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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊健志 | |
dc.contributor.author | "Pei-Chi, Chiu" | en |
dc.contributor.author | 邱佩琦 | zh_TW |
dc.date.accessioned | 2021-06-08T04:13:54Z | - |
dc.date.copyright | 2010-08-19 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22215 | - |
dc.description.abstract | AtMAPRs 家族蛋白質包含 AtMAPR2、AtMAPR3、AtMAPR4 和 AtMAPR5。在 E1、E2、ATP 和泛素的環境中,GST-AtMAPR3 和 GST-AtMAPR5 的重組蛋白質可接上泛素 (林盈涵, 2009)。由於以上的實驗中沒有作 GST 的負控制組,因此以胞外泛素化修飾實驗分析 GST。經由免疫染色分析發現,GST (26 kD) 上方有一分子量上升之色帶 (35 kD),此結果可推論GST 可能具有自身泛素化活性。進一步利用 pull down assay 分析 GST 與泛素可能之交互作用,並利用軟體模擬 GST 與泛素交互作用之模式。此外,利用胞外泛素化修飾實驗,分析不同長度之 His-AtMAPR3 (AtMAPR3, AtMAPR3ΔTM 和 AtMAPR3ΔTMΔC) 和 His-AtMAPR5 (AtMAPR5ΔTM 和 AtMAPR5ΔTMΔC)的自身泛素化活性,其中只有 His6-AtMAPR5ΔTM (70-220 a.a.) 觀察到可能具有胞外自身單一泛素化活性。GST是否會影響 His6-AtMAPR5ΔTM (70-220 a.a.) 的自身泛素化活性,還待進一步的實驗驗證。 | zh_TW |
dc.description.abstract | In Arabidopsis, AtMAPR family consists of four members which are AtMAPR2, AtMAPR3, AtMAPR4 and AtMAPR5. In the presence of E1, E2, ATP and ubiquitin, recombinant proteins of GST-AtMAPR3 and GST-AtMAPR5 are modified by ubiquitin. To study the effect of GST on this reaction, purified GST protein was subjuected to in vitro ubiquitination assay. A shifted protein band of 35 kD in addition to the GST (26 kD) appeared on a immunoblot of SDS-PAGE stained with anti-GST antibody. This result suggested that GST alone may be subjected to autoubiquitination. Pull-down analysis was needed to study the possible interaction between GST and ubiquitin. Protein-protein docking was performed to simulate this interaction. On the other hand, the autoubiquitination activity of His-AtMAPR3 (AtMAPR3, AtMAPR3ΔTM and AtMAPR3ΔTMΔC) and His-AtMAPR5 (AtMAPR5ΔTM and AtMAPR5ΔTMΔC) were also examined by in vitro ubiquitination assay, but only His6-AtMAPR5ΔTM (70-220 a.a.) showed auto-monoubiquitinaiton activity. In order to clarify whether GST affects the auto-monoubiquitination activity of His6-AtMAPR5ΔTM (70-220 a.a.) or not, more experiments should be done in the future. | en |
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dc.description.tableofcontents | 謝誌 II
摘要 IV Abstract V 縮寫表 IX 第一章 緒論 1 1.1 泛素-蛋白酶體系統 1 1.1.1 26S 蛋白酶體 1 1.1.2 泛素 (ubiquitin) 2 1.1.3 泛素化修飾 3 1.1.3.1泛素活化酶 (ubiquitin activating enzyme, E1) 4 1.1.3.2泛素銜接酶 (ubiquitin conjugating enzyme, E2) 4 1.1.3.3泛素接合酶 (ubiquitin ligase, E3) 4 1.1.3.4去泛素酶 (deubiquitinating enzymes, DUBs) 7 1.1.3.5 泛素化修飾形式 7 1.1.3.6聚泛素化修飾與單一泛素化修飾之比較 9 1.1.4 泛素結合區塊 (Ubiquitin-binding domains, UBDs) 11 1.1.4.1 