HSL7基因在大腸桿菌中的蛋白質表現和純化以及甲基化活性

HSL7 gene expression in Escherichia coli,protein purification,and methylation activity

廖道學(Tao-Hsueh Liao)

指導教授 ： 陳銘凱

淡江大學/理學院/生命科學研究所碩士班/碩士(2008年)

https://doi.org/10.6846/TKU.2008.00114

摘要

在蛋白質甲基化的轉譯後的修飾反應中，甲基加到polypeptide上，而催化這個反應進行的酵素是甲基轉移酶，甲基轉移酶主要是利用S-adenosylmethionine當做甲基的給予者，這可以修飾在polypeptide的親核性氧、氮、和硫原子上，這會形成甲基酯、甲基氨、甲基醯氨，會在20種氨基酸之中的其中9種氨基酸的側鏈上接上甲基。而其中的2種arginine的甲基化是在arginine的側鍊上形成單一(mono)或雙甲基化(dimethylated)，此反應可以在myelin basic protein上發現，或在nuclear proteins, ribosomal protein上也可發現，而根據以往文獻的報導，HSL7p是一個甲基轉移酶，而且具有in vitro蛋白質甲基化的活性，文獻也指出，HSL7基因在蛋白質表現在Yeast中時所表現出的HSL7p是甲基轉移酶，可以在calf thymus histone H2A 的arginine上形成甲基化，而我們的實驗目的是將HSL7基因用兩種方式在E.coli中做蛋白質表現，且證明它具有蛋白質甲基轉移酶的活性。

關鍵字

甲基轉移酶

並列摘要

In the reaction of protein methylation as a post- translation modification, addition of a methyl group occurs to the polypeptide chain.The enzyme that catalyzes these reaction is termed protein methyltransferase , which mainly utilizes S-adenosylmethionine as the methyl donor and can modify a variety of nucleophilic oxygen,nitrogen and sulfur atoms on the polypeptide chain. These processes result in methyl esters, methyl amines, methyl amides and other derivatives on the side chains of nine of the 20 common amino acids. Two of the arginine methylation are mono- and dimethylated arginine .This reaction can be found in myelin basic protein, nuclear proteins, and ribosomal protein. According to recent papers, HSL7p is a methyltransferase and had in vitro protein methyltransferase activity. The literature indicated that HSL7 gene protein expressed in yeast can methylate the arginine of calf thymus histone H2A.

並列關鍵字

methyltransferase ； S-adenosylmethionine

參考文獻

【1】.Tina Branscombe MIRANDA, Joyce SAYEGH, Adam FRANKEL1, Jonathan E. KATZ, Mark MIRANDA and Steven CLARKE2,Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A.Biochem J.(2006) May 1;395(3):563-70

【2】.Myriam Ruault and Lorraine Pillus1,Chromatin-modifiying enzymes are essential when the Saccharomyces cerevisiae morphogenesis checkpoint is constitutively activated. Genetics. (2006 )Nov;174(3):1135-49. Epub 2006 Sep 1.

【4】.Brian P. Pollack, Sergei V. Kotenko, Wen HeLara S. Izotova, Barry L. Barnoski, and Sidney Pestka,The Human Homologue of the Yeast Proteins Skb1 andHsl7p Interacts with Jak Kinases and Contains Protein Methyltransferase Activity:THE JOURNAL OFBIOLOGICAL CHEMISTRYVol. 274, No. 44, Issue of October 29, pp. 31531–31542, (1999)

【5】. Steven Clarke ,Protein methylation Current Opinion in Cell Biology 1993, 5:977-983

【6】.X J Ma,Q Lu and M Grunstein,A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae,Genes Dev 10 (1996) 1327-1340