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題名 | Immobilization and Solid-State Refolding of 6×His-Tagged Glutathione-S-Transferase Using Metal Affinity Matrices=利用金屬親和凝膠固定化與固態再摺合帶有6×His-Tag之Glutathione-S-Transferase融合蛋白 |
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作 者 | 陳秀美; 陳愷悌; 羅森林; | 書刊名 | Journal of the Chinese Institute of Chemical Engineers |
卷期 | 35:6 2004.11[民93.11] |
頁次 | 頁643-650 |
分類號 | 460.02 |
關鍵詞 | 金屬親和凝膠; 融合蛋白; 鎳離子; Immobilization; Glutathione-S-transferase; Metal affinity matrix; 6×His peptide; Solid-state refolding; |
語文 | 英文(English) |
中文摘要 | 本研究利用整合有鎳離子之金屬親和凝膠探討C端融合有6 × His tag之日本血吸蟲穀胱甘呔轉移酵素(C-terminally 6× His tagged Schistosoma japonicum glutathione-S-transferase,SjGST/His)之親和固定化與固態再摺合。首先於純蛋白之固定化研究發現,以濃度介於5-50μg/mL間之蛋白質溶液與凝膠進行吸附時,所得固定化酵心之比活性最高,且活性回收率佳。固定化酵素儲存於4°C下84天後仍保有原活性;然而於25°C儲存時,可能由於其N端受到蛋白酶分解之故,經過34至50天後,其活性開始急遽下降。此外,針對原生種以及Cys85→Ser、Cys138→Ser和Cys178→Ser等定點突變種之酵素進行熱處理,初步發現所有固定化酵素之熱穩定性均比其自由態之酵素稍高。其次,於固態再摺合之研究發現,先以低濃度之變性蛋白質溶液與凝膠進行吸附後,再多次清洗凝膠並同時逐步地降低洗劑中變性試劑之濃度,如此可有效地得到完整且具活性之SjGST/His酵素,應用此方法亦成功地自細胞內含體中回收活性酵素。 |
英文摘要 | The affinity immobilization of a recombinant Schistosoma japonicum glutathione-S-transferase C-terminally fused with a 6 × His peptide, named SjGST/His, was first investigated using Ni-2+-chelated metal affinity matrices. The immobilized enzyme retained best activity over a 5-50μg/mL range of binding protein concentrations, with a high activity yield. Innobilized SjGST/His remained active during 84 days of storage at 4°C, but its activity started to decline considerably after 34-50 days of storage at 25°C, presumably due to expensive proteolytic degradation of the fixed enzymes beginning at their N-Termini. Following heat treatment of both free and immobilized forms of wild-type and Cys85→Ser, Cys138→Ser, and Cys178→Ser site-directed mulant SjGST/His, all the immobilized forms retained better activity than their free ones did. In addition, the solid-state refolding study revealed that immobilizing denatured SjGST/His onto Ni2+-chelated gels at a low binding concentration, followed by stepwise washing of the resulting gels with gradual reduction of the denaturant, yielded active and intact enzymes at the final native elution. Under the same optimal conditions, active SjGST/His was successfully recovered from solubilized inclusion bodies. This study demonstrates that it is possible to directly and efficiently immobilize and solid-state refold polyHis-tagged proteins using metal affinity matrices. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。