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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳蕙芬 | |
| dc.contributor.author | Pei-I Lin | en |
| dc.contributor.author | 林佩宜 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:23:05Z | - |
| dc.date.available | 2008-07-29 | |
| dc.date.copyright | 2006-07-29 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | 1. 余建泓, 大腸桿菌 ClpQ 蛋白質的C端為負責其單元體間交互作用的區段. 農業化學研究所碩士論文, 2004.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33047 | - |
| dc.description.abstract | 大腸桿菌ClpQY (HslVU)是一個ATP依賴的蛋白酶，ClpQY蛋白酶包含了具ATPase活性的大蛋白分子和一個小分子的peptidase，ClpQY蛋白酶作為一伴隨蛋白質 (Chaperon) 扮演分解錯誤摺疊的蛋白質，也為一催化分解不正常的蛋白質的蛋白酶角色。ClpQ和ClpY分子由六個單元體，形成一六元環狀結構，ClpQ和ClpY分別由兩個六元環形成一像啞鈴形狀的蛋白質複合體，單一ClpY六元環結合在兩個六元環堆疊成ClpQ的兩側。本篇研究重點在ClpQ單元體間的作用如何形成穩定六元環結構 (oligomerization) 和探討ClpQ C端對ClpQY蛋白酶活性的影響。利用綠膿桿菌的clpQ基因部分區段與大腸桿菌的clpQ基因部分區段做融合的建構，以進行作用區段的確認，實驗得知ClpQ C端後段在其單元體間的作用與功能上的重要性比其中的一段二级螺旋結構 (O-helix) 顯得重要；另外再對C端區段進行隨機點突變，並以ClpQY的基質RcsA對cpsB10-lacZ正向調控的系統進行β-galactosidase的活性測試，和以MMS誘導ClpQY基質SulA觀察對細胞生長的影響結果，藉此分析ClpQ的C端是否負責在大腸桿菌中對其本身具有專一性的辨認能力；使用yeast two-hybrid系統偵測ClpQ突變株之間交互作用的情形幫助了解單元體oligomerization的穩定性。結果顯示ClpQ C端上的突變株對ClpQY蛋白酶功能上的影響並未能與其單元體之間的作用能力有一定的正相關。靠近ClpQ C端後段的三個突變位置Asp154 (D154N)，Cys160 (C160A) 和Tyr162 (Y162A)在與ClpQ/ClpQ聚合一穩定結構的環境下使得ClpQY 蛋白酶活性功能受損。 | zh_TW |
| dc.description.abstract | Abstract
The E. coli ClpQY (HslVU) is an ATP-dependent protease that consists of an ATPase large subunit and a peptidase small subunit. The function of ClpQY protease is as a chaperon in refolding of misfolded peptides and as a protease to catalyse the degradation of abnormal proteins as well. Six identical subunits of both ClpQ and ClpY self-assemble into an oligomeric ring and two rings of each subunit form a dumbbell shape complex, two ClpQ rings surrounded by two ClpY single rings. Construction of the fusion gene of P. aeruginosa and E. coli clpQ was used to analyse the C domain and O-helix within C domain of ClpQ for subunits oligomerization and ClpQY protease function. The further C-terminal region of ClpQ is more important in proteolytic function than O-helix which is likely important to ClpQ subunits oligomerization. The C domain region of ClpQ was randomly mutagenized and the single point mutants were selected for investigation of their in vivo function. In addition, The β-galactosidase assay of which the reporter gene (cpsB10 – lacZ) was activated by RcsA, the substrate of ClpQY, and the accumulation of cell division inhibitor, SulA, which increased the sensitivity to MMS induction of E. coli cells were both used to detect ClpQY protease function. Using yeast two-hybrid system, I explore the in vivo protein-protein interactions of the individual mutant subunits of ClpQ involved in self-oligomerization. Several mutants altering ClpQY protease activity were verified. Mutants Asp154 (D154N), Cys160 (C160A) and Tyr162 (Y162A) carrying mutation located in the C– terminal region of ClpQ and have defective proteolytic function based on cpsB10-lacZ expression and MMS test. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:23:05Z (GMT). No. of bitstreams: 1 ntu-95-R92623023-1.pdf: 862024 bytes, checksum: e7040668979b7b34f3410e601d975314 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 壹、 前言 1
