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中文論文名稱 酵母菌IDS2基因在大腸桿菌中的蛋白質表現和純化以及甲基化活性
英文論文名稱 Expression, purification, and methylation activity of yeast IDS2p in Escherichia coli
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 97
學期 2
出版年 98
研究生中文姓名 林榆清
研究生英文姓名 Yu-Chin Lin
學號 696180453
學位類別 碩士
語文別 中文
第二語文別 英文
口試日期 2009-07-23
論文頁數 70頁
口試委員 指導教授-陳銘凱
委員-官宜靜
委員-林賜恩
中文關鍵字 IDS2  啤酒酵母菌  甲基轉移酶  IEF 
英文關鍵字 IDS2  ISaccharomyces cerevisiae  methyltransferase  IEF 
學科別分類 學科別醫學與生命科學生物學
中文摘要 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。
英文摘要 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.
論文目次 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

參考文獻 Chen, D., H. Ma, et al. (1999). "Regulation of transcription by a protein methyltransferase." Science 284(5423): 2174-7.


Chern, M. K., K. N. Chang, et al. (2002). "Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2." J Biol Chem 277(18): 15345-53.


Friesen, W. J., S. Paushkin, et al. (2001). "The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins." Mol Cell Biol 21(24): 8289-300.


Goffeau, A., B. G. Barrell, et al. (1996). "Life with 6000 genes." Science 274(5287): 546, 563-7.


Hitzeman, R. A., F. E. Hagie, et al. (1981). "Expression of a human gene for interferon in yeast." Nature 293(5835): 717-22.


Katz, J. E., M. Dlakic, et al. (2003). "Automated identification of putative methyltransferases from genomic open reading frames." Mol Cell Proteomics 2(8): 525-40.


Malakhova, O. A., M. Yan, et al. (2003). "Protein ISGylation modulates the JAK-STAT signaling pathway." Genes Dev 17(4): 455-60.


Martin, J. L. and F. M. McMillan (2002). "SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold." Curr Opin Struct Biol 12(6): 783-93.


Sia, R. A. and A. P. Mitchell (1995). "Stimulation of later functions of the yeast meiotic protein kinase Ime2p by the IDS2 gene product." Mol Cell Biol 15(10): 5279-87.


van Leeuwen, F., P. R. Gafken, et al. (2002). "Dot1p modulates silencing in yeast by methylation of the nucleosome core." Cell 109(6): 745-56.


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