淡江大學覺生紀念圖書館 (TKU Library)

系統識別號 U0002-3107200523461100
中文論文名稱 酵母菌(Pichia pastoris)的基因表現
英文論文名稱 Gene Expression in Yeast(Pichia pastoris)
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 93
學期 2
出版年 94
研究生中文姓名 秦嘉志
研究生英文姓名 Chia-Chi Chin
學號 692171076
學位類別 碩士
語文別 中文
口試日期 2005-06-15
論文頁數 77頁
口試委員 指導教授-簡素芳
中文關鍵字 α-半乳糖水解酵素  基因表現  表現質體  蛋白質純化  基因重組 
英文關鍵字 gene expression  Pichia pastoris  clones  integrate  oligosaccharide  transformants  enzyme activity 
學科別分類 學科別自然科學化學
中文摘要 α-半乳糖水解酵素(EC.能水解B型紅血球表面抗
表現最多的菌株為pPIC9K-αgal in SMD1168。目前可以表現在細胞內
英文摘要 α-galactosidase(EC. is able to cleave the
terminal α-galactose from surface oligosaccharide chain of
B red blood cells, thus B type RBC can be converted into O type RBC.
In order to gain large quantity of enzyme for this purpose,
we tried to clone the rice α-galactosidase gene into
each of the pPICZαA and pPIC9K vector(Invitrogen®),
and to expression the enzyme in both GS115 and SMD1168 strain.
The transformants from pPIC9K/SMD1168 was obtained.
Expression the transformants in flask culture,
most enzyme activity was found intracellularly (10 mg
per liter culture);while there is about 1 mg per liter
culture of the enzyme activity was secreted in the media.
The secreted enzyme would be easier for further media
purification. From SDS-PAGE, the secreted enzyme revealed
as a major band. We have to try some other culture conditions to improve this results.
論文目次 目錄:
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖表目錄 Ⅴ
索引 Ⅶ
1.α-半乳糖水解酵素(α-galactosidase)的簡介 1
2. α-半乳糖水解酵素的應用 1
3.酵母菌(Pichia pastoris)蛋白質表現系統簡介 6
4.使用嗜甲基酵母菌的優點 10
5.研究目標 12
1.菌種與質體 13
2.培養基 13
3.酵素與受質 14
4.儀器 16

1.培養基配製 17
2.酵母菌表現質體及寄主的選擇 22
3.製備α-半乳糖水解酵素基因 27
4.製備表現質體 31
5.表現質體電導轉型到酵母菌 37
6.測試培養條件 47
7.用SDS-PAGE分析表現的酵素 50
8.紅血球轉型實驗 53
1.製備α-半乳糖水解酵素基因 54
2.製備表現質體 56
3.表現質體電導轉型到酵母菌 59
4.測試培養條件 62
5.用SDS-PAGE分析表現的酵素 70
6.紅血球轉型實驗 71
五、結論與未來展望 72
六、參考資料 75

圖1. 血型抗原糖鏈結構 3
圖2. GL-3在人體的正常分解過程 5
圖3. Pichia pastoris的AOX 1 promoter 7
圖4. Gene insertion at AOX 1,gene replacement at AOX1。 9
圖5. Gene multi-integration 8
圖6.動物細胞與酵母菌表現的糖鏈比較 11
圖7. Zeocin結構 14
圖8. G418結構 15
圖9. pPICZαA and pPIC9K map 24
圖10-a. pPICZαA multiple cloning site 25
圖10-b. pPIC9K multiple cloning site 26
圖11. PCR洋菜電泳 55
圖12. 回收流程 30
圖13.表現質體gene cloning 31
圖14.水解pPICZαA-αgal電泳 57
圖15.水解pPIC9K-αgal電泳 57
圖16.菌種劃盤在α-X-gal plate反應結果 60
圖17.multicopy 洋菜電泳 61
圖18. MeOH對酵母菌培養的菌數和活性表現的影響 62
圖19. SDS-PAGE 分析誘導後培養基結果 70
圖20. 血球凝聚實驗結果 71
圖21: 2 copies gene vector製作 74

表1. 血液中血球跟抗體的凝聚現象 4
表2.Insert基因定序與稻米α-半乳糖水解酵素基因比對 59
表3.不同溫度的誘導結果 64
表4.不同菌數的誘導結果 66
表5.不同培養基的誘導結果 68
表6.不同菌種的誘導結果 69
表7.使用Pichia pastoris表現的酵素 72

中文 ;英文 頁數
α-半乳糖水解酵素;α-galactosidase 1
血凝現象 ;hemagglutination 3
法布瑞氏症 ;Fabry disease 4
嗜甲基酵母菌 ;methylotrophic yeast 6
整合 ;integrate(integration) 7
包覆體 ;inclution body 12
再摺疊 ;refolding 12
轉殖 ;transform 32
勝任細胞 ;competent cell 32
培養 ;incubate 32
熱休克 ;heat-shock 32
篩選 ;select 33
質體分離 ;plasmid DNA isolation 33
洗提 ;elute 33
電導轉型 ;electroporation 39
誘導 ;induce 44
轉型株 ;transformant 45

