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中文論文名稱 酵母菌ALD6基因選殖與多型性、異源表現、純化與酵素特性之探討
英文論文名稱 Cloning and polymorphism of the ALD6 gene and heterologous expression,purification, and catalytic characterization of Ald6p of Saccharomyces cerevisiae.
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 93
學期 2
出版年 94
研究生中文姓名 陳煜群
研究生英文姓名 Yu-Chun Chen
學號 692290322
學位類別 碩士
語文別 中文
口試日期 2005-06-01
論文頁數 160頁
口試委員 指導教授-陳銘凱
委員-王三郎
委員-陳灝平
中文關鍵字 酵母菌ALD6 基因  單核苷  酸多態性  Ald6p 純化  酵素動力 
英文關鍵字 Aldehyde dehydrogenase  Yeast Ald6p  ALD6 gene  Single nulotide polymorphism (SNP) 
學科別分類 學科別醫學與生命科學生物學
中文摘要 自1950 年代,啤酒酵母菌 Aldehyde dehydrogenase (ALDH) 研究至今,已知是種pyruvate dehydrogenase (PDH) bypass 的關鍵酵素。至今,已發現5 個ALDH 成員( ALD 基因)。在細胞質中ALDH 所扮演的角色是在於產生乙醯輔酶 A。根據文獻報告指出此ALD6 基因位在第16 號染色體的 left arm 上, ALD6 基因所轉錄之 Ald6p 即為細胞質的 ALDH。
在本研究中,利用聚合酶連鎖反應 (PCR) 放大欲選殖之ALD6 基因片段。此 DNA 片段大小為 1,506 bp,包含在5'端設計一個限制酶( NdeI)作用反應序列。接著將 ALD6 基因接於pGEM®-T easy vector ,此法稱做TA-Cloning。經質體轉型至 E.coli DH5α取得大量含pGEM-T-ALD6 基因之質體。此外,在pGEM-T-ALD6 質體上含此兩種限制酶( NdeI、SpeI)作用反應序列,透過限制酶反應作用後自pGEM-T-ALD6 質體上得到含有 NdeI 與SpeI 之 ALD6 基因片段。同樣在表現载體 pET-43.1c(+) 也含有此兩種限制酶( NdeI、SpeI)作用反應序列,經過NdeI 與SpeI 作用後,可以得到此兩種限制酶所切下的專一性片段载體DNA。隨後透過接合酶T4 ligase 接合完成表現質體的pET-43-ALD6 的建構。接著再次轉型至 E.coli DH5α取得大量之表現質體 pET-43-ALD6,隨後再轉型送入四種E.coli 表現宿主菌株(BL21(DE3)、BL21pLysS、Rosetta(DE3)、BL21-CodonPlus(DE3)-RIL)以達到大量表現 Ald6p 重組蛋白之目的。在這四種含pET-43-ALD6 質體之表現菌株利用不同的誘發物質、培養溫度、最適培養時間以及初步酵素活性測試結果顯示,以30℃、Lactose 誘導Rosetta(DE3) 最佳,最適條件為誘導後的第12 小時,Ald6p 的反應速率與蛋白質含量皆比其他三種表現菌種來的好。
在已找尋最適表現 Ald6p 重組蛋白之條件後,利用該條件擴大製備 Ald6p。接著進行Ald6p 重組蛋白的純化,本研究使用 Amersham Biosciences 公司純化樹脂:1. Q Sepharose
fast flow 2. Blue Sepharose 6 fast flow。已純化的 Ald6p 利用不同的受質之醛類(Propionaldehyde、4-aminobutyraldehyde、3-aminopropionaldehdye、Betaine aldehyde 等)進行一系列酵素動力分析,求得 Km 與 Vmax 值,藉以了解不同受質之醛類對Ald6p 之間的親合力與催化反應的最大速率。
此外,在ALD6 DNA 序列鑑定分析結果顯示酵母菌菌株 YPH499 Genomic DNA 在2 次獨立的PCR 反應發現皆有5 個相同的突變位置,推測具有單核苷酸多態性 (SNP) 的可能性。為了證明ALD6 之多型性,使用另一種酵母菌菌株 BY4742 的Genomic DNA 進行PCR 反應,經定序結果顯示在YPH499 的突變位置並沒有在 BY4742 出現。因這五個變異皆為啞突變,所以酵母菌ALD6 基因仍是種高度保留序列之DNA。
英文摘要 Since 1950s,Yeast acetaldehyde dehyrogenase (ALDH) has been studied for many years. It is a key enzyme of the pyruvate dehydrogenase (PDH) bypass。Recently, the role of the five members of ALDH family (ALD genes) was found to be the essential task of generating acetyl-CoA in the cytosol. Based on literature, this ALDH is deduced on the left arm of chromosome XVI of Saccharomyces cerevisiae .
