麩胺酸2,3胺基異位酶(Glutamate 2,3-Aminomutase, G2,3-AM)是一種以B6(PLP)與S-adenosyl methionine(SAM)作為輔酵素的蛋白質，其PLP、鐵、硫化物含量，以及催化反應機制所需要SAM皆與離胺酸2,3胺基異位酶(Lysine 2,3-Aminomutase, 2,3-LAM)相同，且兩者有類似的UV-visible光譜圖，推測兩者也擁有類似的催化反應機制。
為了使麩胺酸2,3胺基異位酶未來能夠有更完善的反應機制研究，本篇論文也著重在利用將含有此蛋白質的基因轉殖至E.coli，並找尋有效的大量培養方法，使其破菌後能夠表現蛋白質，最後藉由快速液相層析儀純化出麩胺酸2,3胺基異位酶。
由實驗結果顯示，在大量培養時利用Lysogeny broth(LB)作為培養基，且利用加入多量的FeCl3與在第16小時時加入IPTG誘發蛋白質，最後藉由快速液相層析儀透過疏水性層析法與陰離子交換層析法成功地純化出麩胺酸2,3胺基異位酶。

Glutamate 2,3-AAminomutase (G2,3-AM) is a protein with B6 (PLP) and S-adenosyl methionine (SAM) as coenzymes. The PLP-, SAM, iron-, and sulfide-content of glutamate 2,3-Aminomutase and requirement for SAM are the same as lysine 2,3-aminomutase (2,3-LAM). Also that the UV-visible spectrum of glutamate 2,3-aminomutase is similar to lysine 2,3-aminomutase. So we speculate that both have similar catalytic reaction mechanisms.
In order to enable more research about reaction mechanisms of glutamate 2,3-aminomutase to be completed and proved in the future, this thesis focuses on that transform gene which contains this protein into E.coli, and then find some effective method of culture, so that the protein can be expressed after breaking the bacteria. The glutamate 2,3-aminomutase was purified by FPLC at the end.
The results show that Lysogeny broth (LB) was used as the medium for culture, and the protein was induced by adding IPTG at 16th hour and a large amount of FeCl3 at the beginning of culture. Finally, glutamate 2,3-aminomutase can be purified successfully by using FPLC with Phenyl and QFF.

謝誌　　　i
摘要　　　ii
Abstract　　　iii
目錄　　　iv
圖目錄　　　vi
第一章 緒論　　　1
1.1 研究背景　　　1
1.2 研究目的　　　1
第二章 文獻回顧　　　3
2.1 蛋白質　　　3
2.1.1 酶　　　3
2.1.2 輔酶　　　4
2.2 麩胺酸 2,3 胺基異位酶　　　4
2.2.1 G23AM簡介　　　5
2.2.2 G23AM之輔酵素　　　6
2.2.3 G23AM之反應機制　　　8
第三章 實驗儀器與原理　　　11
3.1 層析法　　　11
3.1.1 快速液相層析儀(Fast Performanse liquid chromatography, FPLC)　　　11
3.2 電泳膠片分析法　　　14
3.3 紫外光-可見光分光光度法　　　16
第四章 實驗材料與方法　　　19
4.1 製備菌種　　　19
4.1.1 轉殖大腸桿菌(E.coli DH5α)　　　19
4.1.2 轉殖大腸桿菌(E.coli BL21(DE3))　　　20
4.2 養菌　　　21
4.2.1 劃盤　　　21
4.2.2 小量培養　　　22
4.2.3 大量培養　　22
4.2.4 收菌　　　23
4.3 破菌　　　23
4.4 純化　　　26
4.5 SDS-PAGE 電泳分析　　　29
第五章 實驗結果與討論　　　33
5.1 養菌-生長曲線及蛋白質電泳表現　　　33
5.1.1 以LB為培養液，量測不同時間加入IPTG的生長曲線　　　33
5.1.2 以LB/TB為培養液　　　35
5.1.3 增加接種的小培液/提高FeCl3濃度　　　36
5.1.4 在加入IPTG時補充2X/4X FeCl3　　　37
5.2 純化　　　38
5.2.1 Phenyl疏水交換層析　　　38
5.2.2 Q-FF陰離子交換層析　　　39
第六章 結論與總結　　　43
參考文獻　　　45

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