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  • 學位論文

啤酒酵母菌ARP1p、GIS1p重組蛋白之轉譯後修飾活性之探討

Study of the post-translational modification activity of recombinant ARP1p, GIS1p of Saccharomyces cerevisiae

指導教授 : 陳銘凱

摘要


蛋白質的修飾和轉錄調控,一直以來都是細胞分子生物學研究的重要課題,蛋白質的作用,作為細胞傳遞訊息的一種方式,尤其在轉譯後的修飾,參與著蛋白質調控的任務,這些修飾作用包含:甲基化(methylation)、去甲基化(demethylation)、乙醯化(acetylation)、去乙醯化(deacetylation)、磷酸化(phosphorylation)、泛素化(ubiquitination)、糖基化(glycosylation),目前已知部分蛋白質甲基轉移酶(methyltransferase)和去甲基轉移酶(demethyltransferase)扮演細胞調控的重要角色,但是其功能和詳細作用,所知依然有限。 根據兩篇不同的文獻資料,我們分別以啤酒酵母菌(Saccharomyces cerevisiae)基因YHR129c、YDR096w作為研究目標,再利用重組蛋白技術,分別表現重組蛋白質在大腸桿菌(Escherichia coli)中,並且設計前後帶有His-tag,純化出重組蛋白,配合去除所表現基因的酵母菌為受質,來研究其甲基化和去甲基化作用。

並列摘要


Protein modification and transcriptional regulation are two areas in cellular and molecular biology. Protein expression is the way of conveying the message of DNA. Modification of the protein includes methylation, demethylation, phosphorylation, acetylation, deacetylation and ubiquitination.Methyltransferase and demethyltransferase play an important role in cell regulation. According to different references, we selected Saccharomyces cerevisiae gene YHR129c and YDR096w as research targets. Using recombinant protein technology, expression of recombinant protein was made in Escherichia coli. We designed the fusion protein with His-tag, and-purified the recombinant protein. Using △YHR129c and △YDR096w yeast the source of substrate and S-adenosyl-L-methionine as co-substrate, I study methylation and demethylation of the respective recombinant proteins.

參考文獻


【1】 Malakhova, O. A., M. Yan, et al. (2003). "Protein ISGylation modulates the JAK-STAT signaling pathway." Genes Dev 17(4): 455-60.
【2】 Martin, J. L. and F. M. McMillan (2002). "SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold." Curr OpinStruct Biol 12(6): 783-93.
【3】 Lee, D. Y., C. Teyssier, et al. (2005). "Role of protein methylation in regulation of transcription." Endocr Rev 26(2):147-70.
【4】 van Leeuwen, F., P. R. Gafken, et al. (2002). "Dot1p modulates silencing in yeast by methylation of the nucleosome core." Cell 109(6): 745-56.
【5】 Friesen, W. J., S. Paushkin, et al. (2001). "The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins." Mol Cell Biol 21(24): 8289-300.

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