In our studies, we amplified cloning the ALD6 gene by polymerase chain recitation (PCR)。The size of this DNA fragment is 1,506bp,containing a restriction enzyme ( NdeI) reaction site in DNA 5’-terminal.The ALD6 gene was cloned to to pGEM®-T easy vector by TA-Cloning. Through plasmid transformation to E.coli DH5α, we obtained the pGEM-T-ALD6 plasmid DNA in preparative amount. We can then get a specific ALD6 gene fragment by NdeI、SpeI restriction enzymes from pGEM-T-ALD6. Expression vector pET-43.1c(+) was used to contruct pET-43-ALD6 by inserting ALD6 gene fragment to the(NdeI、SpeI)restriction enzyme reaction sites on pET-43.1c(+).
Mini preparation of pET-43-ALD6 plasmid DNA was obtained by transformation to E.coli strain DH5α, then transformed into four strains of E.coli expression hosts: BL21(DE3)、BL21pLysS、Rosetta(DE3)、BL21-CodonPlus(DE3)-RIL), We used different inducer agents, incubation times and temperature, and enzyme activity assay to determined the optimal growth conditions. The results show:At 30℃,lactose induced Rosetta(DE3) is the best of the four strains tested . The optimal time 12 hr after induction.The reaction rate and induced amount of Ald6p recombinant protein are also optimal under these conditions.
We then used the optimal conditions for large-scale preparation of Ald6p followed by purification.The following resins (Amersham Biosciences company) are used for purification:1. Q Sepharose fast flow 2. Blue Sepharose 6 fast flow. Purified Ald6p was characterized on different aldehydes (Propionaldehyde,4-aminobutyraldehyde, 3-aminopropionaldehyde, betaine aldehyde, etc.). km,Vmax values were determined in order to understand the affinity and highest reaction rates with different aldehydes.
In addition, from two independent PCRs using yeast strain YPH499 genomic as template, we found the ALD6 DNA sequence has five SNP sites by DNA sequence analysis. To verify, yeast strain BY4742 genomic DNA was used for PCR reaction. No SNPs were detected in BY4742. Since all these five SNPs are silent mutations, the ALD6 gene sequence is highly conserved in Yeast
論文目次 授權書
論文口試委員審議通過委員簽名表
致謝 ……………………………………………………………………… I
中文摘要 ………………………………………………………………… II
英文摘要 ……………………………………………………………… III
目錄 …………………………………………………………………… IV
表目錄 ………………………………………………………………… VII
圖目錄 …………………………………………………………………… X

