Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3907
標題: 以界面活性劑提升固定化金屬親和吸附材進行蛋白質復性效率之研究
Detergent-assisted protein renaturation with immobilized metal affinity adsorbents
作者: 張智瑋
Chang, Chih-Wei
關鍵字: protein renaturation;蛋白質復性;artificial chaperone;IMAC;界面活性劑;固定化金屬親和吸附材
出版社: 化學工程學系所
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摘要: 
中文摘要

以大腸桿菌大量生產重組差向異構酶,其中大部分的差向異構酶是以內函體(inclusion body)的形式存在。本研究以尿素(8M urea, 0.1M phosphate)溶解系統於固定化金屬親和吸附材復性(IMAC)中添加假伴隨物(artificial chaperone),探討對於相對可溶蛋白值回收率、相對比活性和總活性之影響。
實驗中使用的假伴隨物(artificial chaperone)系統為(CTAB, β-CD)與(Triton X-100, β-CD)。而蛋白質復性的流程,首先將溶解的內函體溶液以固定化金屬親和吸附材(Ni-NTA)進行吸附,再加入含有界面活性劑的復性緩衝溶液,使變性蛋白質與界面活性劑形成複合物,最後加入含有β-CD的復性緩衝溶液,移除和蛋白質結合的界面活性劑,此時差向異構酶在固定化金屬親和吸附材上重新摺疊復性。比較於不同的復性方式更可顯示AC-IMAC的優點,它可抑制蛋白質聚集現象,增加蛋白質的正確摺疊,而本實驗以artificial chaperone(Triton X-100,β-CD)的添加有較佳的效果,可得到差向異構酶總活性63%,不但優於直接稀釋法的18% 和artificial chaperone的 23%,也高於IMAC的 32%。

Abstract

Over-expression of recombinant GlcNAc 2-epimerse in Escherichia coli system was formed with inclusion body. In this study, the artificial chaperone-assisted immobilized metal affinity adsorbents (AC-IMAC) was applied to urea solubilization systems for investigating the effect of relative soluble protein recovery and relative specific activity. The artificial chaperone system was composed of detergent (cetyltrimethylammonium bromide (CTAB) or Triton X-100) and β-cyclodextrin (β-CD).In the refolding process, denatured protein, which was bound to Ni-NTA through the histidine tag in the IMAC column, and then was mixed by refolding buffer containing detergent to form a protein-detergent complex. Finally, it was a refolding buffer containing β-CD was added to remove the detergent from the bound protein and initiated in situ refolding in the column. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC (Triton X-100, β-CD) led to a total enzyme activity of 63%, in which was not only much higher than direct dilution (18%) and AC-assisted refolding (23%) in bulk solutions, but also superior to its partner, IMAC (32%).
URI: http://hdl.handle.net/11455/3907
其他識別: U0005-1708201117310200
Appears in Collections:化學工程學系所

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