啤酒酵母菌SEE1p在大腸桿菌中的蛋白質表現,純化及甲基化活性

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Title:	啤酒酵母菌SEE1p在大腸桿菌中的蛋白質表現,純化及甲基化活性
Other Titles:	SEE1p of Saccharomyces cerevisiae : protein expression in Escherichia coli, purification and methylation activity
Authors:	簡晏生;Jian, Yan-sheng
Contributors:	淡江大學生命科學研究所碩士班陳銘凱;Chern, Ming-kai
Keywords:	啤酒酵母菌;甲基轉移酶;S-腺苷甲硫氨酸;Saccharomyces cerevisiae;methyltransferase;S-adenosyl-L-methionine
Date:	2010
上传时间:	2010-09-23 16:10:05 (UTC+8)
摘要:	人類基因體計劃完成後，生命科學的研究慢慢從基因體學轉變到蛋白質體學的領域。蛋白質的修飾及轉錄調控成為兩個重要研究主題。蛋白質的甲基化在這兩部份佔了重要的地位，此為一種常見的轉譯後修飾行為，目前已知部分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還所知有限，依然有許多蛋白質甲基化所需的轉移酶尚未找到。對於SEE1這段不確定的酵母菌開放讀碼框(open reading frame;ORF)，假定它能在細胞質裡轉錄出一個28 kDa大小的功能性蛋白質。再經由序列的比對，我們推測它是一株S-腺苷甲硫胺酸甲基轉移脢(S -adenosyl-L-methionine methyltransferase)。因此我們利用重組蛋白技術，將啤酒酵母菌中的SEE1基因構築到大腸桿菌(Escherichia coli ; E.coli)中並使其分別表現重組蛋白，並利用His-tag融合純化法純化出所需之重組蛋白，並以去除SEE1基因的啤酒酵母菌(簡稱ΔSEE1)為受質，以具有放射性的S-腺苷甲硫氨酸（S-adenosyl-L-methionine）為輔基質來測定See1p重組蛋白之活性。並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，以質譜儀定序出其可能受質產物。 As humen genome project is finished, the research of life science has changed its paradigm from genomics to proteomics gradually. The study of protein modifications and transcriptional regulation has started to dominate the research headlines. Protein methylation plays a central role in both of these fields, and it is a post-translational 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 methyltransferases for protein methylation that remains to be identified . The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein,recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. We constructed SEE1 into E. coli to express See1p . We used His-tag column to purify See1p. The protein extract from ΔSEE1 yeast strains was mixed with See1p 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 identify the substrates.
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