[[alternative]]Study of saccharomyces cerevisiae TEF1p : expression in escherichia coli,and purification

碩士[[abstract]]第一部分研究啤酒酵母菌中ALD4p對粒線體型態所造成之影響，我們分別將建構之ALD4-GFP-pYes2和不含ALD4基因序列之pYes2轉形至酵母菌BJ2168，在誘導其蛋白質表現並利用Mito-Tracker Red FM將粒線體染色後，透過螢光顯微鏡觀察過度表現ALD4p對粒線體型態的影響，結果發現當過度表現ALD4p時，顆粒狀的粒線體比例確實明顯上升，管柱狀的粒線體比例明顯下降，藉由流式細胞儀確認ALD4-GFP蛋白確實大量表現，接著將點突變後的ALD4C324S-GFP-pYes2轉形至酵母菌BJ2168，誘導其蛋白質表現並利用Mito-Tracker Red FM將粒線體染色後，藉由螢光顯微鏡觀察失去活性的ALD4p對粒線體型態之影響。 第二部份我們將啤酒酵母菌中之TEF1基因與pGEX-4T-1酶接後轉形至大腸桿菌BL21中，並表現TEF1-GST融合蛋白，再利用GST純化所需之重組蛋白，而在SDS膠片分析時，TEF1-GST融合蛋白無法大量表現於上清液中，GST純化也無法將其有效地單獨分離出來，而仍含有其它分子量的蛋白質，可能會影響後續之研究，因此我們將已經建構好之TEF1-GST基因再接上帶有His-tag融合蛋白基因之載體pET28c，希望在利用GST純化前，先藉由His-tag純化將欲得到之TEF1-GST-His蛋白與其他可能會影響後續研究之蛋白分離後，再使用GST純化，以提高其純度，並且將表現系統換成酵母菌以使融合蛋白大量表現於上清液。[[abstract]]In part I, we transformed yeast strain BJ2168 with ALD4-GFP-pYes2 and pYes2, and induced protein expression with galactose. We stained yeast mitochondrial by Mito-Tracker Red FM and observed the morphological change under fluorescence microscopy. We found that mitochondria and ALD4-GFP fluorescence overlap partially, indicating that mitochondrial morphological changes may be due to activity of ALD4p. Then we used flow cytometery to analyzed the ALD4-GFP protein quantitatively, and confirmed the expression of ALD4-GFP protein. We also inactivated ALD4p catalytic activity to verify it being the activity rather than the overexpression protein which effects morphological change of mitochondria. In part II, we expressed TEF1-GST fusion protein after constructing TEF1-pGEX-4T-1 into E.coli. TEF1-GST fusion protein could not expressed in supernatant numerous, and still had other protein after we used GST column to purified, and those proteins might affect subsequent research. So we constructed and expressed TEF1-GST-pET28c and used His-tag column to purified before used GST column to avoided those proteins. We wanna constructing TEF1-GST-His-pYES2 into yeast to change expression system, and improve about fusion protein expression in supernatant.[[tableofcontents]]目錄 中文摘要 ................................................................................................................................... I 英文摘要 ................................................................................................................................ III 致謝 .......................................................................................................................................... V 目錄 ........................................................................................................................................ VI 第一章 緒論 ............................................................................................................................. 2 第二章 材料方法 ..................................................................................................................... 4 實驗材料 ............................................................................................................................... 4 菌株 .................................................................................................................................. 4 載體(Vector) ..................................................................................................................... 4 引子(Primer) ..................................................................................................................... 4 純化套件組(Kit) ............................................................................................................... 4 酵素 .................................................................................................................................. 4 實驗藥品 ........................................................................................................................... 5 實驗儀器 ........................................................................................................................... 6 實驗方法 ............................................................................................................................... 6 啤酒酵母菌BJ2168 genomic DNA萃取......................................................................... 6 ALD4基因之引子設計 .................................................................................................... 7 聚合酶鏈鎖反應（Polymerase Chain Reaction, PCR）大量複製目標基因-ALD4 ...... 7 瓊脂凝膠電泳法 ( agarose gel electrophoresis ) ............................................................. 7 PCR產物切膠純化........................................................................................................... 8 PCR產物兩端補adenine residue ..................................................................................... 