一、 伴隨蛋白質和蛋白酶 1 二、 ClpQY two-component protease 2 三、 ClpQY蛋白酶結構 3 四、 ClpQY蛋白酶與基質的研究 4 (1) SulA (suppressor of ultraviolet sensitivity A) 5 (2) RcsA (regulator of capsule synthesis A) 5 (3) RpoH (σ32) 5 五、 近期相關研究 6 六、 研究目的 6 貳、 研究方法 7 一、實驗材料 7 (一) 菌株及質體 7 (二) 培養基 8 (三) 藥品及試劑 9 (四) 分析軟體 9 (五) 實驗相關核酸引子 9 (六) 器材設備 9 二、實驗方法 10 (一) Mini-preparation of plasmid DNA from bacteria 10 (二) Midi-preparation of plasmid DNA from bacteria 10 (三) Purification of plasmid DNA from sliced gel (Gel-M) 10 (四) Purification of PCR products 10 (五) 基因選殖 (Gene cloning) 10 1. 聚合酶連鎖反應 10 (1) ClpQ hybrid of E. coli and P. aeruginosa DNA片段增幅 11 (2) ClpQ＊ C端隨機突變DNA片段增幅 11 (3) 定點突變clpQ＊ (Gln131Leu/Asp/Ala, Ser125) 的DNA片段增幅 13 2. 限制酶作用 (Digestion) 14 (1) 載體製備 14 (2) 選殖基因製備 14 (3) 接合作用 (Ligation) 14 (4) 轉形作用 (Transformation) 14 勝任細胞的製備 (Competent cells) 14 E. coli transfomation 15 Heat shock transformation 15 Electroporation transformation 16 TSS-Transformation 16 (5) 選殖質體之確認 17 Yeast transformation 17 3. ClpQ C 端突變株篩選 18 4.以cpsB10-lacZ 間接偵測RcsA表現 18 5. Methylmethansulfonate (MMS) 之抗性分析 19 6. LacZ 報導基因於yeast two-hybrid系統中偵測 20 β-galactosidase的活性測試 20 Leu2 expression: Growth test 21 LacZ expression: X-gal test 22 7. SDS蛋白質膠體電泳 22 8. 西方式雜交法 (Western Blotting) 24 (1) 轉印 (Transfer) 24 (2) 免疫雜交 (Hybridization) 25 (3) 免疫呈色反應 (Detection) 26 Hybond ECL (Amersham) 套組 (包括Solution I及Solution II) 26 參、 研究結果 27 一、 以大腸桿菌和綠膿桿菌之雜合蛋白來確認ClpQ C端為分子聚合和ClpQY蛋白酶功能的作用區段 27 1.大腸桿菌和綠膿桿菌ClpQ雜合蛋白與ClpY蛋白酶功能之測試 27 2.大腸桿菌和綠膿桿菌ClpQ雜合蛋白與ClpY蛋白酶之MMS生長測 28 3.LacZ and leu2 expression in yeast two-hybrid system 28 二、 建構ClpQ C端缺失突變株以及功能之測試 29 三、 ClpQ 特定區段 (C端) 隨機點突變 29 1.突變株的篩選 29 2.突變株功能之測試 30 3. ClpQ 點突變株 Gln131 (Proline/Leucine/Aspartate/Alanine) 功能測試 30 4. ClpQ突變蛋白分子單元體聚合 31 肆、 討論 32 一、 以大腸桿菌和綠膿桿菌之雜合蛋白來確認ClpQ C端為分子聚合和ClpQY蛋白酶功能的作用區段 32 二、 ClpQ 特定區段 (C端) 隨機點突變 34 伍、 參考文獻 38 表次 表一、 本研究所使用的菌株及質體 45 表二、 引子對列表 46 表三、 大腸桿菌與綠膿桿菌ClpQ雜合突變蛋白於AC3112中分解基質SulA的MMS生長測試 48 表五、 ClpQ缺失突變株於AC3112中分解基質SulA的MMS生長測試 50 表六、 ClpQ C端隨機點突變株於AC3112分解基質SulA MMS生長測試 51 表七、 ClpQ (Gln131) 點突變蛋白單元體之間的交互作用 52 表八、 ClpQ (Gln131) 點突變株於AC3112分解基質SulA MMS生長測試 53 表九、 ClpQ點突變蛋白單元體間之交互作用情形 54 圖次 圖一、 大腸桿菌ClpQ分子功能區塊圖譜 56 圖二、 大腸桿菌ClpQ胺基酸序列圖譜 57 圖三、 建構大腸桿菌與綠膿桿菌clpQ突變選殖質體 58 圖四、 建構clpQ C端隨機點突變選殖質體 (pBAD33 clpQ*) 59 圖五、 細菌中報導基因cpsB10-lacZ 的表現 60 圖七、 大腸桿菌與綠膿桿菌ClpQ雜合突變蛋白於AC3112中ClpQY蛋白酶活性對cpsB10-lacZ表現的影響 62 圖八、 ClpQ缺失突變株於AC3112中ClpQY蛋白酶活性對cpsB10-lacZ表現的影響 63 圖九、 ClpQ C端隨機點突變株於AC3112中ClpQY蛋白酶活性對cpsB10-lacZ表現的影響 64 圖十、 ClpQ (Gln131) 點突變株於AC3112中ClpQY蛋白酶活性對cpsB10-lacZ表現的影響 65 圖十一、 pBAD24載體上大腸桿菌與綠膿桿菌雜合突變蛋白西方式雜交 66 圖十二、 pB42AD融合ClpQ突變蛋白之西方式雜交 67 圖十三、 pBAD33載體上ClpQ突變蛋白西方式雜交 68 圖十四、 ClpQY蛋白酶3D預測結晶結構圖 69 圖十五、 ClpQ單元體部分區段3D預測結晶結構圖 70 附表(圖) 附表一、 E. coli/P. areuginosa ClpQ 雜合蛋白單元體間交互作用 71 附表二、 ClpQ缺失突變蛋白單元體間交互作用 72 附圖一、 ClpQY蛋白酶四個六元環複合體結構 73 附圖二、大腸桿菌ClpY分子功能區塊圖譜 74 附圖三、 大腸桿菌ClpQY蛋白酶結晶結構圖 75 附圖四、 酵母菌雙雜交系統中報導基因的表現 76 | |
| dc.language.iso | zh-TW | |
| dc.title | 大腸桿菌ClpQ蛋白分子之間的聚合和其C端的功能 | zh_TW |
| dc.title | ClpQ subunit oligomerization and its C domain function in Escherichia coli | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳建德,蔡珊珊,李昆達,林乃君 | |
| dc.subject.keyword | 蛋白酶,大腸桿菌,綠膿桿菌,伴隨蛋白,基質, | zh_TW |
| dc.subject.keyword | ClpQY,HslVU,protease,chaperon,E. coli,P. areuginosa,ClpQ,RcsA,SulA, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-23 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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