第一章 緒論
1.1 S.cerevisiae Aldehyde dehydrogenase 簡介 ………………….…. 1
1.2 S.cerevisiae Aldehyde dehydrogenase 家族 ………………….….. 2
1.3 S.cerevisiae Ald4p、Ald6p在酒精發酵過程中與醋酸形成之關係 .. 4
1.4 S.cerevisiae ALD6 基因相關研究 …………………………………. 6
1.5 分子選殖表現酵母菌 Ald6p 與酵素特性分析 …….......……..… 8
1.6 Betaine aldehyde dehydrogenase (BALD) 為非專一性酵素 …... 9
1.7 S.cerevisiae ALD2 與 ALD3 基因參與β-Alanine 生合成 .....…. 11
1.8 S.cervisiae ALD6 基因研究動機 ………..…………………..……. 13
第二章 選殖、單核苷酸多態性試驗
2.1 簡介 ……………..…………………………………………….……. 16
2.1.1 儀器設備 ………………………………………………….……. 17
2.1.2 重組菌種 ………………………………………………….……. 19
2.1.3 菌種儲存 ………………………………………………….……. 20
2.1.4 菌種馴養 ………………………………………………….……. 20
2.1.5 培養基之配製 …………………………………………….……. 20
2.1.6抗生素溶液配製 …………………………………………..……. 21
2.1.7 ALD6 基因引子(primer)設計 ………………………….……. 22
2.1.8 Yeast gemonic DNA 的製備 …………………………….……. 22
2.1.9 聚合酶連鎖反應(polymerase chain reaction) ………..……. 24
2.1.10 DNA電泳分析 (Agarose gel electrophoresis) ..…………. 27
2.1.11 DNA純化 …………………………………………………..……. 29
2.1.12 限制酶之剪切反應(digestion)…………………………….. 31
2.1.13 DNA 接合反應 ………………………………………………...... 33
2.1.14 TA Cloning …………………………………………………...... 33
2.1.15 勝任細胞的製備 (Competent Cell) .……...….…………..…. 38
2.1.16 質體轉型 (Transformation)……………………………….…. 40
2.1.17 質體的抽取 (Plasmid Extraction) ……………………….... 41
2.1.18 DNA定序 ………………………………..…………………..… 43
2.1.19 酵母菌ALD6 基因單核苷酸多態性試驗……………………..... 46
2.2 結果與討論 ……………………………………………………....… 48
2.2.1 PCR 製備含 NdeI ALD6 基因片段結果 …………………….…. 48
2.2.2 TA Cloning 結果 …………………………………………….…. 50
2.2.3初步抽取pGEM-T-ALD6 質體確認結果 ……………………....… 51
2.2.4 DNA 定序結果 ……………………………………………….…... 52
2.2.5單核苷酸多態性(SNP)實驗結果 …………………………...… 53
第三章 pET-43-ALD6 質體建構與 Ald6p 重組蛋白表現
3.1 簡介 ……………………………………………………………....… 58
3.1.1 建構pET-43-ALD6 表現質體 ……………………………….….. 59
3.1.2初步誘導表現Ald6p重組蛋白 …………………………….….... 69
3.1.3 蛋白質電泳分析(SDS-PAGE)………………………………...... 71
3.1.4重組菌種表達能力研究 ……………………………………….… 77
3.1.5 重組菌種(E.coli)生長曲線試驗 …………………………....... 80
3.1.6 Aldehyde dehydrogenase 酵素活性測試 ……...…………..…. 81
3.1.7 蛋白質定量測試 ……………………………………………....… 84
3.2 結果與討論 …………………………………………………….…... 86
3.2.1 pET-43.1c 質體製備結果 ………………………………….…... 86
3.2.2 pGEM-T-ALD6質體製備結果 ……………………………….…… 87
3.2.3製備NdeI與SpeI 之pET-43.1c表現載體片段 ….…..…….… 87
3.2.4製備含NdeI與SpeI之ALD6基因片段 ……………………..…. 88
3.2.5 pET-43-ALD6 質體與限制酶剪切反應確認 …………………… 89
3.2.6初步誘導表現Ald6p重組蛋白結果 ……………………………. 92
3.2.7菌種表達能力研究探討 …………………………………………. 93
第四章 Ald6p 純化與酵素活性分析
