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系統識別號 U0002-0408200814031800
中文論文名稱 HSL7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 HSL7 gene expression in Escherichia coli,protein purification,and methylation activity
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 96
學期 2
出版年 97
研究生中文姓名 廖道學
研究生英文姓名 Tao-Hsueh Liao
學號 695180462
學位類別 碩士
語文別 中文
口試日期 2008-07-15
論文頁數 60頁
口試委員 指導教授-陳銘凱
委員-譚鳴輝
委員-林賜恩
中文關鍵字 甲基轉移酶 
英文關鍵字 methyltransferase  S-adenosylmethionine 
學科別分類 學科別醫學與生命科學生物學
中文摘要 在蛋白質甲基化的轉譯後的修飾反應中,甲基加到polypeptide上,而催化這個反應進行的酵素是甲基轉移酶,甲基轉移酶主要是利用S-adenosylmethionine當做甲基的給予者,這可以修飾在polypeptide的親核性氧、氮、和硫原子上,這會形成甲基酯、甲基氨、甲基醯氨,會在20種氨基酸之中的其中9種氨基酸的側鏈上接上甲基。而其中的2種arginine的甲基化是在arginine的側鍊上形成單一(mono)或雙甲基化(dimethylated),此反應可以在myelin basic protein上發現,或在nuclear proteins, ribosomal protein上也可發現,而根據以往文獻的報導,HSL7p是一個甲基轉移酶,而且具有in vitro蛋白質甲基化的活性,文獻也指出,HSL7基因在蛋白質表現在Yeast中時所表現出的HSL7p是甲基轉移酶,可以在calf thymus histone H2A 的arginine上形成甲基化,而我們的實驗目的是將HSL7基因用兩種方式在E.coli中做蛋白質表現,且證明它具有蛋白質甲基轉移酶的活性。
英文摘要 In the reaction of protein methylation as a post- translation modification, addition of a methyl group occurs to the polypeptide chain.The enzyme that catalyzes these reaction is termed protein methyltransferase , which mainly utilizes S-adenosylmethionine as the methyl donor and can modify a variety of nucleophilic oxygen,nitrogen and sulfur atoms on the polypeptide chain. These processes result in methyl esters, methyl amines, methyl amides and other derivatives on the side chains of nine of the 20 common amino acids. Two of the arginine methylation are mono- and dimethylated arginine .This reaction can be found in myelin basic protein, nuclear proteins, and ribosomal protein. According to recent papers, HSL7p is a methyltransferase and had in vitro protein methyltransferase activity. The literature indicated that HSL7 gene protein expressed in yeast can methylate the arginine of calf thymus histone H2A.

論文目次 目錄
致謝………………………………………………………………………………………….………....Ⅰ
中文摘要…………………………………………………………………….………………………….Ⅱ
英文摘要…………………………………………………….………………………………………….Ⅲ
縮寫檢索表………..……………………………………………………………………………………Ⅳ
目錄………………………………………….………………………………………………………….Ⅴ
圖目錄………..…………………………………………………………………………………………Ⅶ
1.序論……………………………………………………………………………………………………1
1.1研究動機…………………………………………………………………………………………….2
2.材料和方法………………………….…………………………………………………………………3
2.1設計primer …………………………………………………………………………………………3
2.2聚合酶鏈反應……………………………………………………………………………………….3
2.3 DNA 瓊膠電泳分析………………….……………………………………………………………4
2.4將 切下的DNA瓊膠進行純化…………………………………..…………………………………5
2.5在純化的產物兩端補A…………………………………….…………………….…………………6
2.6 TA cloning………………………………………………….…………………………………..…..7
2.7 將TA cloning 產物transformation至DH5α…………………...………………………………..7
2.8藍白篩選………………………………………………………….…………………………………8
2.9菌種保存……………………………………………………….……………………………………9
2.10抽Plasmid……………………………………………….…………………………………………9
2.11以限制酶切 plasmid確認………………………….……………………………………………10
2.12Ligation………………………………………...……………………………………..………….12
3. 以E. coli表現HSL7蛋白質..……………………..…………………………………………………15
3.1 BL21(DE3)的蛋白質表現………………………………………………………..………………15
3.2 tRNA380和Rosetta的誘導…………………………………………………….…………………16
3.3超音波破菌步驟………………………………………………………...…………………………17
