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標題: 竹嵌紋病毒三重疊基因區第三轉譯蛋白之表現、純化及特性分析
Overexpression, Purification, and Characterization of the Triple-Gene-Block Protein 3 of Bamboo Mosaic Virus
作者: 周遠霖
Chou, Yuan-Lin
關鍵字: Bamboo Mosaic Virus
Triple-Gene-Block Protein 3
6 kDa membrane protein
出版社: 生物化學研究所
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摘要: 竹嵌紋病毒(Bamboo mosaic virus, BaMV)三重疊基因區(Triple gene block, TGB)所轉譯出的三個蛋白,會參與病毒核酸在宿主胞間的轉移,因此這三個TGB蛋白被稱為移動蛋白。TGB蛋白協助病毒核酸轉移的詳細機轉,目前仍有待解答。本研究的目的是希望藉由對TGBp3生化特性的瞭解,來進一步說明這個蛋白對病毒在宿主胞內移動的可能功能。由胺基酸序列比對分析,推測TGBp3是一個N-端含有疏水性穿膜區域的6 kDa膜蛋白。相對於TGBp1和TGBp2,TGBp3之表現及純化均屬不易,因此一直缺乏此蛋白生化特性相關的研究資料。經比對E. coli密碼子的使用率後,我發現TGBp3中有25%胺基酸密碼為E. coli細胞之稀有密碼子。因此,我將TGBp3 cDNA中的稀有密碼,改換成E. coli常用之密碼,或以能表現這些稀有密碼子 tRNA之E. coli Rosetta(DE3) / pLysSRARE 菌株來生產TGBp3蛋白。E. coli Rosetta(DE3) / pLysSRARE所生產的TGBp3蛋白以TALON親和層析及Tricine SDS-PAGE切膠回收的方式,加以純化,並經蛋白N-端定序無誤。為了進一步證實TGBp3的確為膜蛋白,並研究其生化特性,我利用micelle-vesicle transition的原理製備含有TGBp3的蛋白脂質體(proteoliposome)。製備的蛋白脂質體樣品經Ficoll gradient離心、銀染及西方墨點分析,結果顯示TGBp3和磷脂質在gradient中之分佈是重疊的。進一步利用鹼、尿素及高鹽,對含有TGBp3的脂質體進行化學處理,發現TGBp3主要以嵌入的方式存在於脂質體的雙層膜上,亦即TGBp3可以成功的被重組在蛋白脂質體中。未來,我將繼續分析TGBp3在膜上的拓樸特性,同時探討TGBp3是否與viral RNA、TGBp1、TGBp2或coat protein(CP)等BaMV移動元件具有交互作用能力。
The triple gene block (TGB) of Bamboo mosaic virus (BaMV) encodes three movement proteins, TGBp1, TGBp2 and TGBp3, which are involved in cell-to-cell movement of viral RNA. However, detailed mechanism of the three TGB proteins to assist viral movement remains to be uncovered. The purpose of this study is to analyze the biochemical properties of TGBp3 as well as the possibility of interaction between TGBp3 and other viral movement components. Amino acid sequence analysis of TGBp3 revealed that it is a 6 kDa protein with an N-terminal hydrophobic transmembrane segment and a hydrophilic C-terminal region. Moreover, about 25% of the amino acid codons in TGBp3 are rarely used by E. coli. Therefore, E. coli Rosetta (DE3) / pLysSRARE carrying plasmid-borne tRNA complementary to the rare codons was used as host for TGBp3 expression. In addition, mutant TGBp3 with the rare codons in the whole coding sequence replaced with the frequently used ones was created. The overexpressed TGBp3 was purified by TALON immobilized metal affinity chromatography and a subsequent gel purification protocol. The identity of the purified protein was confirmed by N-terminal sequencing to be TGBp3. To confirm that TGBp3 is really an integral membrane protein, a micelle-vesicle transition theory was adopted to reconstitute TGBp3-containing proteoliposomes. Ficoll gradient centrifugation of the reconstituted sample revealed that TGBp3 was able to co-migrate with phospholipid, suggesting that it is membrane associated. Chemical treatments of TGBp3-containing liposomes revealed that most of TGBp3 is integrated in lipid-bilayer. In the future, the membrane topology of TGBp3 and the interaction between TGBp3 and other viral movement components will be studied.
其他識別: U0005-3107200622402500
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