https://scholars.lib.ntu.edu.tw/handle/123456789/64198
標題: | 低倍率之溶菌?復性 Low Dilution Refolding for Lysozyme |
作者: | 冼祐安 Sheim, Yu-An |
關鍵字: | 溶菌?蛋白質復性;直接稀釋法;二硫代蘇糖醇;lysozyme;protein refolding;direct dilution;DTT | 公開日期: | 2012 | 摘要: | 變性蛋白質的復性通常是利用不同方法移除變性成分進而使其恢復活性。傳統的直接稀釋法是利用大量的復性劑,稀釋變性成分對蛋白質的作用。本實驗則藉由簡單的氧化還原反應,即利用GSSG以四倍稀釋與變性劑中所殘餘的DTTRed反應降低DTTRed對蛋白質的破壞。由實驗結果顯示採用低倍率直接稀釋法亦可得由如同高倍率稀釋之效果,因而進一步探討因變性殘於的DTTRed與GSSG於復性環境中對復性之影響。 低倍率直接稀釋溶菌酶突破傳統高倍率直接稀釋的方法,在實驗中除了展示四倍稀釋對復性之高效率,更確立DTTRed、GSSG共同存在可提供良好的復性環境。由實驗結果檢視活性回復率受復性系統中的變因包括DTTRed與GSSG及氧化還原試劑對活性之影響。例如因DTTRed過量而無法呈現活性之原因歸為還原態DTT控制所致;並將所有操作溶菌酶濃度依其受復性劑成分影響所呈現的活性回復結果,區分為氧化還原控制區、氧化還原至聚集體過度區、聚集體控制區。於尿素2M可將氧化還原區之溶菌酶,添以適當的GSSG濃度復性可得80%之復性回收率。例如經四倍稀釋後於2M尿素中,最終溶菌酶濃度小於1.5g/L之活性回復率皆可達到80%以上。 聚集體控制區之溶菌酶因濃度過高,故於復性環境中加入尿素可有效地抑制聚集體產生而提升此區溶菌酶的復性回復率,例如3g/L的溶菌酶於2M尿素之環境活性回復率僅有60%,但於3M尿素中則可達70%以上。 Protein refolding is mainly carried out by eliminating denaturant through various methods. Direct dilution method is a traditional one by diluting the denaturant with a large amount of refolding buffer. However, this research added GSSG to react with carry-over DTTRed which was a crucial hinderence to successful protein refolding. The experimental results indicated that low dilution refolding method was as good as, if not better than, that of high dilution refolding method with this strategy. Therefore, this research further discussed the impacts of refolding environmental factors including GSSG and carry-over DTTRed. According to the experiments, there was a relationship between refolding environments and lysozyme activity recovery depending on its concentrations. Namely, it was classified into three regions, including redox control region, transition region, and aggregation control region. For example, in redox control region, the activity of lysozyme up to final concentration of 1.5g/L would regain 80% in 2M urea condition with appropriate GSSG refolding buffer. In addition, lysozyme of higher concentration in aggregation control region usually needed the help of urea to minimize aggregation, and thus obtain better activity recovery. For instance, the activity recovery of the 3g/L final lysozyme concentration by 4-times dilution in 2M urea condition was merely 60%; nevertheless, the activity recovery could be enhanced to 70% in 3M urea. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/252183 |
顯示於: | 化學工程學系 |
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