3.4以18℃誘導的結果………………………………………………………...………………………18
3.5將Rosetta以sorbitol-betaine誘導……………………………………...…………………………19
3.6純化所需的條件和試劑…………………………………………...………………………………20
3.7純化步驟…………………………………………………………...………………………………21
4.將HSL7基因接到pMAL-c2x vector上…………………………...………………………...………23
4.1BL 21(DE3)的蛋白質表現……………………………………..…………………………………27
4.2tRNA380和Rosetta的蛋白質表現………………………………..………………………………28
4.3BL21(DE3),tRNA380和Rosetta的蛋白質純化………………………….………………………29
4.4純化步驟…………………………………………………………...………………………………29
4.5用BugBuster純化…………………………………………………………………………………30
4.6改成以18℃來誘導……………………………………………………………………………….31
4.7抽取細胞核……………………………………………………………………………………….32
4.8以Hydroxyapatite進行純化………………………………………………………………………33
5.甲基化活性測試…………………………………………………………………………………….36
6.實驗結果…………………………………………………………………………………………….38
6.1蛋白質表現結果………………………………………………………………………………….38
6.2細胞核分離結果………………………………………………………………………………….39
6.3Histone純化結果…………………………………………………………………………………39
6.4放射線標定結果………………………………………………………………………………….39
7討論……………………………………………………………………………………………………40
7.1蛋白質表現討論………………………………………………………………………………….40
7.2細胞核分離討論………………………………………………………………………………….40
7.3Histone純化討論…………………………………………………………………………………40
7.4放射線標定討論………………………………………………………………………………….41
8.參考文獻…………………………………………………………………………………………….42
9.附圖………………………………………………………………………………………………….45

圖目錄
附圖1:PCR完HSL7的電泳圖…………………………………………………..…45
附圖2:HSL7接在pGEMT-easy vector,再以EcoRI切………………………….45
附圖3:pET28c vector以EcoRI切一刀的圖,以NdeI切一刀……………….……46
附圖4:接在pGEMT-easy vector上的 HSL7,以EcoRI和NdeI切兩刀的圖…....46 附圖5: pET28c vector以CIAP切5端磷酸根的圖…………………...…………..47
附圖6:將HSL7和pET28c進行Ligation的圖…………………………………….47
附圖7:將HSL7和pET28c Ligation,再以EcoRI切一刀,大小為7800bp的圖….48
附圖8: 將HSL7和pET28c Ligation,再以EcoRV切…………………..…………48
附圖9:HSL7接到pMAL-c2x,再以EcoRV切兩刀…………………..…………….49
附圖10:以18℃誘導的蛋白質表現圖………………………...……………………49
附圖11:以50 ml離心管純化的HSL7p………………………...………………….50
附圖12:以4管50 ml離心管所純化的HSL7p…………………………………….50
附圖13: 改變條件所得HSL7p…………………….………………………………51
附圖14:HSL7接到pMAL-c2x,以30℃誘導的蛋白質表現圖………..…………..51
附圖15: HSL7接到pMAL-c2x,以LB和LB+sorbitol的蛋白質表現圖………..52
附圖16: HSL7接到pMAL-c2x的純化圖………………………………..………..52
附圖17:以 bugbuster純化的圖…………………………………...……………….53
附圖18: HSL7接到pMAL-c2x以18℃誘導後,進行蛋白質純化的圖……..……53
附圖19:抽取出的細胞核直接加熱100℃煮破的圖……………………….……….54
附圖20:甲基化活性測試,在壓片之前的電泳圖…………………….…………….54
附圖21:放射線標定的圖片…………………………………………………..……..55
附圖22:同上圖,放射線標定圖片…………………………………………………..55
附圖23: 抽取出的細胞核,尚未經Dounce homogenizer拉過的圖片…………..56
附圖24: 以Dounce homogenizer拉過的細胞核圖片,可得到較多的細胞核…..56
附圖25:以Hydroxyapatite純化的圖………………………………………………57
附圖26:基因型…………………………………………………………………...….57
附圖27:pET28c vector map………………………………………………………...58
附圖28:pMAL-c2x vector map………………………………………………….…58
附圖29:在未壓片前的電泳圖……………………………………………….………59
附圖30:放射線標定後的圖………………………………………………….………59
附圖31:將附圖30曝光後的圖…………………………………………………..…60


參考文獻 參考文獻
【1】.Tina Branscombe MIRANDA, Joyce SAYEGH, Adam FRANKEL1, Jonathan E. KATZ, Mark MIRANDA and Steven CLARKE2,Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A.Biochem J.(2006) May 1;395(3):563-70
【2】.Myriam Ruault and Lorraine Pillus1,Chromatin-modifiying enzymes are essential when the Saccharomyces cerevisiae morphogenesis checkpoint is constitutively activated. Genetics. (2006 )Nov;174(3):1135-49. Epub 2006 Sep 1.
