蛋白質會由許多種方式來修飾，其中一種為蛋白質的甲基化，而形成蛋白質甲基化的酵素為甲基轉移酶，甲基轉移酶是將S-adenosylmethionine (S-腺甲硫氨酸)所提供的甲基轉移到蛋白質上。蛋白質的甲基化作用是一種轉譯後修飾常發生於polypeptide chain，它會調節生物的生理作用，包括DNA、RNA的代謝、蛋白質的生合成及訊息傳導。

   Ime2p是只在啤酒酵母菌（Saccharomyces cerevisiae）進行減數分裂時才會表現的protein kinase，而Ime2p會刺激早期、中期、晚期的減數分裂基因的表現，IDS2則是刺激Ime2p作用的基因,兩者之間在功能上相互影響。

    而根據以往文獻的報導，IDS2p具有甲基轉移酶之特徵序列。我們將啤酒酵母菌（Saccharomyces cerevisiae）IDS2基因建構到大腸桿菌的Rosseta 菌株中作蛋白質表現，利用His-tag融合純化出所需要的IDS2p，並及將△IDS2菌株之蛋白質抽取液作受質，加入氚化甲基S-腺苷甲硫氨酸[C3H3]S-adenosy-L-methionine一起進行甲基化反應，接著將受質蛋白質加以分離。將受質蛋白質以直立式凝膠電泳isoelectric focusing (IEF)進行第一維的分離,然後將分離的受質蛋白質取下進行SDS-PAGE的第二維電泳，最後我們將分離出的受質蛋白質取下進行質譜儀的鑑定分析。

Proteins can be modified in several ways by the addition of methyl groups from S-adenosylmethionine. Methylation regulates a variety of biological functions including DNA and RNA metabolism, protein synthesis and signal transduction.

Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression. IDS2 (for IME2-dependent signaling) has a functional interaction with Ime2p.

According to recent studies, the sequence IDS2, is homologous to a methyltransferase. In our studies, we constructed the sequence IDS2 of Saccharomyces cerevisiae and overexpressed IDS2p in E. coli Rosetta (DE3) strains, and purified IDS2p with His-tag. The protein extract from △IDS2 strain was methylated with the cosubstrate [C3H3]S-adenosy-L-methionine prior to separation. The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel, and we cutted off the spot with labeled protein and separated the protein on SDS-PAGE. Finally the labeled protein was extracted from SDS-PAGE and identified by mass spectrometry.

