啤酒酵母菌(Saccharomyces cerevisiae)之ALD2 (YMR170C )及ALD3 (YMR169C)，是以二縱排重覆(two tandem-repeated)的ORFs存在其第13號染色體中。這兩個蛋白質，在胺基酸序列的比較上，呈現出87%的同等性及91%的相似性，同時此二蛋白在特徵上被認為是屬於細胞質壓力誘導型(cytoplasmic stress-inducible )-醛類脫氫酶(aldehyde dehydorgenase, ALD)之同功異構酶(isoforms)。根據Navarro-Avino等學者的研究指出，ALD3p可藉由NAD+當輔酶(coenzyme)，並具有以acetaldehyde及betaine aldehyde為受質之酵素活性 (Navarro-Avino et al., 1999)，但是至目前為止，其尚無詳細的酵素動力學被測定出來；此外ALD2p的酵素活性亦尚未被表達與分析之。本研究為了產生具有酵素活性的ALD2p及ALD3p，我們利用真核細胞同源表現系統，將ALD2與ALD3基因構築到具有Glutathione S – Trans ferase (GST)基因的表現載體pEG-KT，利用4 % galactose誘導此二重組蛋白(GST-ALD2、GST-ALD3)在Saccharomyces cerevisiae (BJ2168)中大量表現，並利用GST親合性層析法純化此二蛋白。研究結果得知，我們所建構表現的重組型GST-ALD2、GST-ALD3具有催化propioaldehyde、γ-aminobutyraldehyde、3-aminopropioaldehyde，及betaine aldehyde等不同醛類受質之酵素活性。

Saccharomyces cerevisiae ALD2 (YMR170C) and ALD3 (YMR169C) are two tandem-repeated ORFs on Chromosome XIII. The amino acid sequence comparison of these two proteins shows significant similarity with 87% identities and 91% positives, and they were characterized as the cytoplasmic stress-inducible isoforms of aldehyde dehydorgenase (ALD). In the previous studies, assays with over-expressed Ald3p showed that this protein is NAD+ linked and active with acetaldehyde and betaine aldehyde. However, none of detailed kinetics has been determined. Neither has ALD2p been expressed and assayed. In this thesis, in order to produce large amount of biologically active Ald2p and Ald3p, the eukaryotic homologous expression system was applied, and the full-length of ALD2 and ALD3 gene was constructed into pEG-KT expression vector. Then the vector was transformed into Saccharomyces cerevisiae (BJ2168), and the transformants were able to over-express recombinant GST-ALD2 and GST-ALD3 by 4% galactose. To study the purified ALD2p and ALD3p, GST affinity chromatography strategy was used. The activity of clarified cell extract was examined by propioaldehyde. And the recombinant protein was characterized by betaine aldehyde, γ-aminobutyraldehyde and 3-aminopropioaldehyde.

