Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3877
標題: 建立INP-INT膜上表現系統並應用於D-Hydantoinase 生產之研究
Development of the INP-INT expression system and its application on D-hydantoinase production
作者: 劉姿岑
Liu, Tzu-Tsen
關鍵字: ecombinant protein;重組蛋白質;ice nucleation protein;intein;hydantoinase;surface display;冰核蛋白;Intein;Hydantoinase;表面表現
出版社: 化學工程學系所
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摘要: 
生產重組蛋白質以往皆利用大腸桿菌可以短時間內快速生長的特性生產目標蛋白,但是純化蛋白質過程瑣碎耗時,並且蛋白質經大量表現後也有可能會產生沒有蛋白質活性(包涵體,inclusion body)的問題,增加生產的困擾。本研究運用細胞表面表現系統直接生產胞外目標酵素,利用經基因剪切修飾後之膜蛋白「冰核蛋白」(ice nucleation protein, INP) ,接上Intein (INT)斷裂蛋白質與目標蛋白重組成融合蛋白,此蛋白可以表現在細胞表面,並且利用Intein斷裂蛋白質對pH值敏感性,進行自我剪切,則D-Hydantoinase (Dht)會從融合蛋白中分離而溶解在Cleavage buffer中。重組蛋白誘導條件為:將含有表現Dht質體的E.coli 在37 ºC,200 rpm下培養,當菌體量達OD600為0.6時,加入IPTG 使其最後濃度為0.05mM,於15 ºC誘導表現24小時。收集經誘導後的菌體,再懸浮於Cleavage buffer (pH6)中,在25 ºC,100 rpm下進行斷裂反應。經HPLC分析後,在搖瓶發酵液中可獲得活性約0.225 U/ml的Dht酵素。利用TotalLab v2.01分析SDS- PAGE中可獲得48.7 %的純度。

The production of recombinant proteins usually employs E. coli due to its fast growth and easy manipulation. However, the purification of the protein is time-consuming and complicated. Meanwhile, over expression of the recombinant protein would result in inactive protein (i.e. inclusion body) and hinder the protein production.
In this study, the cell surface expression system was used to directly produce extracellular enzyme. The genes of the truncated ice nucleation protein (INP) along with intein (INT)and target protein, here is D-Hydantoinase (Dht), were fused together to construct an INP-INT-Dht gene, which was able to produce fusion protein on cell membrane surface.
The induction conditions for E. coli with the constructed plasmid to produce fusion protein are as follows: E.coli was incubated at 37 ºC and 200 rpm till OD600 reached 0.6. Then, IPTG was added to the broth and the induction was conducted at 15 ºC for 24 h. The cell was harvested and resuspended in the cleavage buffer of pH6, 25 ºC, and 100 rpm for 24 h. A centrifugation was performed to remove the cell from the buffer and the supernatant was collected. The harvested Dht with the activity of 0.225 U/ml and a purity of 48.7% was obtained.
URI: http://hdl.handle.net/11455/3877
其他識別: U0005-0908201118591600
Appears in Collections:化學工程學系所

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