9 TA-cloning ........................................................................................................................ 9 轉形至大腸桿菌DH5α( transformation ) ........................................................................ 9 大腸桿菌質體萃取 ......................................................................................................... 10 ALD4基因片段與酵母菌表現載體pYes2酶接(ligation) ........................................... 11 啤酒酵母菌BJ2168勝任細胞製備 ............................................................................... 11 轉形至啤酒酵母菌BJ2168 ............................................................................................ 11 啤酒酵母菌BJ2168誘導 ............................................................................................... 12 啤酒酵母菌BJ2168破菌 ............................................................................................... 12 SDS膠體電泳 ................................................................................................................. 13 酵母菌BJ2168粒線體染色 ........................................................................................... 13 ALD4點突變 .................................................................................................................. 14 第三章 結果與討論 ............................................................................................................... 16 ALD4-pGEM-T質體建構 .................................................................................................. 16 ALD4-pYes2質體建構 ...................................................................................................... 16 ALD4-pYes2轉形至啤酒酵母菌BJ2168並誘導蛋白質表現 ........................................ 17 ALD4-pGEM-T質體再建構 .............................................................................................. 17 ALD4-GFP-pYes2質體建構 .............................................................................................. 18 啤酒酵母菌BJ2168粒線體型態變化 ............................................................................... 18 ALD4C324S-GFP-pYes2質體建構 ................................................................................... 19 過度表現ALD4C324S後啤酒酵母菌BJ2168粒線體型態變化 .................................... 19 第四章 結論 ........................................................................................................................... 20 圖與表 .................................................................................................................................... 21 圖一 PCR大量複製ALD4基因片段之瓊脂凝膠電泳分析圖....................................... 21 圖二 PCR產物DNA純化和補adenine residue之瓊脂凝膠電泳分析圖 ..................... 22 圖三 限制酶反應確認ALD4-pGEM-T質體之瓊脂凝膠電泳分析圖 ........................... 23 圖四 ALD4-pGEM-T、pYes2質體分別進行限制酶反應之瓊脂凝膠電泳分析圖 ...... 24 圖五 限制酶反應確認ALD4-pYes2質體之瓊脂凝膠電泳分析圖................................ 25 圖六 以30℃誘導ALD4p蛋白質表現量之SDS膠片分析圖 ....................................... 26 圖七 PCR大量複製不含終止碼的ALD4基因片段之瓊脂凝膠電泳分析圖 ............... 27 圖八 PCR產物DNA純化和補adenine residue之瓊脂凝膠電泳分析圖 ..................... 28 圖九 限制酶反應確認ALD4-pGEM-T質體之瓊脂凝膠電泳分析圖 ........................... 29 圖十 ALD4-pGEM-T、GFP-pYes2質體分別進行限制酶反應之瓊脂凝膠電泳分析圖 ............................................................................................................................................ 30 圖十一 限制酶反應確認ALD4-GFP-pYes2質體之瓊脂凝膠電泳分析圖 ................... 31 圖十二 誘導ALD4蛋白質過度表現之粒線體型態差異 ............................................... 32 圖十三 誘導ALD4蛋白質過度表現之粒線體型態統計圖 ........................................... 33 圖十四 以流式細胞儀對ALD4-GFP蛋白質綠螢光強度之偵測圖 .............................. 34 圖十五 限制酶反應確認ALD4C324S-GFP-pYes2質體之瓊脂凝膠電泳分析圖 ........ 35 圖十六 誘導ALD4C324S蛋白質過度表現之粒線體型態差異 .................................... 36 圖十七 誘導ALD4C324S蛋白質過度表現之粒線體型態統計圖 ................................ 37 表一 大量複製ALD4基因片段之PCR溫度、時間設定表 ......................................... 38 表二 TE/LiOAc/DTT buffer之成分表 ............................................................................. 39 表三 PTLAD buffer之成分表 .......................................................................................... 40 表四 大量複製ALD4C324S-GFP-pYes2質體之PCR溫度、時間設定表 .................. 41 第一章 緒論 ........................................................................................................................... 43 第二章 材料方法 ................................................................................................................... 44 實驗材料 ............................................................................................................................. 44 菌株 ................................................................................................................................ 44 載體(Vector) ................................................................................................................... 