4.1 簡介 ………………………………………………………………. 114
4.1.1 Ald6p純化 ……………….……………………………………. 115
4.1.2 酵素動力學試驗 ………………………………………………. 122
4.2 結果與討論 ………………………………………………………. 128
4.2.1 Q Sepharose fast flow 純化 Ald6p 探討 …………...…… 128
4.2.2 Blue Sepharose 6 fast flow 純化 Ald6p 探討 ……….… 133
4.2.3 Ald6p 酵素動力分析結果 ……………………………………. 137
第五章 綜合討論與結論 …………………………………………….. 146
第六章 參考文獻 ……………………………………………………. 149

表 目 錄
表1.1 酵母菌 Aldehyde dehydrogenase 基因家族 …………..…. 3
表2.1 研究中使用之大腸桿菌重組菌種及基因型 ……………..... 19
表2.2 LB (Luria-Bertani) 液態培養基組成成份 …………..… 21
表2.3 LB (Luria-Bertani) 固態培養基組成成份 …………..… 21
表2.4 抗生素溶液配製 ………………………….………….….…. 22
表2.5 酵母菌 gemonic DNA 製備相關藥品配製方法與組成…..... 23
表2.6 PCR 反應之各項成分 …………………………….………… 26
表2.7 DNA 電泳分析相關藥品配製方法與組成 ………………….. 28
表2.8 PCR ALD6 基因限制酶剪切反應成分組成 ………….…….. 32
表2.9 DNA 接合反應之組成 …………..……….………………….. 33
表2.10 PCR 產物3'末端加入dATP 反應之各項成分與組成 …... 36
表2.11 TA Ligation 之各項成分組成與用量.………….………… 37
表2.12 TA Cloning 相關藥品配製方法與組成 ……….....………. 37
表2.13 勝任細胞製備之相關藥品配製方法與組成 …….……….. 39
表2.14 酵母菌 ALD6 基因 SNP PCR 獨立反應次數 ……………. 46
表2.15 SNP PCR 反應各項成分與組成 …………………………... 47
表 2.16 #1、#2 菌種含有pGEM-T-ALD6 質體之序列比對分析
結果 ……………………………...………………………... 53
表3.1 pET-43.1c 質體確認限制酶反應成分 ……………….…... 60
表3.2 pGEM-T-ALD6 質體確認限制酶反應成分…………....…….. 61
表3.3 pET-43.1c SpeI 限制酶反應之各項成分組成與用量 .….. 64
表3.4 pET-43.1c NdeI 限制酶反應之各項成分組成與用量 …... 64
表3.5 pGEM-T-ALD6 SpeI 限制酶反應之各項成分組成與用量…. 65
表3.6 pGEM-T-ALD6 NdeI 限制酶反應之各項成分組成與用量 ... 66
表3.7 pET-43-ALD6 質體建構接合反應 …………………………. 67
表3.8 pET-43-ALD6 質體確認限制酶反應成分 …………………. 68
表3.9 Ald6p 誘導相關藥品配製方法與組成 ……………………. 70
表3.10 製備 SDS-PAGE 膠體溶液配方 ………………………..…. 73
表3.11 SDS-PAGE 膠體溶液相關藥品配製方法與組成 ……….…. 75
表3.12 培養溫度與誘導物質誘導生產Ald6p 操作組合 ……..…. 79
表3.13 Cell Lysis buffer ………………………………………. 80
表3.14 Aldehyde dehydrogenase 活性測試反應液組成 …...…. 82
表3.15 Aldehyde dehydrogenase 活性測試反應用量 …………. 83
表3.16 Dye binding reagent 反應液組成 ……………….…..…. 85
表3.17 蛋白質定量試驗反應液用量 ……..……………………..…. 85
表 3.18 四種重組菌種 30℃,0.5% Lactose 誘導 Ald6p 表現
活性計算結果 ………..…………………………………...…. 113
表4.1.1 Q Sepharose fast flow Buffer A ……………….…. 120
表4.1.2 Q Sepharose fast flow Buffer B ……………….…. 120
表4.2.1 Blue Sepharose 6 fast flow Binding Buffer ….…. 121
表4.2.2 Blue Sepharose 6 fast flow Elution Buffer …….. 121
表4.3.1 Propionaldehyde Ald6p 酵素動力試驗反應液用量 …. 124
表 4.3.2 Propionaldehyde + MgCl2 Ald6p 酵素動力試驗反應液
用量 ………..……………………………………………….. 124
表4.4.1 AB-ald Ald6p 酵素動力試驗反應液用量 ………...….. 125
