- 學位論文
變性劑各成份在透析操作中移除方式的設計對溶菌酶復性效果之影響
The Effect of Urea and Dithiothreitol Removal on Refolding of Lysozyme by Dialysis
摘要
以化學方法進行蛋白質復性操作為一多變因之系統。其中還原態DTT與溶菌酶之雙硫鍵及穀胱甘肽氧化還原對皆會發生氧化還原反應，造成原濃度組成隨時間增加而改變，對蛋白質中的雙硫鍵鍵結與復性結果皆會帶來差異及影響。於是使用透析方法改變透析液組成，使透析袋內的變性溶菌酶溶液的成份產生變化，並藉由質傳的計算，得知各變性劑組成在透析過程中的濃度變化情形，故可單獨探討各變性劑成份的作用力對溶菌酶摺疊過程的關聯性。 實驗中發現尿素並非是影響失活溶菌酶發生復性的最主要條件，尿素僅控制蛋白質結構中的疏水性作用力，避免錯誤摺疊產生聚集體，其中在復性緩衝液中最適當的尿素使用濃度為2M。透析時須待還原態DTT完全移除後，才開始測得活性上升的趨勢。在改變變性劑移除順序的實驗中，發現無論先移除尿素或是還原態DTT皆會令復性效果明顯下降。若復性緩衝液中僅添加GSSG時，透析袋內含有還原態DTT的組別復性效果較佳；而當透析袋內不含還原態DTT時，在復性緩衝液中添加GSH，可令活性回復率上升。因此當袋內含有還原態DTT時能與GSSG發生氧化還原反應，產生GSH修正錯誤摺疊的雙硫鍵，提升復性效果。 在本研究中，我們發現若提高GSSG的比例可促使復性速率增加，原使用0.3mM GSSG及3mM GSH作為復性緩衝液成份時，復性1mg/ml溶菌酶8.5小時後得到22.8%之活性回復率；但改用1.8mM GSSG為復性緩衝液成份時，復性8.5小時後可提升至55.1%。並使用兩段式透析作為改良，第一次透析以批次透析法操作，快速降低尿素濃度至2M，接著以連續式透析法透析，將剩餘尿素濃度完全移除，使溶菌酶完全摺疊。以[GSSG]=1.8mM、[GSH]=0.36mM的復性緩衝液進行二段式透析，復性19小時後可得到約88%的活性回復率。因此，採用批次及連續式的二段式透析組合方式復性，除了設備簡易成本便宜外，可大幅縮短透析時間使實驗更有效率，並可避免聚集體產生，最後可得到良好的復性效果。
並列摘要
DTT, one of the widely used denaturants, can interact with oxidized glutathione (GSSG) and the disulfide-containing protein via the thiol-disulfide interchange reaction. This interchange reaction makes protein refolding very complicated and difficult to analyze. Changes in the concentrations of reduced DTT (DTTred), GSSG, and glutathione (GSH) during the process of refolding have considerable impact on the yield of refolding. In this study, renaturation of hen egg-white lysozyme via dialysis was investigated with aid of mass transfer coefficients to understand the changes of urea and DTTred concentrations in the denatured protein solution. We found that urea does not play a crucial role in the refolding of denatured lysozyme due to the fact that urea could only control the intermolecular hydrophobic interaction to suppress protein aggregation. The denatured lysozyme could not be refolded by the removal of urea and an enhancement in the activity of denatured lysozyme was observed upon complete removal of DTTred by dialysis. When GSSG was added in the refolding buffer solution, the yield of activity recovery would be significantly increased if the DTTred still remained in the mixture within the dialysis bag. However, in the case with not DTTred left in the mixture within the dialysis bag, the addition of GSH was found to improve the refolding yield through the disulfide rearrangement mechanism with the involvement of reducing and oxidized reagents. It was also observed that the renaturation of denatured lysozyme was accelerated by increasing the concentration of GSSG in the refolding buffer. For example, when performing dialysis refolding on 1mg/ml denatured lysozyme for 8.5 h, around 22.8% of refolding yield was observed for the refolding buffer of 0.3mM GSSG and 3mM GSH, while ~55.1% of refolding yield was obtained when the refolding buffer contained 1.8mM GSSG. To increase the refolding yield of protein refolding, attempts were made to develop a refolding strategy for dialysis refolding process. First, remove urea rapidly from 8M to 2M by a batch dialysis operation, and then remove urea completely by continuous dialysis method. As a result, the formation of intermediates would be more stable and tend to refold into the native structure. We were able to increase the refolding yield of 1mg/ml denatured lysozyme to 88% after 19 h by using the refolding buffer containing 1.8mM GSSG and 0.36mM GSH. It was concluded that the removal of denaturants from denatured lysozyme solution by batch/continuous dialysis could effectively result in a superior refolding performance.