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    题名: I.酵母菌假設性甲基轉移酶YJR129c蛋白質受質之鑑定分析. II.重組酵母菌醛類去氫酶ALD6p之非降解表現與一步純化
    其它题名: I. Analysis of the protein substrate for putative methyltransferase YJR129c of saccharomyces cerevisiae. II. Non-degraded expression and one-step purification of recombinant ALD6p of saccharomyces cerevisiae
    作者: 夏維志;Shiah, Wei-jyh
    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: 啤酒酵母菌醛類去氫酶;甲基轉移酶;等電點具焦電泳;陰離子交換樹脂;Saccharomyces cerevisiae;aldehyde dehydrogenase;methyltransferase;isoelectric focusing(IEF);anion exchange.
    日期: 2009
    上传时间: 2010-01-11 02:30:11 (UTC+8)
    摘要: I.啤酒酵母菌( Saccharomyces cerevisiae )的開放讀碼框 YJR129Cp 具有甲基轉移酶之特徵序列,且在資料庫中與幽門螺旋菌( Helicobacter pylori )的核糖體蛋白L11甲基轉移酶最為相似,因此本研究以假設性甲基轉移酶 YJR129Cp為酵素,透過 ΔYJR129Cp 之全細胞粗抽蛋白質為受質,以及二維電泳原理,找出 YJR129Cp 在啤酒酵母菌細胞中的受質,目前在本研究以初步蛋白質鑑定此甲基化受質為 Elongation Factor 2(EF-2) 之後會純化出此蛋白質以確定此假設性甲基化受質EF-2是否為YJR129Cp之甲基化受質。並在確定受質後希望進一步分析 YJR129Cp 甲基轉移酶將受質甲基化修飾所具有的生理意義。


    II.在之前學長的研究重組酵母菌醛類去氫酶 ALD6p 並轉型至大腸桿菌( Escherichia coli )的表現載體 BL21,以達到大量誘導表現重組蛋白之目的。但由於之前一直存在目標蛋白質降解之問題,故我們透過降低培養時的溫度以改善目標蛋白質降解問題。另外也利用陰離子交換樹脂Q SepharoseTM Fast Flow ( Amersham Biosciences )採用反向吸入欲純化之樣品,正向洗出雜蛋白質並搭配目標蛋白質pI之適當 pH 值的 Buffer 以達到一步純化( one-step purification )之目的。
    I.The ORF of YJR129Cp of S. cervisiae has a close match with the methyltransferase in database, therefore, in this research the putative methyltransferase YJR129Cp is used as enzyme to find the substrate of YJR129Cp of S. cervisiae by means of the substrate of ΔYJR129Cp of cell lysate as well as 2-D electrophoresis procedure.

    Thus far, the translation factor 2 (EF-2) has been identified as a methylation substrate forYJR129Cp in S. cervisiae by proteomics analysis. Afterward, EF-2 will be purified to make sure the substrate of putative methyltransferase YJR129Cp. Also, after it is confirmed we want to analyze further that methyltransferase YJR129Cp methylates substrate in a way with the physiological significance.

    On the other hand, this research is proceeded with isoelectric focusing (IEF) and SDS-PAGE in a separate manner. For this reason, we are able to congregate a large quantity of focus bands after IEF is analyzed. And we can extract protein from some of them and then concentrate the protein to carry out SDS-PAGE. In this way, even though they are only lightly exposed, the exposed focus bands are still enough by combination of multiple bands. Therefore, we can gather a plenty of protein samples to benefit proteomics analysis.

    II.In the previous research the recombinant ALD6p of S. cervisiae has been overexpressed in E. coli strain. However, the degradation problem persisted. We solved this problem by way of decreasing the temperature of induction. Moreover, we used Q SepharoseTM Fast Flow ( Amersham Biosciences ) and reversed the flow direction of elution relative to that of sample loading, and also referred to ALD6p pI value to select for suitable buffer pH to fulfill the purpose of one-step purification.
    显示于类别:[生命科學研究所] 學位論文


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