表4.4.2 AB-ald + MgCl2 Ald6p 酵素動力試驗反應液用量 …. 125
表4.5.1 AP-ald Ald6p 酵素動力試驗反應液用量 ……………. 126
表4.5.2 AP-ald + MgCl2 Ald6p 酵素動力試驗反應液用量 ….. 126
表4.6.1 Bet-ald Ald6p 酵素動力試驗反應液用量 …………... 127
表4.6.2 Bet-ald + MgCl2 Ald6p 酵素動力試驗反應液用量 ... 127
表4.5 Ald6p 各階段之純化結果 ………………………………... 136
表4.6 Ald6p 酵素動力測試結果 ………………………………... 145

圖 目 錄
圖1.1 酵母菌 PDH bypass 代謝途徑 ……………………………...... 5
圖1.2 酵母菌甘油產生反應方程式 ………………………..………... 7
圖 1.3 酵母菌 Aldehyde dehydrogenases (ALDHs)相似胺基酸序列 .. 8
圖 1.4 天然產生基本醛類之結構 ………………………………...…. 10
圖 1.5 酵母菌 Pantothenic acid 產生之代謝途徑 ……...……...…. 13
圖 2.1 TA cloning 概念圖 …..............................................................…. 34
圖 2.2 Promega pGEM ® -T Easy Vector 質體圖 ...........................…. 35
圖 2.3.1 線上BLAST2 輸入已知ALD6 基因核酸序列 ....……….….. 44
圖 2.3.2 線上BLAST2 開啟廠商鑑定結果之序列檔案 ……….…..... 45
圖 2.3.3 線上BLAST2 開啟廠商鑑定結果之序列檔案 ……….…..... 45
圖 2.3.4 線上BLAST2 分析結果之畫面 ……………………….…..... 45
圖 2.4 PCR annealing 溫度測試電泳結果圖 …………………....... 48
圖 2.5 PCR 1.5 kb ALD6 基因片段電泳結果圖 ……….……….…...... 49
圖 2.6 PCR 1.5 kb ALD6 基因片段純化電泳結果圖 ……..………...... 49
圖 2.7 PCR 1.5 kb ALD6 基因片段限制酶剪切反應電泳結果圖 ........ 50
圖 2.8 ALD6 TA cloning 藍白篩選 …….……………………....….….. 51
圖 2.9 pGEM-T-ALD6 質體抽取進行限制酶切割及電泳分析結果圖I . 52
圖 2.10 YPH499 第二次PCR 1.5 kb ALD6 基因片段電泳結果圖 …... 54
圖 2.11 YPH499 第二次PCR 1.5 kb ALD6 基因片段純化與限制酶剪
切反應電泳結果圖 ……...…………………………..………... 54
圖 2.12 BY4742 第一次與第二次PCR 1.5 kb ALD6 基因片段電泳結
果圖 …….….………………..……………………...….……… 55
圖 2.13 BY4742 第一次與第二次PCR 1.5 kb ALD6 基因片段純化與
限制酶剪切反應電泳結果圖 ……..………………………… 55
圖 2.14 YPH499、BY4742 之pGEM-T-ALD6 質體EcoRI 限制酶剪切反
應電泳分析結果圖 ……..…………………………………… 56
圖 3.1 pET-43.1 質體圖 ………………………………….……….…… 62
圖 3.2 ALD6基因於pET-43 表現質體選殖位置 …….…………..…... 63
圖 3.3 Aldehyde dehydrogenase 反應方程式 …………………..…… 81
圖 3.4 pET-43.1c 質體抽取與限制酶剪切反應電泳分析結果圖 …… 86
圖 3.5 pGEM-T-ALD6 質體抽取與限制酶剪反切應電泳分析結果圖II . 87
圖 3.6 切膠純化前NdeI/ SpeI 剪切pET-43.1c 質體結果圖 ………… 88
圖 3.7 切膠純化後,純化取得pET-43.1c ( NdeI, SpeI) 5,786 bp
DNA 片段 …………………………..………………………….… 88
圖 3.8 切膠純化前NdeI/ SpeI 剪切pGEM-T-ALD6 質體結果圖 …...… 89
圖 3.9 切膠純化後,純化取得pGEM-T-ALD6 ( NdeI/ SpeI) 1.5 kb
DNA 片段………………………………………………….……… 89
圖 3.10 NdeI 限制酶剪切pET-43-ALD6 質體質體電泳圖 ………...… 90
圖 3.11 EcoRI 限制酶剪切pET-43-ALD6 質體電泳圖 ………….....… 91
圖 3.12 初步 IPTG 誘導 Ald6p SDS-PAGE 電泳結果 …….............… 92
圖 3.13 37℃,0.4 mM IPTG 誘導誘導後第12 小時四種重組菌種
Ald6p 表達果 .........................................................................… 96
