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dc.description.abstract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。zh_TW
dc.description.abstractThe 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.en_US
dc.description.tableofcontents中文摘要 i ABSTRACT ii 目 錄 iii 圖目錄 v 表目錄 vi 縮寫字對照表 vii 第一章、緒 論 1 1.1 前 言 1 1.2 研究動機 4 第二章、文獻回顧 5 2.1 細胞表面系統 5 2.2 革蘭氏陰性菌(gram-neigative bacteria) 7 2.3 冰核蛋白(ice nucleation protein) 9 2.4 蛋白質內含子(intein) 13 2.5 增強型綠螢光蛋白(Enchanced gree fluorescence protein,EGFP) 16 2.6 胜肽(GRA) 16 第三章、實驗材料與方法 17 3.1實驗設備 17 3.2實驗藥品 19 3.3實驗用試劑 21 3.4 實驗流程 27 3.5 重組質體方法 28 3.5.1菌株與質體 28 3.5.2 菌種保存與培養方法 30 3.5.3 大腸桿菌質體DNA抽取 31 3.5.4 電泳分析(Agarose gel electrophoresis ) 32 3.5.5 DNA purification回收 32 3.5.6 剪切反應(Digestion)、黏接反應(Ligation) 33 3.5.7 勝任細胞的製備與轉形作用(Transformation) 34 3.5.8 重組質體之建構 35 3.5.9 Colony PCR 43 3.6 目標蛋白分析方法 43 3.6.1 蛋白質電泳(SDS-PAGE)分析 43 3.6.2 綠螢光蛋白(EGFP)螢光值分析 44 3.6.3 綠螢光蛋白(EGFP)純度分析 44 3.7 在細胞中萃取目標蛋白方法 45 3.7.1 重組蛋白INP-INT-EGFP於Cleavage Buffer反應 45 第四章、結果與討論 46 4.1 建構重組質體pINP-INT-EGFP及其表現 46 4.1.1 建構重組質體pINP-INT-EGFP 46 4.1.2 重組質體pINP-INT-EGFP表現 50 4.2 在不同誘導時間對螢光蛋白質(EGFP)表現影響 52 4.3 不同pH值對螢光蛋白(EGFP)生產之影響 54 4.4 Intein尾端所外加之GRA序列片段移除後對螢光蛋白(EGFP)生產之影響 56 4.5 螢光蛋白(EGFP)生產之最適化條件 58 第五章、結論與展望 61 5.1 結論 61 5.2 未來展望 63 參考文獻 64zh_TW
dc.subjectRecombinant proteinen_US
dc.subjectSurface displayen_US
dc.subjectIce nucleation proteinen_US
dc.titleComparison of INP-INT surface display system in various E. coli host cells for EGFP productionen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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