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  • 學位論文

探討精胺酸甲基化對於CNBP功能的影響與建構在大腸桿菌中大量表現哺乳類動物精胺酸甲基化蛋白質的系統

Functional studies of arginine methylation of cellular nucleic acid binding protein And Establishment of a system to overexpress mammalian arginine-methylated proteins in Escherichia coli

指導教授 : 李娟

摘要


第一部分 Cellular nucleic acid binding protein (CNBP) 為分子量18.4 kDa 之單股核酸結合蛋白。CNBP在結構上,同時具有7個Cys-Cys-His-Cys (CCHC) 鋅手指 (zinc finger domains) 和一個 arginine/glycine (RG) rich區域。實驗室先前的研究結果顯示,CNBP可與PRMT1發生直接的交互作用,而且GST-CNBP重組蛋白可以被PRMT1甲基化。在細胞實驗中,辨識精胺酸甲基化的抗體可以辨認全長的 FLAG-CNBP 蛋白質,然而RG rich deletion (ΔRG) 的 FLAG-CNBP則無法被此抗體所辨認。在本篇研究中我們觀察了HeLa細胞內源性及外源性的CNBP在細胞中的分佈情形,結果發現不論細胞處理AdOx或是CNBP RG deletion與否,CNBP在細胞中的分佈情形皆沒有顯著的變化,因此我們認為CNBP的RG rich區域和蛋白質精胺酸甲基化修飾並不影響CNBP在細胞中的表現位置。在CNBP的RG rich 區域中含有R25,R27,R30,R32以及R34 一共5個arginine,我們製備了His-CNBP-RK1 (R25K , R27K) 以及His-CNBP-RK2 (R30K , R32K , R34K) 兩種突變的重組蛋白來進行 in vitro 蛋白質甲基化實驗,根據實驗結果推測CNBP主要的甲基化修飾位點是在R25或R27上。進一步探討精胺酸甲基化是否會影響CNBP與RNA的結合能力,結果發現,以AdOx抑制了甲基化後的FLAG-CNBP與RNA的結合力較強。 我也以CNBP重組蛋白做進一步的RNA結合能力分析,然而E. coli系統中並不會表現PRMT,直接純化出來的CNBP重組蛋白是不會受到甲基化修飾的,因此我嘗試使用co-transformation的方式將帶有PRMT1以及CNBP基因的質體同時送入E. coli中,再從E. coli中純化出已受甲基化修飾的CNBP重組蛋白。使用CNBP重組蛋白進行RNA結合實驗的結果發現,已受甲基化修飾的CNBP重組蛋白與RNA的結合力有大幅下降的趨勢。這些研究結果有助於初步的了解精胺酸甲基化對於CNBP蛋白質的功能性調控。 第二部份 由於精胺酸甲基轉移酶 (Protein arginine methyltransferase, PRMT) 所催化的精胺酸甲基化修飾作用只存在於真核生物中,然而想要從真核生物中取出已受甲基化修飾的蛋白質難度相當的高。因此我們想利用原核生物容易培養且能夠生產大量標的蛋白的優勢,嘗試在大腸桿菌中建立能夠生產甲基化修飾蛋白的系統。我們選用實驗室經常使用的哺乳類PRMT1為甲基轉移酶以及其已知受質Fibrillarin RG-rich (GAR)區域作為甲基接受蛋白來建立雙表現系統,以pET-28b載體上原有的T7 promoter誘導His6-GAR表現,並以pGEX-4T1系統的tac promoter做為雙表現載體的第二組啟動子,誘導GST-PRMT1表現。以此系統純化所得的His6-GAR能夠被精胺酸甲基化專一性抗體ASYM24所辨識,說明了這個表現系統的可行性,期望將來能夠運用此系統來生產實驗室所需的甲基化蛋白。

並列摘要


1. Cellular nucleic acid binding protein (CNBP) is a single-stranded nucleic acid binding protein with seven Cys-Cys-his-Cys (CCHC) zinc fingers and an arginine / glycine (RG) rich region between zinc finger 1 and 2. Previous studies showed direct interaction of CNBP with PRMT1and methylation of recombinant CNBP by PRMT1. Full-length but not RG deletied (ΔRG) FLAG-CNBP protein can be recognized by methylarginine-specific antibodies. In this study, we analyzed the distribution of endogenous and exogenous CNBP in HeLa cells. Regardless of the treatment of a methylation inhibitor AdOx or the RG deletion, there were no significant changes of CNBP distribution in cells. Therefore, the CNBP RG rich region and protein arginine methylation modification does not affect the localization of CNBP in cells. We further explored whether arginine methylation can affect the RNA binding capacity of CNBP. AdOx treatment appeared to strengthen the RNA binding of FLAG-CNBP. Recombinant His6-CNBP proteins expressed from E. coli cells co-transformed with plasmids expressing GST-PRMT1 or not were prepared as arginine-methylated or unmethylated protein. Purified His6-CNBP protein can be recognized by methylarginine-specific antibodies. Non-methylation His6-CNBP appeared to strengthen the RNA binding. There are five arginines (R25, R27, R30, R32, and R34) in the RG rich area of CNBP. We prepared His6-tagged CNBP-RK1 (R25K, R27K) and RK2 (R30K, R32K, R34K) mutant protein for in vitro protein methylation. The results suggest that the major CNBP methylation sites are R25 or R27. These findings contribute to the understanding of arginine methylation of CNBP. 2. Isolation of a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies can be laborious and difficult. In this study we developed an expression system to produce recombinant proteins enriched in NG,NG-asymmetry dimethylarginines. We first put a mammalian type I protein arginine methyltransferase gene (PRMT1) on a plasmid under the control of the Escherichia coli methionine aminopeptidase (MAP) promoter for constitutive expression, and also the protein targeted for arginine methylation on the same plasmid behind a T7 promoter for inducible expression of His6-tagged proteins. Fibrillarin RG-rich (GAR) segment was used as the model protein to examine this expression system. There are fifteen arginines within the arginine-glycine-rich motif of GAR near the N terminus of fibrillarin that can be methylated. We could not get successful modification of the recombinant His6-GAR with either human or rat PRMT1 under the control of the MAP promoter or the tac promoter on the same plasmid. Nevertheless, we obtained arginine-methylated recombinant His6-GAR whenPRMT1 was expressed behind the tac promoter with GST-fusion as in its original configuration in the pGEX-PRMT1 plasmid. Recognition of His6-GAR by methylarginine-specific antibodies indicated that we successfully established an expression system in E. coli to overproduce mammalian proteins that can be methylated by PRMT1. The arginine methylated proteins can be used in future studies for further characterization of the function of arginine methylation of the proteins.

並列關鍵字

CNBP arginine-methylated proteins

參考文獻


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