圖 3.14 37℃,0.5% Lactose 誘導後第12 小時四種重組菌種
Ald6p 表達結果 .....................................................................… 97
圖 3.15 30℃,0.4 mM IPTG 誘導誘導後第12 小時四種重組菌種
Ald6p 表達結果 .....................................................................… 98
圖 3.16 30℃,0.5% Lactose 誘導後第12 小時四種重組菌種
Ald6p 表達結果 .....................................................................… 99
圖 3.17 20℃,0.5% Lactose 與 0.5% Lactose + 0.4 mM IPTG
誘導後第12 小時 BL21(DE3)、BL21pLysS Ald6p 表達結果 . 100
圖 3.18 20℃,0.5% Lactose 與 0.5% Lactose + 0.4mM IPTG
誘導後第12 小時 Rosetta(DE3)、BL21-CodonPlus(DE3)-RIL
Ald6p 表達結果 …….....……………………………..........… 101
圖 3.19 20℃,0.5% Lactose 與 0.5% Lactose + 0.4 mM IPTG
誘導後第14 小時BL21(DE3)、BL21pLysS Ald6p 表達結果 ... 102
圖 3.20 20℃,0.5% Lactose 與 0.5% Lactose + 0.4 mM IPTG
誘導後第14 小時Rosetta(DE3)、BL21-CodonPlus Ald6p
表達結果 …………………………………………….…….… 103
圖 3.21 30℃,0.5% Lactose 誘導生長曲線 ……..……….…...… 105
圖 3.22 30℃,0.5% Lactose 誘導四種重組菌種生長曲線 .….… 106
圖 3.23 30℃,0.5% Lactose 誘導四種重組菌種細胞萃取液
(CFX)酵素活性測試 ………………………………..……..… 107
圖 3.24 30℃,0.5% Lactose 誘導BL21(DE3)第12、14 小時
Ald6p 表現結果 …………………………….…………..…… 108
圖 3.25 30℃,0.5% Lactose 誘導BL21pLysS 第12、14 小時
Ald6p 表現結果 ……………………………………...……… 109
圖 3.26 30℃,0.5% Lactose 誘導Rosetta(DE3)第12、14 小時
Ald6p 表現結果 ………………………………..….………… 110
圖 3.27 30℃,0.5% Lactose 誘導BL21-CodonPlus(DE3)-RIL
第12、14 小時Ald6p 表現結果 ………….…………………... 111
圖 3.28 30℃,0.5% lactose 誘導後第12 小時四種重組菌種酵素
活性測試比較 ……………………………………………..… 112
圖 4.1 測量反應初速 ……………………….………...……………… 122
圖4.2 以受質濃度對速率做圖 ……..……...………….……………
圖 4.3 Q Sepharose fast flow 純化Ald6p 初步活性測試範圍 ….. 129
圖 4.4 Q Sepharose fast flow 純化Ald6p 細部活性測試範圍 …... 130
圖 4.5 Q Sepharose fast flow 純化Ald6p 初步電泳分析結果圖 .. 131
圖 4.6 Q Sepharose fast flow 純化Ald6p 細部電泳分析結果圖 .. 132
圖 4.7 Blue Sepharose 6 fast flow 純化Ald6p 結果圖 …….…….... 134
圖 4.8 Blue Sepharose 6 fast flow 純化Ald6p 電泳分析結果圖I . 135
圖 4.9 Blue Sepharose 6 fast flow 純化Ald6p 電泳分析結果圖II . 135
圖 4.10 Propionaldehyde 雙倒數圖 ………………………….….... 138
圖 4.11 Propionaldehyde + MgCl2 雙倒數圖 ………………….....… 138
圖 4.12 AB-ald 雙倒數圖 ……………………….……………….... 140
圖 4.13 AB-ald + MgCl2 雙倒數圖 ………………….…………….... 140
圖 4.14 AP-ald 雙倒數圖 …………..…………….……………….... 142
圖 4.15 AP-ald + MgCl2 雙倒數圖 ………………….…………….... 142
圖 4.16 Bet-ald 雙倒數圖 ……………………….……………….... 144
圖 4.17 Bet-ald + MgCl2 雙倒數圖………………….…………….... 144
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