding. Then combining this method with chaperone solvent plug strategy could achieve complete recovery of denatured proteins, both mass and activity recoveries.
Furthermore, we also investigated how aggregate formation as well as renaturation yield varied with the tubing dimension (diameter or length) of sample loop in the SEC refolding process. It was found that a sample with large volume and low concentration was preferable for refolding process.
In addition, via a gradient of redox buffers through the SEC, oxidative refolding was achieved under kinetic control the disulfide bond formation. Employing the renaturation buffer system with different redox constituents and comparison with batch dilution, we demonstrated that the porous matrix packing in SEC could assist protein refolding in the absence of reduced and oxidized glutathione (GSH/GSSG).
Subjects
大小排阻層析法
復性
Size Exclusion Chromatography
Protein Refolding
Type
thesis
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