殘留之二硫代蘇糖醇與尿素對溶菌酶復性之影響

Abstract

蛋白質進行復性程序前需先進行變性溶解的步驟，故在進入復性程序時，變性程序中所存在的各種藥劑會一起進入復性系統中，對於復性結果產生影響。96孔盤活性測試具有可快速且大量獲得溶菌酶活性的優點，因此，本實驗經由直接稀釋法進行溶菌酶的復性，並搭配96孔盤活性測試（0.8g/L的基質，10秒/90秒的測量時間，基質對於溶菌酶之體積比例為200μL比10μL，待測溶菌酶濃度0.01g/L~0.1g/L），探討變性程序中還原態二硫代蘇糖醇（DTTRed）的殘留對於溶菌酶復性所造成之影響，也由復性程序中尿素濃度的改變，探討復性環境中尿素濃度對於溶菌酶性活性回復率之影響。 大小排阻層析法（Size Exclusion Chromatography，SEC）可測出溶菌酶與DTTRed進行變性24小時後所殘留之DTTRed，只要知道初始DTTRed與初始溶菌酶濃度（Lyi），即得知氧化態DTT（DTTOxi）濃度而進一步算出殘留之DTTRed。在直接稀釋法結果中，溶菌酶之活性回復率隨著殘留DTTRed濃度不同呈現三種趨勢：(a)氧化還原對控制區 (b)聚集體控制區 (c)殘留DTTRed控制區，調整各控制區內的最大影響因子即可使得活性回復率提升。活性回復率與溶菌酶的濃度為反比關係，加入尿素可有效減少聚集體並提升活性回復率，1M尿素使得最終復性溶菌酶小於0.16g/L時有80%以上之活性回復率；2M尿素使得最終復性溶菌酶小於0.5g/L時有75%以上之活性回復率；3M尿素所提供之疏水性作用力過多，但可有效提升1g/L以上最終復性溶菌酶的活性回復率。實驗結果顯示，聚集體形成極快，變性溶菌酶接觸復性液瞬間就有可能因聚集體的產生降低活性回復效果，當最終尿素濃度為1M且初始變性溶菌酶高於10g/L時，初期聚集體的量使得活性回復率下降；最終尿素濃度為2M時，只要初始尿素濃度大於1.76M，初始變性溶菌酶在25g/L以下，復性初期形成的聚集體不會對活性回復率產生影響。

Before the renaturation process, proteins need to be denatured and dissolved. Thus, when entering renaturation process, the denaturing chemicals are carried over into the refolding system and affected the performance of renaturation process. In this investigation, we used the direct dilution method to refold lysozyme, and measured the activity recovery by an efficient 96 well microplate method (0.8g/L Micrococcus lysodeikticus, 10s/90s measuring time, substrate and lysozyme volume ratio of 200μl and 10μl, for the lysozyme concentration range 0.01g/L~0.1g/L.). We examined the effect of the carried-over DTTRed from the denaturation process on the refolding performance. The effect of urea on the refolding of lysozyme was also explored by varying the concentration of urea in the refolding condition. Size exclusion chromatography (SEC) was applied to determine the carried-over DTTRed after lysozyme denaturation. With the initial concentration of DTTRed and lysozyme（Lyi）, we could calculate the concentration of DTTOxi by the equation , and accordingly the concentration of the carried-over DTTRed. From the results of direct dilution method, the relationship among lysozyme activity recovery, lysozyme concentration, and carried-over DTTRed concentration could be divided into three regions including the redox control region, aggregate control region, and DTTRed control region. An improvement in the activity recovery could be achieved through the proper regulation of the contributing factors in each region. Generally speaking, the activity recovery was inversely proportional to the lysozyme concentration and the addition of urea could reduce the aggregation. 1M of urea helps to recover the activity of lysozyme up to final concentration of 0.16g/L with more than 80% yield. 2M of urea could recover the enzyme activity up to 0.5g/L with the yield more than 75%. Although 3M of urea resulted in stronger hydrophobic interaction, it could still recover the activity of lysozyme of final concentration more than 1g/L efficiently. According to our experimental results, the rapid formation of aggregates would occur as soon as the denaturated lysozyme was in contact with the refolding buffer. With 1M of final concentration of urea and 10g/L of initial concentration of lysozyme, the amount of initial aggregates would lead to a reduction in activity recovery. However, a better inhibition of initial aggregate formation was observed when the final concentration of urea was 2M and initial concentration of urea was above 1.76M. We believe our work may contribute to a better design of protein refolding processes.