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    题名: 啤酒酵母菌 ABP140 及 NNT1 重組蛋白之活性及受質研究
    其它题名: Study of the activity and substrates of recombinant ABP140p and NNT1p of saccharomyces cerevisiae
    作者: 李政彥;Lee, Chung-yen
    贡献者: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    关键词: 啤酒酵母菌;甲基轉移酶;S-腺苷甲硫氨酸;ABP140;NNT1;Saccharomyces cerevisiae;methyltransferase;S-adenosyl-L-methionine;ABP140;NNT1
    日期: 2009
    上传时间: 2010-01-11 02:30:42 (UTC+8)
    摘要: 隨著人類基因體計劃的完成,生命科學的研究從基因體學漸漸轉變成蛋白質體學的領域,其中又以蛋白質的修飾及轉錄調控兩部分為主要研究指標。蛋白質的甲基化在這兩部份佔了重要的位置,它是一種常見的轉譯後修飾行為,已知有些蛋白質甲基化扮演重要的細胞調控角色,然而大部分的蛋白質甲基化所產生的功能目前還所知有限,目前依然有許多蛋白質甲基化所需的轉移酶尚未找到。

    啤酒酵母菌( Saccharomyces cerevisiae )的 ABP140 和 NNT1 基因具有甲基轉移酶之特徵序列。本研究為探討 ABP140 和 NNT1 是否具有甲基轉移酶活性及其可能的受質,本研究將啤酒酵母菌中的 ABP140 及 NNT1 基因分別構築到大腸桿菌( Escherichia coli )中,並使其分別表現重組蛋白,利用His-tag融合純化法分別純化出所需之 ABP140 及 NNT1 重組蛋白,並分別以去除 ABP140 及 NNT1 基因的啤酒酵母菌( 以下簡稱 ΔABP140 及 ΔNNT1 )為受質,以具有放射性的S-腺苷甲硫氨酸( S-adenosyl-L-methionine )為輔質來分別測定ABP140 及 NNT1 重組蛋白之活性。利用等電點聚焦電泳( Isoelectric Focusing electrophoresis ; IEF )和十二基硫酸鈉-聚丙烯醯胺膠體電泳( Sodium dodecyl sulfate–polyacrylamide gel electrophoresis ; SDS-PAGE )方式分離可能受質後,以質譜儀鑑定出其可能受質產物。本研究證實 NNT1 確實具有甲基轉移酶活性,而 ABP140 重組蛋白目前尚無法利用本實驗方法證實其具有甲基轉移酶活性。
    As humen genome project is finished, the research of life science has changed its paradigm from genomics to proteomic from genomic gradually. The study of protein modifications and transcriptional regulation has strated to dominate the research head -lines. Protein methylation plays a central role in both of these fields, and it is a post -translation modification of frequent occurrence. Although in many cases the roles of protein methylation are poorly understood, some have been known to play regulatory roles in the cell . Up to now, there are still many protein methyltransferase of protein methylation that remains to be identified .

    The sequences of ABP140 and NNT1 of Saccharomyces cerevisiae match the se -quences of methyltransferase in the database. In this study, we planned to find the activity and substrate of ABP140p and NNT1p. We constructed ABP140 and NNT1 into E. coli to express ABP140p and NNT1p. We used His-tag column to purify ABP140p and NNT1p. The activity is tested by a reaction containing ABP140p and NNT1p, protein extract from ΔABP140 and ΔNNT1 yeast strains and the cosubstrate S-adenosyl-L-methionine of which the methyl being transferred is radioactive. We used isoelectric focusing electrophoresis ( IEF ) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate substrates being methylated. We then used MALDI-TOF to identified the substrates. The result showed the NNT1 is a protein methyltransferase. However our method can not see the activity of ABP140p.
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