復性緩衝液的組成對蛋白質摺疊與聚集體生成之影響

Abstract

摘要 本實驗以直接稀釋法進行長時間蛋白質復性，探討復性液組成變化對於蛋白質摺疊結構及聚集體生成的影響。運用分析方法包括活性測定及280 nm的吸收值測量，直接或間接計算各種狀態之蛋白質量。並使用穿透式電子顯微鏡觀察蛋白質的聚集形態，及Congo Red染色法討論蛋白質摺疊結構，藉此瞭解蛋白質摺疊路徑中分子內作用力與分子間作用力之關係。 實驗中首先以Tris-HCl緩衝液，或加入1.2mM GSH，或加入1.2mM GSSG進行稀釋法復性，觀察復性液中各成份影響蛋白質摺疊，發現於Tris-HCl緩衝液和加入GSH環境下復性的蛋白質，分子間作用力為主導蛋白質摺疊主要因素，而在加入GSSG環境下復性時，分子內與分子間作用力產生相互競爭。其次調整復性液中GSH與GSSG之組成比例(固定總濃度2[GSSG]+[GSH]=3.6mM)，控制A280/A260比例為0.1~0.3，且添加低濃度尿素進行長時間(六天)蛋白質復性，結果發現低濃度尿素可延緩聚集體形成的速率，並於不同時間下形成各類的聚集體﹔添加2M尿素可完全抑制聚集體生成，使反應逐漸朝原態結構方向反應；當復性液中A280/A260比值為0.3（即[GSSG]/[GSH]=1.2mM/1.2mM）時，蛋白質活性回收率由65%(復性四小時)逐漸增加為96%(復性六天後)，並由比較結果得知復性液中添加GSH可增加10%左右之活性回收率。

Abstract This study involved the investigations of lysozyme refolding and aggregates refolding by direct dilution. The renaturation of active protein and aggregate was determined by activity measurement, absorbance at 280nm, together with Congo Red binding assay, ThT fluorescence, TEM and optical measurement. To understand the competition behavion between the protein refolding and aggregation, we investigated the effect of different constituents of renaturation buffer on refolding performance by direct dilution method. Experiments results showed that low concentration urea addition in the renaturation process could suppress protein aggregation to stimulate the pathway of correct refolding at high protein concentration. Chang [2004] proved that the maximum refolding efficiency occurred in 60 minute with renaturation buffer while maintaining [GSSG]/[GSH]≧1 the ratio A280/A260 above 0.3 for various protein concentrations. Subsequently, the refolding experiments of renaturation buffer with various combination of [GSSG]/[GSH] and urea concentration were carried out for 6 days. The results showed that activity recovery increased gradually from 65% to 96% by 10-fold dilution at the concentration 1.2mM/1.2mM [GSSG]/[GSH] and 2M Urea of renaturation buffer during 6day incubation.