謝誌	I
中文摘要	II
英文摘要	III
縮寫表  IV
目錄 V
圖、表目錄  VIII
第一章 緒論 1
第二章 ALD2、ALD3之基因選殖及重組基因之次選殖(subcloning)13
第一節 檢體來源與聚合酶鏈反應(PCR)複製處理 13
2.1.1菌種來源 13
2.1.2萃取酵母菌(S. cerevisiae)genomic DNA 13
2.1.3聚合酶鏈反應(PCR)複製ALD2及ALD3基因 14
2.1.4 PCR產物之限制酶切割確認 15
2.1.5洋菜膠體電泳	16
2.1.6 PCR產物的純化 16
2.1.7 PCR產物之3’末端補“A”鹼基 16
第二節 ALD2與ALD3之基因選殖(TA- cloning)	17
2.2.1 DNA的接合(DNA ligation)	18
2.2.2大腸桿菌之質體轉型 (E. coli - DH5α)	19
2.2.3 Colony PCR及電泳分析	20
2.2.4少量質體DNA的備製	20
2.2.5 DNA定序分析	21
第三節 重組基因之次選殖(subcloning)	24
2.3.1建構選殖基因之BamHI及SmaI限制酶切割位	24
2.3.2純化Target DNA fragment	25
2.3.3 ALD2、ALD3選殖基因與pEG KT 載體的接合	26
2.3.4大腸桿菌之質體轉型 (E. coli-DH5α)	26
2.3.5 Colony PCR及電泳分析	26
2.3.6少量質體DNA的備製	26
2.3.7 DNA定序分析	27
2.3.8大量質體DNA的快速備製	27
第四節 實驗結果與討論	27
   2.4.1 PCR產物與限制酶切割確認	27
   2.4.2 Transformed大腸桿菌之colony PCR確認	28
   2.4.3 定序分析	28
   2.4.4 選殖基因之限制酶BamHI及SmaI切割位的建構	30
   2.4.5 重組基因次選殖之Cloning -PCR及限制酶切割之確認 30
第三章 同源表現與純化重組蛋白 37
第一節 同源表現(homologous expression) 38
      3.1.1酵母菌之細胞轉型(Electroporation method, Bio-Rad)	38
      3.1.2表現載體之誘導	40
第二節純化重組之GST - ALD2與GST - ALD3蛋白	42
      3.2.1提取酵母菌粗抽液蛋白	42
      3.2.2蛋白純化－GST親合性吸附法	43
      3.2.3 SDS-PAGE蛋白質電泳分析	44
      3.2.4 Coomassie Blue染色	46
      3.2.5蛋白質定量 (Bradford protein-binding assay)	47
第三節實驗結果與討論	48
  3.3.1 啤酒酵母菌之pEG KT-ALD transformation efficiency 48
   3.3.2 表現載體誘導時間之檢測	49
  3.3.3 融合蛋白之純化	50
第四章 酵素活性之分析	54
第一節 Ald2p與Ald3p活性之分析	54
第二節 實驗結果與討論	55
第五章 綜合討論與結論	63
參考文獻	66
附表(一) 實驗用的DNA引子	73
附表(二) 實驗用的菌種及質體	74
附錄(一) ALD2與ALD3蛋白質胺基酸序列之比較	75
附錄(二) pGEM®-T Easy Vector DNA圖示	76
附錄(二) pEG-KT (shuttle vector)載體之圖譜	77
圖1-1	Betain – Choline Pathway	4
圖2-1	ALD2-PCR產物與限制酶切割之結果	31
圖2-2	ALD3-PCR產物與限制酶切割之結果	32
圖2-3	TA cloning-PCR之電泳結果	33
圖2-4	選殖基因與表現質體以限制酶BamHI、SmaI切割後之結果	34
圖2-5	重組基因次選殖之colony PCR電泳結果	35
圖2-6	重組基因(pEG KT-ALD2、pEG KT-ALD3)以限制酶BamHI、SmaI切割後之結果	36
圖3-1	pEG KT-ALD2與pEG KT-ALD3表現載體誘導時間之檢測 52
圖3-2	pEG KT-ALD2及pEG KT-ALD3在酵母菌BJ2168中，經
4% galactose誘導表現後，以10% SDS-PAGE分析純化
recombinantGST-ALD3、GST-ALD3各步驟之結果	53
圖4-1	純化前、後的ALD2蛋白，以丙醛為受質之活性分析	59
圖4-2	純化前、後的ALD3蛋白，以丙醛為受質之活性分析	60
圖4-3	比較ALD2及ALD3 crude lysate於不同pH值之緩衝液系統中，以四種醛類、NAD+為受質之反應後，NADH的生成量	61
圖4-4	比較ALD2及ALD3 crude lysate於不同pH值之緩衝液系統中，以四種醛類、NADP+為受質之反應後，NADPH的生成量	62
表4-1	ALD2與ALD3之融合蛋白以丙醛為受質之比活性	58

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