I.酵母菌YNL035C 在大腸桿菌中的蛋白質表現，純化及甲基化活性 II.酵母菌NNT1p自甲基化活性探討

I. Selfmethylation activity of yeast NNT1p II. YNL035Cp of Saccharomyes cerevisiae: expression in Escherichia coli, purification, and methylation activity

魏政育(Cheng-Yu Wei)

指導教授 ： 陳銘凱

淡江大學/理學院/化學學系碩士班/碩士(2011年)

https://doi.org/10.6846/TKU.2011.00025

摘要

蛋白質會由許多種方式來修飾，其中ㄧ種為蛋白質甲基化，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-腺甲硫胺酸 (S-adenosylmethionine)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，他會調節生物的生理作用，包括DNA、RNA的代謝、蛋白質的生成及訊息傳導。已知有些蛋白質甲基化扮演重要的調控角色，然而大部份的蛋白質甲基化所產生的功能還所知有限，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 calsbergenesis)的YNL035C基因具有甲基轉移酶之特徵序列，將其構築到大腸桿菌(Escherichia coli)中，使其表現重組蛋白，利用His-tag純化法純化出所需蛋白YNL035Cp，再以去除掉YNL035C基因的啤酒酵母菌為受質，以具有放射性的S-腺甲硫胺酸 (S-adenosylmethionine)為輔質測定其活性。以目前實驗的結果並未觀察到YNL035Cp具有甲基轉移之活性。而NNT1p經之前學長實驗結果顯示出可能具有甲基轉之活性，並懷疑可能為自甲基化。所以將NNT1p進行突變使其失去活性，經由具有放射性的甲基轉移反應結果指出，NNT1p可能具有甲基轉移之活性，並可能為自甲基化。

關鍵字

啤酒酵母菌 ； 甲基轉移酶 ； S-腺苷甲硫氨酸

並列摘要

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. 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 methyltrnasferases for protein methylation that remains to be identified.
The sequence of YNL035C of Saccharomyces cerevisiae matches the sequences of methyltransferase in the database. In this study, We constructed YNL035C into E.coli to express YNL035Cp. We planned to find the activity and substrate of YNL035C. We used His-tag column to purify YNL035Cp. The activity is tested by a reaction containing YNL035Cp , protein extract from ΔYNL035C yeast strains and the consubstrate S-adenosyl-L-methionine of which the methyl being transferred is radioactive. However our method could not see the activity of YNL035Cp. The experimental results of the previous senior student showed the NNT1p had the activity of methyltransferase and may be self-methylated. So we mutated NNT1p for the elimination of activity. According to the result of the radioactive methyl transfer reaction, NNT1p may have the activity of methyltransferase for self-methylation.

並列關鍵字

Saccharomyces cerevisia ； Methyltransferase ； S-adenosyl-L-methionine

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