【3】.Jin-Hyung Lee, Jeffry R. Cook, Brian P. Pollack, Terri Goss Kinzy, David Norris, and Sidney PestkaHsl7p, the Yeast Homologue of Human JBP1,Is a Protein Methyltransferase,Biochemical and Biophysical Research Communications 274, 105–111 (2000)
【4】.Brian P. Pollack, Sergei V. Kotenko, Wen HeLara S. Izotova, Barry L. Barnoski, and Sidney Pestka,The Human Homologue of the Yeast Proteins Skb1 andHsl7p Interacts with Jak Kinases and Contains Protein Methyltransferase Activity:THE JOURNAL OFBIOLOGICAL CHEMISTRYVol. 274, No. 44, Issue of October 29, pp. 31531–31542, (1999)
【5】. Steven Clarke ,Protein methylation Current Opinion in Cell Biology 1993, 5:977-983
【6】.X J Ma,Q Lu and M Grunstein,A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae,Genes Dev 10 (1996) 1327-1340
【7】.ATSUSHI FUJITA,AKIO TONOUCHI,TAKATOSHI HIROKO,FUMIKA INOSE,TAKEYUKI NAGASHIMA,RIKA SATOH,AND SHIGEKO TANAKA,HSL7p,a negative regulator of Ste20p protein kinase in the Saccharomyces cerevisiae filamentous growth-signaling pathway(1999) Proc.Natl.Acad.Sci.USA Vol.96,pp.8522-8527
【8】.MARK J.SHULEWITZ,CARLA J.INOUYE,AND JEREMY THORNER,HSL7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieve Tyrosine Phosphorylation of Cdc28 Protein Kinase in Saccharomyces cerevisiae(1999),MOLECULAR AND CELLULAR BIOLOGY,p.7123-7137
【9】.R.KUCHARCZYK,R.GROMADKA,A.MIGDALSKI,P.P.SLONIMSKI AND J.RYTKA,Disruption of Six Novel Yeast Genes Located on Chromosome II Reveals One Gene Essential for Vegetative Growth and Two Required for Sporulation and Conferring Hypersensitivity to Various Chemicals(1999),Yeast 15,987-1000
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【13】. Victor J. Cid, Mark J. Shulewitz. Kent L McDonald, and Jeremy Thorner, Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle(2001), Molecular Biology of the Cell Vol. 12, 1645–1669
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【18】. Jonathan E. Katz, Mensur Dlakic, and Steven Clarke, Automated Identification of Putative
Methyltransferases from Genomic Open Reading Frames(2003), The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org
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【24】. Kathleen M. Dorgan , Whitney L. Wooderchak , DonRaphael . Wynn Erin L. Karschner , Joshua F. Alfaro , Yinqiu Cui ,Zhaohui Sunny Zhou , Joan M. Hevel , An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases(2006), doi:10.1016/j.ab.2006.01.004
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