Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96365
標題: 利用大腸桿菌共表現阿拉伯芥RuvB-like核酸解旋酶並進行生化特性鑑定
Characterization of RuvB-like DNA helicases of Arabidopsis thaliana co-expressed in E. coli
作者: 莊宗憲
Tsung-Hsien Chuang
關鍵字: 阿拉伯芥
RuvB-likes
AAA+ domain
pCDF-Duet1
Arabidopsis thaliana
RuvB-likes
AAA+ domain
pCDF-Duet1
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摘要: RuvB-like 1(RuvBL1)和RuvB-like 2 (RuvBL2)是屬於Superfamily 6 (SF6)的核酸解旋酶,具有AAA+ (ATPase associated with various cellular activities) domain可以水解ATP並從中獲得能量。SF6解旋酶通常會形成六聚體,RuvBL1和RuvBL2甚至會交錯排列形成兩個六聚體重疊的構造。RuvBLs廣泛存在於多種蛋白質複合體與核酸蛋白複合體中,參與和DNA相關甚至不相關的諸多活動,在酵母菌、線蟲和果蠅中已被證實為生物細胞的必需基因,在哺乳動物細胞的研究上亦被高度重視,但在植物方面,卻少有相關的研究。本實驗擬分析阿拉伯芥RuvBLs的生化特性,檢查其是否具有ATPase與DNA helicase等活性。阿拉伯芥具有一個RuvBL1和兩個RuvBL2基因,分別命名為RuvBL1、RuvBL2-3和RuvBL2-5,兩個RuvBL2蛋白在序列上有高達九成的相似度。先前的研究發現RuvBL1和RuvBL2-5具有相似的表現時空關係,而RuvBL2-3則異於兩者,因此推測前述兩者可能會形成蛋白質複合體。在本實驗中我們採用pCDF-Duet1作為共表現RuvBLs蛋白質的質體,並使用E. coli做為表現蛋白的系統。另外,為了在活性測試中有對照組,我們也設計了一組針對活性中心(walker B domain)突變的蛋白質,稱其為catalytically-inactive (CI) form,理論上不具有ATPase的活性。由於His-tag僅重組在RuvBL2-5的N端,是透過帶有S-tag的RuvBL1與RuvBL2-5形成複合體後,再一起由Ni-NTA共同純化。進一步利用蛋白質變性洗脫的方式搭配SDS-PAGE鑑定兩者的比例,發現僅有少數的RuvBL1與RuvBL2-5形成複合體。在這樣的情況下我們仍嘗試測定ATPase及Helicase的活性,結果發現具有活性的RuvBLs與CI form相比,並沒有觀察到明顯的差異。即使我們將S-tag自RuvBL1上移除,以及將His-tag由RuvBL2-5的N端移動到C端,期望減少複合體組合時的空間阻礙,活性測試的結果依然沒有改善。為了測定RuvBLs真正的酵素活性,我們需要改善RuvBL1的穩定度以及表現量,並且考慮是否要改變蛋白質的表現系統。
RuvB-like 1 (RuvBL1) and RuvB-like 2 (RuvBL2) are members of the superfamily 6 (SF6) helicase that contain core AAA+ (ATPase associated with various cellular activities) domain for coupling chemical energy provided by ATP hydrolysis. The SF6 helicases were found to form hexamer or doubled hexamers with alternated RuvBL1 and RuvBL2. They are present in various protein and nucleoprotein complexes and play important roles in cellular processes including activities unrelated to DNA. Although RuvBLs are essential for viability in yeast, nematode, fruit fly, and are speculated to be also essential in mammalian cells, only very few studies were involved in plants. Here we aim to characterize roles of RuvBL1/2 in Arabidopsis via biochemical analysis. Arabidopsis thaliana possesses one RuvBL1 but two RuvBL2 genes, named as RuvBL1, RuvBL2-3, and RuvBL2-5. Previous studies revealed that the RuvBL1 share similar expression profiles and protein localizations with RuvBL2-5, but not with RuvBL2-3, suggesting protein complex formation between RuvBL1/2-5. Here we employed pCDF-Duet1 vector for co-expression of both proteins in E. coli. To have negative controls in enzyme activity assays, we generated a paralleled construct in which both proteins were mutated on the walker B domain and were catalytically-inactive (CI) theoretically. As the His-tag was fused only at the N-terminus of RuvBL2-5, the co-purified RuvBL1-S-tag proteins were caused by forming a protein complex with RuvBL2-5 in E. coli. Further selective denaturing elution together with SDS-PAGE analysis revealed only a very small amount of RuvBL1 retained by RuvBL2-5. Both ATPase and helicase activities performed for the RuvBL1/2-5 active forms were not significantly higher than that of the inactive form. Although the S-tag was removed from RuvBL1 and the His-tag was moved from the N- to the C-terminus of RuvBL2-5 to avoid steric hindrance in complex formation, their enzyme activities were not improved. To pursuit true enzyme activities, we need to improve the protein stability and quantity of RuvBL1 and will consider to change their expression systems.
URI: http://hdl.handle.net/11455/96365
文章公開時間: 2019-08-24
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