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標題: 不同宿主細胞對INP-INT膜上表現系統生產EGFP之影響
Comparison of INP-INT surface display system in various E. coli host cells for EGFP production
作者: 陳啟銘
Chen, Chi-Ming
關鍵字: 重組蛋白;Recombinant protein;冰核蛋白;內含子;綠螢光蛋白;細胞表面表現;Surface display;Ice nucleation protein;Intein;EGFP
出版社: 化學工程學系所
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前人所開發的細胞表面表現系統可直接生產胞外目標蛋白質,利用經修飾後之膜蛋白「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可進行自我剪切之特性,使得目標蛋白可從融合蛋白中分離而溶解在反應液中。
本研究主要探討此膜上表現系統於三種宿主細胞,ER2566、DH1、BL21(LPS free)中生產EGFP的差異,一併比較在不同pH值、不同的融合蛋白誘導時間以及不同斷裂時間下對EGFP產量以及純度的影響。
實驗結果顯示在相同的條件下,誘導時間在24小時EGFP產量以及純度表現比9小時佳。三種不同宿主細胞以大腸桿菌株ER2566表現最佳,在菌體經24小時誘導後,於pH6之條件下反應4小時可得EGFP純度62.5%;於pH7之條件下反應24小時可得EGFP最高產量82.3 mg/L。而在反應液 pH6與pH7之條件下,EGFP產物之產量與純度差異皆不顯著;然於pH7之條件下其產量較高,而於pH6之條件下其純度較高。
將原本系統中 intein 尾端所加之GRA序列片段移除後,其EGFP之產量降為原本之1/3倍,且純度亦降低,顯示GRA序列片段對於目標蛋白質之生產具有正面助益。

The cell surface expression system was constructed to produce extracellular enzyme in E. coli directly. The genes of the truncated ice nucleation protein (INP) along with intein (INT) and target protein, i.e., enchanced green fiuorescence protein (EGFP), were fused together to construct an INP-INT-EGFP gene, which was able to produce protein anchoring on cell membrane surface.
The performance of the production for EGFP with three different host cells, E. coli ER2566, DH1, BL21 (LPS free) was studied in surface expression system. The effects of different induction time, cleavage time and pH values of cleavage buffer on the yield and purity for EGFP were discussed.
From the experiments, it shows that under the same conditions, better performance was obtained for the yield and purity of EGFP under 24 h induction. Among the three different host cells, E. coli strain ER2566 had a better performance than the others. After 24 h induction with the cleavage condition of pH6 for 4 h, 62.5% purity can be obtained. The production of EGFP reached 82.3 mg/L under the cleavage condition of pH7 for 24 h. A higher yield of EGFP was achieved under pH7, whereas a higher purity was achieved under pH6.
The effect of intein segment with or without the additional GRA fragment on EGFP production was also compared. The result shows that with the GRA fragment in the terminal of intein, the yield and purity of EGFP production were both higher than that without GRA fragment. This indicates that the fragment of GRA has a positive effect on the production of the target protein.
其他識別: U0005-2108201213522900
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