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研究生:孫維聲
研究生(外文):Wei-Sheng Sun
論文名稱:大腸桿菌中的YgfZ蛋白質功能之探討
論文名稱(外文):Characterization of YgfZ in Escherichia coli
指導教授:許萬枝許萬枝引用關係
指導教授(外文):Wan-Jr Syu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:48
中文關鍵詞:YgfZ蛋白質磯松素氧化壓力
外文關鍵詞:YgfZ proteinplumbaginoxidative stress
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磯松素是一種由白花丹屬的藥用植物中所淬取出的天然分子。在實驗中磯松素經常用來產生超氧陰離子。但在蛋白質體學的分析中,發現在大腸桿菌中,對磯松素作用產生反應的蛋白質種類,與其他產生超氧陰離子的化合物處理下影響的蛋白質種類不全然相同。先前的研究指出,磯松素的刺激會活化mar/sox操控群中的基因。大部份mar/sox操控群中的基因都擁有與細菌對抗氧化壓力有關的功能;唯獨有一個ygfZ基因是功能未知的。我們發現當大腸桿菌失去ygfZ基因的功能時,對磯松素的耐受性會大幅降低。因此,ygfZ基因在細菌中是對抗磯松素毒性所必須的。而且我們發現,ygfZ基因剔除的大腸桿菌突變株的表現型可以透過質體額外表達ygfZ基因來回復;若是額外表達緩解超氧陰離子毒性的sodA基因則無此效果。這代表磯松素在細菌中可能產生除了超氧陰離子以外的毒性。在結構上,YgfZ蛋白質中具有高度保留性的fingerprint區塊,對於YgfZ蛋白質對抗磯松素毒性具有相當的重要性。更進一步探討此一區塊的性質,我們發現其中的C228半胱氨酸具有最主要的影響力。我們的研究結果假設這個胺基酸的功能可能是用來提供一個形成氫鍵的位置,藉此YgfZ蛋白質可以與其它分子互動。基於YgfZ蛋白質可能透過與其它蛋白質或輔因子的互動來進行其功能的假設,我們比較了以非變性方式,被YgfZ蛋白質與被失去fingerprint區塊的YgfZ蛋白質所共同純化出來的蛋白質種類。經由質譜儀定序蛋白質序列,我們發現了一個會與YgfZ蛋白質一起被純化出來蛋白質,PadR,它被預測為一個轉錄調節因子。雖然YgfZ蛋白質和PadR蛋白質之間的關係仍不清楚,但我們在細菌雙雜合系統中,觀察到它們之間有微弱的交互作用。PadR蛋白質是否對YgfZ蛋白質對抗磯松素毒性的功能有所貢獻,仍需要進一步研究。
Plumbagin is a natural compound found in herbal plants of Plumbago genus. It is commonly used as a superoxide generator. And proteomic analysis of the proteins responsive to plumbagin in E. coli has indicated the difference of plumbagin from other superoxide generator. In a previous study, plumbagin was found to activate mar/sox regulon genes. Most of the mar/sox regulon genes are functionally related to the oxidative stress response. Among the up-regulated genes, there is a function unknown gene, ygfZ. Loss of ygfZ function makes E. coli more sensitive to plumbagin. Thus, ygfZ is essential for the detoxification of plumbagin in bacteria. And the phenotype of ygfZ knock-out strain can be fully complemented by ectopically expressed ygfZ but not sodA that is known to resolve the superoxide stress. This indicates that plumbagin may generate toxic effect other than superoxide. Structurally, a highly conserved fingerprint region was found in the YgfZ homolog and this region appeared to be important for the function in anti-plumbagin activity. Further investigation on the fingerprint region has revealed that residue C228 is the most critical in fingerprint region. A hypothesis is that it may function by providing a hydrogen bond to interact with other molecules. Basing upon an assumption that YgfZ may exert its function by interaction with other proteins or cofactors, we compared molecules natively co-purified with YgfZ and those with the fingerprint deleted YgfZ. After mass-spectroscopy determination, a unique protein co-eluted with YgfZ was identified to be predicated transcriptional regulator, PadR. Although the relationship between YgfZ and PadR remains unknown, they showed weak interaction in bacterial two-hybrid assay. Whether PadR adds significance towards the function of YgfZ in anti-plumbagin activity remains to be explored.
CONTENT I
ABSTRACT 1
中文摘要 2
INTRODUCTION 3
MATERIALS AND METHODS 8
BACTERIAL STRAINS, GROWTH CONDITIONS AND CHEMICALS 8
PLASMIDS 8
PRIMERS FOR MOLECULAR CLONING 9
SITE-DIRECTED MUTAGENESIS 9
COMPETENT CELL PREPARATION AND TRANSFORMATION 10
INHIBITION ZONE ASSAY 10
MINIMUM INHIBITION CONCENTRATION (MIC) ASSAYED BY LIQUID CULTURE METHOD 11
SDS-PAGE AND WESTERN BLOTTING ASSAY 11
BACTERIAL TWO-HYBRID ASSAY 12
��-GALATOSIDASE ACTIVITY ASSAY (MILLER ASSAY) 13
NICKEL-NTA COLUMN PROTEIN PURIFICATION 14
ACETONE PRECIPITATION 15
COOMASSIE BLUE STAINING 15
ELECTROSPRAY IONIZATION (ESI) TANDEM MASS SPECTROMETRY (MS/MS) 16
RESULTS 17
CONFIRMATION OF THE PHENOTYPE OF YGFZ- STRAIN 17
YGFZ AND SODA MUTUALLY IRREPLACEABLE IN THE ANTI-PLUMBAGIN ACTIVITY 17
THE SODA- STRAIN SENSITIVE TO SUPEROXIDE BUT NOT THE YGFZ- STRAIN 18
THE AMINO ACID SEQUENCE ALIGNMENT OF YGFZ HOMOLOGUES 19
FINGERPRINT REGION OF YGFZ ASSOCIATED WITH THE PLUMBAGIN DETOXIFICATION 19
DETERMINING THE MOST INFLUENTIAL RESIDUE IN THE FINGERPRINT REGION OF YGFZ IN THE PLUMBAGIN DETOXIFICATION 20
POSSIBLE ROLE OF C228 RESIDUE IN YGFZ 21
NO INTERACTION FOUND BETWEEN YGFZ AND PREVIOUSLY IDENTIFIED PLUMBAGIN RESPONSIVE PROTEINS BASED ON BACTERIAL TWO-HYBRID ASSAY 22
NATIVE PURIFICATION OF YGFZ 23
PROTEIN IDENTIFICATION ANALYSIS BY ESI- TANDEM MASS SPECTROMETRY 24
BACTERIAL TWO-HYBRID ASSAY OF YGFZ AND PADR 24
DISCUSSION 26
FIGURES 30
FIGURE 1 30
FIGURE 2 31
FIGURE 3 32
FIGURE 4 33
FIGURE 5 34
FIGURE 6 35
FIGURE 7 36
TABLES 37
TABLE 1.THE RESULT OF BACTERIAL TWO-HYBRID ASSAY BETWEEN YGFZ AND CANDIDATE PROTEINS. 37
TABLE 2. PRIMERS FOR MOLECULAR CLONING AND MUTAGENESIS 38
REFERENCES 39
APPENDIX 42
APPENDIX 1 42
APPENDIX 2 43
APPENDIX 3 44
APPENDIX 4 45
APPENDIX 5 46
APPENDIX 6 47
APPENDIX 7 48
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