泛素結合區塊-泛素的交互作用 12 1.2 模式植物:阿拉伯芥 (Arabidopsis thaliana) 16 1.3 AtMAPR 家族 17 1.4 研究動機與方向 18 第二章 材料與方法 20 2.1 儀器設備 20 2.2 實驗材料 22 2.2.1 載體 (Vectors) 22 2.2.2 菌種 24 2.3 實驗藥品 25 2.3.1 一般化學藥劑 25 2.3.2 酵素 25 2.3.3 培養基 25 2.4 DNA 相關之基本操作方法 26 2.4.1 質體DNA 之抽取 26 2.4.1.1 質體 DNA 小量製備 26 2.4.1.2 質體 DNA 中量製備 27 2.4.2 DNA 限制酶切割反應 (Restriction enzyme digestion) 28 2.4.3 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 28 2.4.3.1選殖目標基因 28 2.4.3.2 菌落檢定 (colony PCR) 或一般PCR反應 29 2.4.4 DNA 瓊脂糖膠體電泳 (Agarose gel electrophoresis) 29 2.4.5 DNA之定量 30 2.4.6 大腸桿菌之質體轉形-熱衝擊轉形法 (heat-shock transformation) 30 2.4.7 大腸桿菌菌種保存 30 2.5 蛋白質相關之基本操作方法 31 2.5.1 蛋白質電泳檢定 31 2.5.1.1 SDS 膠體電泳 31 2.5.1.2 梯度膠體電泳 33 2.5.1.3 膠片染色法-Coomassie Brilliant Blue R-250 (CBR)染色法 35 2.5.1.4 膠片乾燥法 36 2.5.2 蛋白質轉印 36 2.5.3 免疫呈色 36 2.5.4 Bradford 蛋白質定量法 37 2.5.5 各目標基因重組蛋白質表現與純化 38 2.5.5.1 重組蛋白質建構 38 2.5.5.2 HsUBE1 表現與純化 39 2.5.5.3 His-tagged之重組蛋白質表現與純化 41 2.5.5.4 GST-tagged 之重組蛋白質表現與純化 42 2.5.6 胞外泛素化修飾(In vitro ubiquitination assay) 43 2.5.7 Glutathione S-transferase (GST) pull down assay 44 2.5.8 LC-MS/MS 分析 45 2.5.8.1 In-gel digestion 45 2.5.8.2 LC-MS/MS 分析 45 第三章 結果與討論 47 3.1 GST-AtMAPR3 與 GST-AtMAPR5 之胞外自身泛素化活性 (in vitro autoubiquitination activity) 47 3.1.1 胞外泛素化修飾分析 (In vitro ubiquitination assay) 47 3.2 探討 GST-tag 與泛素之關係 48 3.2.1 GST 之 E3-independent 胞外自身泛素化活性測定 48 3.2.2以 pull down assay 分析GST-泛素交互作用 49 3.2.3 GST 與泛素交互作用模式預測 49 3.2 AtMAPRs 之 E3-independent 自身泛素化活性測定 50 3.2.1 His-tag AtMAPR3 胞外自身泛素化活性測定 51 3.2.1.1 His10-AtMAPR3 可能不具有胞外自身泛素化活性 51 3.2.1.2 His6-AtMAPR3ΔTM (70-233 a.a.) 可能不具有胞外自身泛素化活性 52 3.2.1.3 His6-AtMAPR3ΔTMΔC (70-167 a.a.) 可能不具有胞外自身泛素化活性 52 3.2.2 His-tag AtMAPR5 胞外自身泛素化活性測定 53 3.2.2.1 His6-AtMAPR5ΔTM (74-220 a.a.) 可能具有胞外單一自身泛素化活性 53 3.2.2.2 His6-AtMAPR5ΔTMΔC (74-167 a.a.) 可能不具有胞外自身泛素化活性 54 3.3 AtMAPRs 之潛在自身泛素化活性 54 3.3.1 胞外泛素化修飾分析 His-tag AtMAPRs 不具自身泛素化活性之原因 54 3.3.2 GST-tag 對 AtMAPRs 自身泛素化之影響 55 3.3.3 AtMAPR2 與泛素交互作用模式預測 56 3.3.4 AtMAPRs 為 E2 受質的可能性 57 3.4 結論 57 第四章 未來展望 60 4.1 探討AtMAPRs 是否具有自身泛素化活性 60 4.2 尋找 GST 和 AtMAPRs 的 UBD 序列 60 4.3探討 AtMAPRs 與不同 E2 之間的專一性 61 4.4 AtMAPRs於內噬體的泛素化相關研究 61 參考文獻 63 圖與表 71 附錄 83 | |
dc.language.iso | zh-TW | |
dc.title | 泛素化修飾分析方法之再探討 | zh_TW |
dc.title | Studies of analysis method of ubiquitination | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇仲卿,王愛玉,陳佩燁,章為皓 | |
dc.subject.keyword | 泛素化, | zh_TW |
dc.subject.keyword | ubiquitination, | en |
dc.relation.page | 105 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2010-08-15 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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