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|標題:||Refolding of proteins with continuous fed-batch processes
大腸桿菌表現系統是目前生產異源蛋白的最佳途徑，本研究是利用大腸桿菌生產N-乙醯胺-D-葡萄糖-2-差向異構酶，但是利用此法生產重組蛋白質會有一個瓶頸，即是大量的重組蛋白會出現在胞內不可溶的部分，也就是所謂的包涵體(inclusion body)。實驗設計先以pH=8.5的20 mM Tris-buffer內含6M GdnHCl將包涵體溶解，再經由稀釋法逐漸降低變性劑濃度以達到復性效果。本研究中探討直接稀釋和連續饋料稀釋對差向異構酶復性的影響，並針對氧化改組系統、小分子添加劑(L-Arginine、Glycerol、PEG)、鹽類三方面來改善可溶蛋白回收率及活性。根據實驗結果，在低流速時復性效果最佳，且加入小分子添加劑的確有提高復性產率的效果，但是添加鹽類則無法達到提升活性的效果。
The Escherichia coli expression system is so far the best way for the over expression of recombinant proteins. In this study, GlcNAc 2-epimerase was expressed in Escherichia coli . However, one of the major bottleneck in utilizing E. coli for over-expressing recombinant proteins arises from the inclusion body formation. The over expressed recombinant proteins appear in an insoluble form. The epimerase from the purified inclusion bodies was solubilized in 20 mM Tris-HCl buffer at pH 8.5 with strong protein denaturant such as 6 M guanidinium hydrochloride or 8 M urea. Subsequently, urea or guanidinium hydrochloride is removed gradually by dilution to allow refolding of the denatured proteins. This study mainly investigated the feasibility of refolding denatured epimerase in direct or fed-batch operation . In this study, we added GSH and GSSG to promote the activity of refolded epimerase and improve the soluble protein recovery utilizing an oxido-shuffling system .On the other hand, we also add low molecular additives such as L-Arginine, Glycerol, PEG , and salts to improve the soluble protein recovery and activity. Experiment results indicated that the refolding in the fed-batch operation at low velocity resulted in higher recovery of enzyme activity. And it is indeed that adding the low molecular additives improve the soluble protein recovery and activity. However, it is recommended that salts addition is not necessary for refolding buffer.
|Appears in Collections:||化學工程學系所|
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