本研究藉由改變溫度的培養或使用不同的誘導因子，找出誘導生產蛋白質的最適化條件，以Eschericha coli合成的重組蛋白質OraSE及GlmE為探討對象。OraSE蛋白質可先後經由疏水性管柱及金屬螯合親和層析管柱分離，得到純的蛋白。另一蛋白質GlmE，也可經由疏水性管柱及陰離子交換管柱分離，達到純化的目的。並比較在不同溫度方式及誘導因子的培養下，所獲得蛋白質產量的差異。

Optimization of the production for recombinant proteins, OraSE and GlmE, was investigated by using several defferent growth conditions in this stusy . OraSE protein was purified by Phenyl – Sepharose HP and Ni2+ - NTA column ; GlmE protein was purified by Phenyl – Sepharose HP and Q – Sepharose FF. Significant difference in protein yield was observed when induction temperature and inducer were changed .

中文摘要.................................................I
英文摘要................................................II
目錄..................................................III
圖目錄...................................................IV
表目錄.....................................................V

第一章	序論............................................1
1.1 前言.................................................1
1.2  T7基因表系統.........................................1
1.3  誘導因子乳糖及IPTG...................................2
1.4  乳糖操縱組 ( The lac operon )..................................3

第二章	實驗材料.....................................5
2.1 實驗藥品.........................................5
2.2 各式溶液組成......................................6
2.3 儀器設備..............................................7

 第三章  OraSE蛋白質最適化生產條件探討................8
3.1  OraSE介紹.....................................................8
3.2  實驗方法.......................................................8
  3.2.1菌種培養....................................................8
  3.2.2 OraSE蛋白質的純化..........................................9
3.3  結果與討論....................................................12
  3.3.1 管柱層析...................................................12
  3.3.2 不同條件下OraSE蛋白質濃度比較.............................15

 第四章  GlmE蛋白質最適化生產條件探討.............................18
4.1  GlmE蛋白質介紹...............................................18
4.2  實驗方法......................................................18
  4.2.1 菌種培養...................................................18
  4.2.2 GlmE蛋白質的純化...........................................19
4.3  結果與討論....................................................23
  4.3.1 管柱層析...................................................23
  4.3.2 不同條件下GlmE蛋白質濃度比較..............................26

第五章	結論.......................................................29

參考資料...........................................................31

圖目錄

圖 1-1乳糖結構式……………………………………………………..2
圖 1-2 IPTG結構式…………………………………………………….2
圖 1-3 乳糖操縱組示意圖……………………………………………..4
圖 3-1 OraSE蛋白質過完疏水性管柱Phenyl-Sepharose HP後層析.圖………………………………………………………………12
圖 3-2 OraSE蛋白質過完Ni2+-NTA column所得之層析圖………..13
圖 3-3 OraSE蛋白質純化過程的電泳膠片………………………….14
圖 3-4 不同OD值加入誘導因子所得濕菌重 ( 2 L )……………….17
圖3-5 不同OD值加入誘導因子單位細胞OraSE蛋白質的量……..17
圖4-1 GlmE蛋白質過完疏水性管柱Phenyl-Sepharose HP後之層析圖………………………………………………………………23
圖 4-2 GlmE蛋白質過完陰離子交換管柱所得之層析圖…………..24
圖 4-3 GlmE蛋白質純化過程的電泳膠片……………………………25
圖 4-4 不同條件下所得GlmE濕菌重………………………………27
圖 4-5 不同條件下所得GlmE蛋白質產量………………………….28




表目錄

表3-1  OD值0.7時不同誘導因子的蛋白質表現量………………..15
表3-2  OD值0.8時不同誘導因子的蛋白質表現量…………………16
表4-1  以37℃培養所得GlmE蛋白質產量.........................................26
表4-2  以降溫方式培養所得的GlmE蛋白質產量…………………..27

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