44 引子(Primer) ................................................................................................................... 44 純化套件(Kit) ................................................................................................................. 44 酵素 ................................................................................................................................ 44 實驗藥品 ......................................................................................................................... 45 實驗儀器 ......................................................................................................................... 46 實驗方法 ............................................................................................................................. 46 啤酒酵母菌BY4742 genomic DNA萃取 ..................................................................... 46 TEF1、TEF1-GST基因之引子設計 ............................................................................. 47 聚合酶鏈鎖反應（Polymerase Chain Reaction, PCR）大量複製目標基因 ............... 47 瓊脂凝膠電泳法 ( agarose gel electrophoresis ) ........................................................... 47 PCR產物切膠純化......................................................................................................... 48 PCR產物兩端補adenine residue ................................................................................... 48 TA-cloning ...................................................................................................................... 49 轉形至大腸桿菌DH5α( transformation ) ...................................................................... 49 大腸桿菌質體萃取 ......................................................................................................... 50 利用限制酶確認基因片段 ............................................................................................. 50 TEF1基因片段與大腸桿菌表現載體pGEX-4T-1酶接(ligation) ............................... 51 轉形至大腸桿菌BL21 ................................................................................................... 51 大腸桿菌BL21誘導 ...................................................................................................... 51 超音波破菌 ..................................................................................................................... 52 SDS膠體電泳 ................................................................................................................. 52 第三章 結果與討論 ............................................................................................................... 53 TEF1-pGEM-T質體建構 ................................................................................................... 53 TEF1-pGEX-4T-1質體建構 .............................................................................................. 53 TEF1-pGEX-4T-1質體於大腸桿菌BL21中之蛋白質表現 ........................................... 54 TEF1-pGEX-4T-1質體再建構 .......................................................................................... 54 TEF1-GST-pET28c質體建構 ............................................................................................ 55 第四章 結論 ........................................................................................................................... 56 圖與表 .................................................................................................................................... 57 圖十八 PCR大量複製TEF1基因片段之聚脂凝膠電泳分析圖 .................................... 57 圖十九 PCR產物DNA純化和補adenine residue之瓊脂凝膠電泳分析圖 ................. 58 圖二十 限制酶反應確認TEF1-pGEM-T質體之瓊脂凝膠電泳分析圖 ........................ 59 圖二十一 TEF1-pGEM-T、pGEX-4T-1質體分別進行限制酶反應之瓊脂凝膠電泳分析圖 ........................................................................................................................................ 60 圖二十二 限制酶反應確認TEF1-pGEX-4T-1質體之瓊脂凝膠電泳分析圖 ................ 61 圖二十三 TEF1-GST蛋白表現量SDS分析圖 ............................................................... 62 圖二十四 TEF1-GST蛋白利用GST純化後之蛋白質表現量SDS分析圖 ................. 63 圖二十五 PCR大量複製TEF1-pGEX-4T-1基因片段之瓊脂凝膠電泳分析圖 ........... 64 圖二十六 PCR產物DNA純化和補adenine residue之瓊脂凝膠電泳分析圖 ............. 65 圖二十七 限制酶反應確認TEF1-GST-pGEM-T質體之瓊脂凝膠電泳分析圖 ........... 66 圖二十八 TEF1-GST-pGEM-T、pET28c質體分別與限制酶反應之瓊脂凝膠電泳分析圖 ........................................................................................................................................ 67 圖二十九 限制酶反應確認TEF1-GST-pET28c質體之瓊脂凝膠分析圖 ..................... 68 表五 大量複製TEF1基因片段之PCR溫度、時間設定表 ........................................... 69 參考資料 ................................................................................................................................ 70[[note]]學號: 600160534, 學年度: 10