目錄
謝誌......................................................I
中文摘要................................................III
英文摘要.................................................IV
目錄......................................................V
簡字表...................................................IX
第一章	前言...............................................1  
1.1酵母菌......................................................................................................................1
1.2 蛋白質的轉譯後修飾.............................................................................................2
1.2.1蛋白質甲基化...................................................................................................2
1.2.2 甲基轉移酶......................................................................................................3
1.2.3 關於IDS2.........................................................................................................3
1.3 實驗目的.................................................................................................................4
第二章	實驗材料...........................................5
2.1 菌種.........................................................................................................................5
2.2培養基......................................................................................................................5
2.3 緩衝液.....................................................................................................................6
2.4 膠片的配製.............................................................................................................8
2.5 實驗藥品與材料.....................................................................................................9
第三章	實驗方法..........................................11
3.1 目標基因YJL146W基因片段之製備................................................................11
3.1.1 野生型酵母菌BY4742 genomic DNA 的製備............................................11
3.1.2 目標基因YJL146w 的Primer之設計.........................................................12
3.1.3 複製放大目標基因 YJL146w .....................................................................12
3.1.4 以洋菜膠電泳進行PCR產物之確認............................................................13
3.1.5 目標基因產物的純化....................................................................................14
3.1.6 A-Tailing…………………………………………………………………….15
3.1.6.1 Gel extraction………………………………………………………….16
3.1.7 基因選殖（TA- cloning）.............................................................................16
3.1.8 勝任細胞之製備............................................................................................18
3.1.9 轉形至勝任細胞............................................................................................18
3.1.10 菌種保存..................................................................................................19
3.1.11 質體DNA的製備.....................................................................................19
3.1.12 使用限制酶處理確認基因片段..............................................................20
3.1.13 定序與序列比對......................................................................................21
3.1.14 表現載體pET28c的製備.........................................................................21
3.1.15 選殖基因的製備......................................................................................22
3.1.16 接合反應..................................................................................................22
3.1.17 轉形至DH5α............................................................................................23
3.2.1 重組質體的蛋白質表現與純化....................................................................23
3.2.2 誘導目標蛋白質表現....................................................................................24
3.2.2.1 添加D-sorbitol .......................................................................................25
3.2.3 生長曲線及各個時間點之蛋白質表現.......................................................26
3.2.4 分析粗抽蛋白質...........................................................................................26
3.2.4.1 高溫煮破法.............................................................................................26
3.2.4.2 超音波破菌法.........................................................................................26
3.2.5 蛋白質SDS-聚丙烯醯胺膠體電泳 ( SDS-PAGE )......................................27
3.2.6 大量表現甲基轉移酶蛋白質........................................................................28
3.2.7 甲基轉移酶蛋白質純化................................................................................28
3.2.7.1 His-tag .....................................................................................................28
3.2.8 蛋白質的濃縮................................................................................................29
3.3 蛋白質甲基化活性測定.......................................................................................30
3.3.1 甲基化受質的製備........................................................................................30
3.3.2 甲基轉移酶酵素活性的測試........................................................................31
3.3.2.1 甲基化反應.............................................................................................31
3.3.2.2 甲基化反應之分析.................................................................................31
3.3.3 利用IEF等電點電泳分析甲基化受質之部位..............................................32
3.3.3.1 樣品前處理.............................................................................................32
3.3.3.2 IEF等電點電泳分析...............................................................................32
3.3.3.3 確認pI值範圍........................................................................................33
3.3.3.4 以cold-SAM 製備大量甲基化受質部位..............................................33
第四章	實驗結果與討論....................................35
4. 重組質體的建構.....................................................................................................35
4.1.1 聚合酶鏈鎖反應（PCR）結果.....................................................................35
4.1.2 T-A ligation 之確認.......................................................................................35
4.1.3 pET28-IDS2重組質體的建構........................................................................36
4.2 蛋白質表現與純化重組蛋白...............................................................................36
4.3 帶有目標蛋白質之重組E. coli之蛋白質表現....................................................37
4.3.1 溫度對目標蛋白質活性的影響....................................................................37
4.3.2 以含有sorbitol 和betaine 的LB 為培養液時之蛋白質表現.....................39
4.3.3 生長曲線與SDS-PAGE分析蛋白表現.........................................................39
4.4 利用His-tag 純化目標蛋白質.............................................................................39
4.4.1 不帶IDS2 基因的E. coli 之蛋白質表現.....................................................40
4.4.2 濃縮目標蛋白質............................................................................................41
4.5 將純化後的酵素進行甲基化反應.......................................................................41
4.5.1 SDS-PAGE 分析............................................................................................41
4.5.2 IEF 等電點聚焦電泳分析.............................................................................42
4.6 甲基化反應之對照組...........................................................................................43
第五章	結論..............................................45
第六章	參考文獻..........................................46
附錄.....................................................64










圖表目錄

圖 1.   PCR目標基因YJL146w之瓊脂凝膠電泳圖..............................................47
圖 2   以EcoR I限制酶切割確認抽出的質體DNA片段.....................................48
圖 3.   YJL146w質體DNA送臺大醫學院基因體中心定序定序結果................52
圖 4.  送入BL21 DE3及Rosetta DE3之後，以限制酶剪切................................53
圖 5.  （1） IDS2p在30℃不同時間點誘導之SDS-PAGE電泳圖.....................54
圖 5.  （2） IDS2p在30℃不同時間點誘導之SDS-PAGE電泳圖.....................55
圖 6.  （1） IDS2p在24℃不同時間點誘導之SDS-PAGE電泳圖......................56
圖 6.  （2） IDS2p在24℃不同時間點誘導之SDS-PAGE電泳圖......................57
圖 7.  （1）IDS2pLB液態培養基添加 sorbitol 和beataine hydrochlori在30℃的生長條件之SDS-PAGE電泳圖....................................................................58
圖 7.  （2）IDS2pLB液態培養基添加 sorbitol 和beataine hydrochlori在30℃的生長條件之SDS-PAGE電泳圖....................................................................59
圖 8.   30℃下誘導之菌體生長曲線圖...................................................................60
圖 9.   IDS2p進行純化後之SDS-PAGE電泳圖...................................................60
圖 10.  不帶IDS2p的pET28c送入Rosetta DE3純化後SDS-PAGE電泳圖.....61
圖 11.  使用蛋白質濃縮管濃縮蛋白質...................................................................61
圖 12.  活性測試之SDS-PAGE電泳圖及底片.......................................................62
圖 13.  活性測試之IEF等電點電泳........................................................................63
圖 14.  IEF等電點電泳更改pI值............................................................................64
圖 15.  切下來的蛋白質條帶進行第2維電泳........................................................65
圖 16.  以不同條件之對照組進行SDS-PAGE電泳圖及底片...............................66

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