Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3444
標題: 差向異構酶包涵體復性最適化研究
作者: 曾資棟
Tzeng, David
關鍵字: inclusion body
包涵體
refolding
renaturation
solubilization
epimerase
GlcNAc 2-epimerase
復性
再摺疊
溶解
差向異構酶
出版社: 化學工程學系
摘要: GlcNAc 2-epimerase was expressed in Escherichia coli in the form of inlcusion bodies. The epimerase from the purified inclusion bodies was solubilized in 100 mM Tris-HCl buffer at pH 12.5 and > 0.3 % SDS had significatly solubilization effect. The optimal conditions for denaturation of epimerase were when inclusion bodies were solubilized in 50mM Tris-HCl buffer. The refolding method is that the solubilized protein was adjusted to neutral pH. Optimal soluble protein recovery is about 65% when the solubilized proteins (8mg/ml) were refolded at pH 8 .In this study,we added GSH and GSSG to promoted activity of refolded epimerase utilizing an oxido-shuffling system .The dimer fractions of refolded epimerase had 30% relative specific activity .
本研究利用大腸杆菌大量生產重組差向異構酶,會在胞內形成大量的包涵體(inclusion bodies),故發展出一個最適的差向異構酶包涵體復性程序,從包涵體回復成具有天然活性的蛋白質。先以探討最適的溶解條件,找出pH 12.5 ,100mM Tris-Buffer和0.3 % SDS 具有高溶解效應。並以pH12.5,50mM Tris-Buffer當做最適溶解劑,得到最佳的溶解蛋白濃度8mg/ml,再以HCl將溶解蛋白質溶液調控至中性進行復性再摺疊程序,可知調控至pH8有最佳的可溶蛋白回收率,並發現形成而大量沒有活性的寡聚體(oligomer),並試圖以溫度、NaCl、添加劑、及氧化系統來改善回收率和再摺疊後無活性之情形。 且本研究應用氧化改組系統(oxido-shuffling system),添加GSH和GSSH來使雙硫鍵正確配對。可得復性差向異構酶二聚體之相對比活性為30%。
URI: http://hdl.handle.net/11455/3444
Appears in Collections:化學工程學系所

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