轉殖(實驗方法) 32
質體分離(實驗方法) 33
電導轉型(實驗方法) 39
分離酵母菌染色體DNA(實驗方法) 45

參考文獻 1.Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine. Genome evolution in yeasts. Nature. 2004 ;430(6995):35-44.
2.Turakainen H, Kristo P, Korhola M. Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions. Yeast. 1994 ;10(12):1559-68.
3.Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G.. The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc. Natl. Acad. Sci. U.S.A. 2002 ;99(22):14422-7.
4.Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature. 2001; 413(6858):848-52.
5.Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura Y. Rice genome research program, national institute of agrobiological sciences, 1-2, kannondai 2-chome. Nature. 2002; 420(6913):312-6.
6.Zhu A, Goldstein J. Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase. Gene. 1994; 140(2):227-31.
7.Zhu A, Monahan C, Zhang Z, Hurst R, Leng L, and Goldstein J. High-level Expression and Purification of Coffee Bean alpha-Galactosidase Produced in the Yeast Pichia Pastoris. Arch. Biochem. Biophys. 1995; 324(1): 65-70.
8.Kim WD, Kobayashi O, Kaneko S, Sakakibara Y, Park GG, Kusakabe I, Tanaka H, Kobayashi H. α-galactosidase from culture rice(Oryza sativa L. var. Nipponbare) cells. Phytochemistry. 2002;61:621-630.
9.Smet B, Hesta M, Seynaeve M, Janssens G, Vanrolleghem P, Wilde RO. The influence of supplementalα-galactosidase and phytase in a vegetable ration for dogs on the digestibility of organic components and phytate phosphorus. Journal of Animal Physiology & Animal Nutrition, 1999;81(1):1-8.
10.Chien SF, Marie LC. The conversion of group B red blood cells into group O by an α-D-galactosidase from taro(Colocasia esculenta). Carbohydrate Research. 1991; 217: 191-200.
11.Zhu A, Leng L, Monahan C, Zhang Z, Hurst R, Lenny L, Goldstein J. Characterization of recombinantα-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. Arch. Biochem. Biophys. 1996; 327( 2): 324-329.
12.Calcutt MJ, Hsieh HY, Chapman LF, Smith DS. Identification, molecular cloning and expression of an α-N-acetylgalactosaminidase gene from Clostridium perfringens. FEMS Microbiology Letters, 2002;214:77-80.
13.Copbell NA, Reece JB, Mitchell LG, Taylor MR. Biology. Benjamin Cummings. 2003; 169.
14.Ohshima T, Murray GJ, Nagl JW, Quirk JM, Kraus MH, Norman W. Barton, Roscoe O. Brady D, Kulkarni AB. Structural organization and expression of the mouse gene encoding α-galactosidase A. Gene. 1995; 166:277-280.
15.Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. Jounal of Molecular Recognition. 2004.
16.Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene. 1991; 105:205-212.
17.Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA. High-level expression of Tetanus toxin fragment c in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology. 1991; 9: 455-460.
18.Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Fuke M, Holden K, Phelps D, Wood P. High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry. 1989 ;28(9):4117-25.
19.Brown TA. Gene Cloning. Stanley Thornes(Publishers). 1995;269-270.
20.Sambrook, Fritsch, Maniatis. Molecular Cloning. Cold Spring Harbor Laboratory. 1990 ; 3:A1-A3.
21.Hagenson MJ, Holden KA, Parker KA, Wood PJ, Cruze JA, Fuke M, Hopkins TR, Stroman DW. Expression of streptokinase in Pichia pastoris Yeast. Enzyme Microbiol. Technol. 1989;11: 650-656.
22.Brandes HK, Hartman FC, Lu TYS, Larimer FW. Efficient Expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis. 1996; J. Biol. Chem. 271: 6490-6496.
23.Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L. High level secretion of glycosylated invertase in the methylotrophic yeast Pichia pastoris. 1987;Bio/Technology 5: 1305-1308.
24.Digan ME, Lair SV, Brierley RA, Siegel RS, Williams ME, Ellis SB, Kellaris PA, Provow SA, Craig WS, Velicelebi G, Harpold MM, Thill GP. Continuous production of a novel lysozyme via secretion from the yeast Pichia pastoris. 1989; Bio/Technology 7: 160-164.
25.Paifer E, Margolles E, Cremata J, Montesino R, Herrera L, Delgado JM. Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences. 1994; Yeast 10: 1415-1419.
26.Guo W, Gonzalez-Candelas L, Kolattukudy PE. Cloning of a new pectate lyase gene pelC from fusarium solani f. sp. pisi (Nectria haematococca, Mating Type VI) and characterization of the gene product expressed in Pichia pastoris. 1995; Arch. Biochem. Biophys 323: 352-360.
27.Gilbert SC, Urk van H, Greenfield AJ, McAvoy MJ, Denton KA, Coghlan D, Jones GD, Mead DJ. Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin. 2005; Yeast 10: 1569 – 1580.
28.Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign gene in Pichia pastoris. Bio/technology. 1993; 11: 905-910
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