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研究生:林耀欽
研究生(外文):YaoChin Lin
論文名稱:竹嵌紋病毒複製酵素之CappingEnzyme重新摺疊對其活性回復的探討
論文名稱(外文):Refolding of the Capping Enzyme of Bamboo Mosaic Virus
指導教授:孟孟孝
指導教授(外文):Menghsiao Meng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:56
中文關鍵詞:竹嵌紋病毒戴帽酵素重新摺疊
外文關鍵詞:Bamboo Mosaic VirusCapping EnzymeRefolding
相關次數:
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竹嵌紋病毒(bamboo mosaic virus)之ORF1能夠轉譯出一大小約155-kDa的複製酵素(replicase),其功能與病毒RNA複製及RNA 5¢端的capping有關。近來已證實全長複製酵素之N端442個胺基酸區域具有guanylyltransferase (GTase)和methyltransferase(MTase)的活性,故稱此N端區域為capping enzyme domain。分別利用酵母菌表現系統及大腸桿菌表現系統表現此capping enzyme domain產生戴帽酵素(capping enzyme)。結果在酵母菌系統中表現的capping enzyme具有GTase與MTase的酵素活性,但蛋白質表現量少且純化回收率低;而在大腸桿菌系統中可表現出大量capping enzyme,但以此表現的蛋白質卻是不具活性的包涵體(inclusion body)。比較此真核跟原核系統對蛋白質活性所造成的差異,可能是因為真核系統中修飾化作用所導致。經過對酵母菌系統所表現的capping enzyme做脂肪酸化檢測及磷酸化對其活性影響的測試,發現此capping enzyme並沒有被脂肪酸化且其磷酸化與否對其活性影響並無絕對地影響;還有根據酵母菌系統所表現的capping enzyme為一胞內膜蛋白質而不是分泌性蛋白質且蛋白質分子量跟預測的相符並沒有大幅地增加,推測此蛋白質亦不被醣基化。基於以上理由推論修飾化作用並不是造成兩系統間蛋白質活性差異的原因,而主要原因可能是蛋白質在大腸桿菌系統中表現時摺疊錯誤或者是大腸桿菌無法提供適當的膜環境給予capping enzyme正確摺疊所引起。所以利用大腸桿菌系統表現出大量capping enzyme,將蛋白質經由變性劑、高溫、高pH的方式處理變性,再經由透析、稀釋、降溫或降低pH來進行重新摺疊,其中並輔以受質GTP及SAM、Ni-NTA resin和不同種類膜的環境,藉由這些物質與蛋白質之間的分子作用力,來幫助蛋白質的重新摺疊,試圖回復該蛋白質應有的酵素活性。希望能藉由此一處理流程能在大腸桿菌系統得到大量且具完整活性的capping enzyme,以利日後蛋白質結晶結構的分析,以期能建立alphavirus-like superfamily capping reaction作用機轉的模型。

Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that is a replicase involved in the replication and formation of the cap structure at the 5¢ end of the viral genome. Previouse study has demonstrated that guanylyltrasferase and methyltransferase activities are in the N-terminal 442 amino acid of the replicase (Li et al., 2001) expressed in Saccharomyces cerevisiae (S. cerevisiae). We used the yeast system or the Escherichia coli (E. coli) system to express the BaMV capping enzyme. Although the yeast-expressed BaMV capping enzyme exhibited enzymatic activities, its expression level was low and its sequent purification was difficult. In contrast, the E. coli-expressed BaMV capping enzyme had high level of expression, but it formed inactive inclusion body. The difference of enzymatic activities between the eukaryotic system and the prokaryotic system probably results from the existence of modification in eukaryotic system. According to the test of lipid modification and the test, the affection of activities by the de-phosphorylation of the yeast-expressed capping enzyme, and the supposition that the capping enzyme is not glycosylated owing to the reasons below: 1.the capping enzyme is not a secreted protein but a membrane protein 2.its molecular weight is not increase enormously out of our forecast, we can conclude that modification in capping enzyme is not the main reason causing the difference of enzyme activities between these two system but the misfolding of the capping enzyme expressed in E.coli system or the lack of proper membrane in E. coli system lead to the misfolding. Using the E. coli system to express a great quantity of BaMV capping enzyme and then BaMV capping enzyme was denatured by detergent, heating and pH treatment. Afterward the protein was refolded by dialysis, diluting, cooling down and adjusting the pH. During the refolding, we also try to use GTP, SAM, Ni-NTA resin and a variety of membrane condition to supply the interactions between molecules to assist the refolding. By refolding via these ways, we try to restore the activity of the capping enzyme expressed in E. coli. Hoping for the acquirement of much and fully active capping enzyme to the later analysis of enzymatic crystal structure, and the establishment of the model of alphavirus-like superfamily capping reaction.

中文摘要 I
英文摘要 II
目錄 IV
圖目錄 VI
表目錄 VII
附表目錄 VIII
壹、前言 1
貳、材料與方法 6
酵母菌系統中蛋白質的誘導表現 6
酵母菌菌體的破碎 6
膜蛋白的粗純化 7
蛋白質的萃取 7
金屬親合性管柱層析 7
蛋白質電泳分析法分析 8
西方點墨法 8
蛋白質的GTase活性分析 9
蛋白質修飾化的檢測-磷酸化的檢測 10
大腸桿菌系統蛋白質的誘導表現 11
大腸桿菌菌體的打破 12
金屬親合性管柱層析 12
蛋白質重新摺疊 13
蛋白質定量 18
參、結果
酵母菌系統中蛋白質的誘導表現 19
酵母菌菌體的打破 19
蛋白質的純化 19
純化蛋白質的活性分析 20
蛋白質修飾化的檢測-磷酸化的檢測 20
大腸桿菌系統蛋白質的誘導表現 21
金屬親合性管柱層析 21
蛋白質重新摺疊 22
肆、討論 27
伍、附圖表 31
陸、參考文獻 48
柒、附錄 52

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