臺灣博碩士論文加值系統

摘要
大腸桿菌表現系統是目前生產異源蛋白的最佳途徑，瓶頸之一是有時會在胞內形成包涵體，大量合成的蛋白因而出現在不可溶部分，目前解決方法是以8 M urea或6 M guanidinium hydrochloride將包涵體溶解，再經由透析，逐漸降低urea或guanidinium hydrochloride的濃度，使變性的蛋白重新摺疊，恢復原來蛋白的結構與活性。本研究利用6×His tag與鎳離子的親和性，探討變性蛋白直接在管柱中進行重新摺疊的可行性，同時藉由個別酵素比活性的測定，追蹤TrxA-GST3融合蛋白重新摺疊的回復率，並與經透析重新摺疊的回復率比較。結果發現TrxA-GST3融合蛋白N端的thioredoxin以透析法或直接在Ni-NTA管柱中進行重新摺疊，其氧化還原活性回復率皆高達50﹪左右，顯示利用Ni-NTA管柱進行變性蛋白的重新摺疊，其回復率有可能達到和透析法相同的回復率。另一方面TrxA-GST3融合蛋白C端的GST-3酵素經過透析，在蛋白濃度0.075 mg/ml時，重新摺疊的回復率可以達到56﹪，然而在Ni-NTA管柱中的回復率，最高只有透析法回復率的六分之一；且透析時蛋白濃度對回復率有顯著的影響，蛋白濃度為0.075-0.185 mg/ml時，GST-3酵素重新摺疊的回復率可達40-56﹪，而蛋白濃度高於或低於此範圍時，回復率就下降至15-25﹪。相較之下，TrxA-GST3融合蛋白N端的thioredoxin經透析重新摺疊的回復率，比較不受蛋白濃度的影響。

Abstract
The Escherichia coli expression system is by far the best way for the over-expression of recombinant proteins. However, one of the major obstacles in utilizing E. coli for over-expressing recombinant proteins arises from inclusion body formation. The over-expressed recombinant proteins appear in an insoluble form. In order to make the recombinant proteins soluble, strong protein denaturant such as 8 M urea or 6 M guanidinium hydrochloride is used. Subsequently, urea or guanidinium hydrochloride is removed gradually by dialysis to allow refolding of the denatured proteins. In this study, by making use of the specific binding between 6×His tag and Ni2+, I explored the possibility of efficient protein refolding upon Ni-affinity chromatography. A chimeric gene transcribed from a T7 promoter for over-expressing a 6×His-tagged TrxA-GST3 fusion protein was constructed. By following the specific activities of thioredoxin and glutathione S-transferase, I could determine and compare the refolding efficiencies quantitatively. Analysis of the results indicated that, either by dialysis or upon Ni-affinity chromatography, the refolding efficiency of the thioredoxin activity could reach as high as approximately 50%. This result suggests that it is possible to achieve the refolding efficiency achievable by dialysis by refolding upon Ni-affinity chromatography. In contrast, while the refolding efficiency of the glutathione S-transferase activity by dialysis could reach 56% (at the protein concentration of 0.075 mg/ml), the refolding in Ni-affinity chromatography was only one-sixth as efficient. Moreover, the recovery of glutathione S-transferase activity by dialysis was apparently influenced by protein concentration. At the protein concentrations of 0.075-0.185 mg/ml, the refolding efficiencies could reach 40-56 %. When the protein concentration was above or below 0.075-0.185 mg/ml, the refolding efficiencies dropped to 15-25 %. On the other hand, the recovery of thioredoxin activity by dialysis was relatively unaffected by protein concentration.

中文摘要 1
英文摘要 2
前言 3
材料方法 8
結果 21
討論 28
參考文獻 33
圖表 38
附錄 50

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