Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3784
標題: 以金屬螯合吸附材進行基因重組蛋白質管柱復性之研究
On-column refolding of a recombinant protein with immobilized metal affinity adsorbents
作者: 李秉忠
Li, Beng-chung
關鍵字: refolding
復性
inclusion body
內函體
出版社: 化學工程學系所
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摘要: 以大腸桿菌大量表現產重組差相異構酶,其中約有80%的差相異構酶是以內函體(inclusion body)的形式獲得,為了使內函體回復具有天然活性的構形並減少蛋白質復性過程中集結現象的發生,故本研究以高pH值(pH=11)與尿素(8 M Urea)此兩種溶解系統於金屬螯合吸附材Ni-NTA之管柱上進行蛋白質復性,分別探討復性梯度時間、靜置時間、鹽濃度、小分添加劑對於可溶蛋白質回收率與相對比活性之影響。研究顯示在高pH值溶解系統中,以50 mM碳酸氫鈉pH=11為最適化溶解內函體條件,且以此條件可回收60%可溶蛋白值,但不具有活性。而在尿素溶解系統中,可溶蛋白回收率偏低約12%上下,但均有10%以上之相對比活性,尤其在濃度梯度時間180分鐘可獲得20%以上之相對比活性。
Over-expression of recombinant GlcNAc 2-epimerse in Escherichia coli system was formed with inclusion body by 80%. In this study, the Ni-NTA immoblilized metal affinity column was applied to high-pH and urea solubilization systems for investigating the effect of soluble protein recovery and relative specific activity on refolding gradient time, stated time, salt concentration and small molecule additives. The results of high-pH solubilization system obtained a optimum denatured condition (50 mM sodium bicarbonate, pH=11) and recovered 60% non-activation epimerase. In urea solubilization system, the recovery of the soluble protein and relative specific activity were equally 12%and 10%, respectively, and more than 20% relative specific activity was gained in the on-column refolding especially by 180 minutes concentration gradient.
URI: http://hdl.handle.net/11455/3784
其他識別: U0005-2907200916023000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907200916023000
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