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研究生:丘仕傑
研究生(外文):Shi-Kit Yau
論文名稱:大腸桿菌中在飽食或肌餓狀態下有差異表現的蛋白質複合體鑑定
論文名稱(外文):Identification of protein complexes differentially expressed in Escheriachia coli under feast and famine conditions.
指導教授:蔡有光
指導教授(外文):Yeou-Guang Tsay
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
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:50
中文關鍵詞:蛋白質複合體琥珀酸脫氫酶α次單元差異表現
外文關鍵詞:protein complexsuccinate dehydrogenase α subunitdifferentially express
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最近幾年,很多研究報告都指出蛋白質複合體是反應細胞功能狀態的關鍵分子,並有證據指出蛋白質複合體的生成可以調節改變細胞功能。大腸桿菌目前是研究生物學最好的模型之一,其在封閉系統內的生長時期,依照生長速率,可以分別為延遲生長、對數生長、停滯以及死亡等四個時期。我們最近成功建立了一個比較蛋白體內蛋白質複合體的平台—膠體過濾層析-二維差異表現蛋白質電泳系統(GFC-2D DIGE),此系統可用以鑑定具差異表現的蛋白質複合體的組成成員。
在這篇論文中,我們首先報告在對數生長期和停滯期的大腸桿菌細胞中,在六百四十萬和三百五十萬道耳吞的次蛋白質體膠體過濾層析分管中,找到十幾個具差異性表現的蛋白質。對於這些差異蛋白質,我們針對其中四個,在胺基端接上Flag標籤,在原細胞表達這些重組蛋白質,以了解這些蛋白質的複合體的特性。於SDS-膠體電泳中,琥珀酸脫氫酶的 (succinate dehydrogenase) α次單元或稱SdhA蛋白,除了合乎其大小的重組蛋白外,另有一泳動較快的形態,可能是這個蛋白的片段。這個蛋白酶的易形成片段的特性,與我們在膠體過濾層析-二維差異表現蛋白質電泳的觀察,不謀而合。於膠體過濾層析分析SdhA重組蛋白的結果發現,完整蛋白與部分片段的四級結構有相當大的差異,因此這個蛋白的一級結構的變化可能與其四級結構的組合有關。而這些蛋白複合體在休眠或活躍的大腸桿菌內,也有些微的差異。因此,琥珀酸脫氫酶α次單元的蛋白質複合體巨集似乎的確受生長狀況的調節。
In recent years, many researches show that protein complexes are the key molecules responsible for cellular functions. Some lines of evidence show that protein complex formation may modulate cellular functional changes. Escherichia coli is probably the best model for biology research. In close system, the population of E. coli may go through four phase, namely lag phase, exponential phase, stationary phase, and death phase. We have previously set up a comparative protein complexomics platform, namely gel filtration chromatography-two-dimensional difference gel electrophoretic analysis (GFC-2D DIGE), which can be used to
facilitate the identification of components in differentially expressed protein complexes.
In this thesis, we first report the documentation of the identities of difference proteins in 6.4- and 3.5-MDa subproteomic gel filtration fractions from E. coli cells at
exponential or stationary phase. Among these difference proteins, we expressed four Flag-tagged recombinant proteins such that their protein complex components could
be characterized. It is intriguing to see that SdhA protein, or succinate dehydrogenase α subunit appeared to have aberrant mobility in SDS-PAGE analyses. This is consistent with the finding that endogenous SdhA has a C-terminal truncated species in dormant cells in GFC-2D DIGE system. When Flag-tagged SdhA was expressed in cells at distinct growth states, gel filtration analyses showed that the profiles of quaternary structures of large and small SdhA protein were quite different. Also, the elution profiles of SdhA complexes were quite different between active and dormant cells. Hence, our results suggest that, while primary structures of SdhA protein may affect the assembly of its quaternary structures, protein complex
assembly of SdhA is also regulated under different growth conditions.
TABLE OF CONTENTS
TABLE OF CONTENTS vii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS x
CHAPTER I: LITERATURE REVIEW 1
1. THE LARGE-SCALE RESEARCH OF E. COLI PROTEIN COMPLEXES 1
2. IDENTIFICATION OF PROTEIN COMPONENTS IN DIFFERNTIALLY EXPRESSED PROTEIN COMPLEXES IN ESCHERICHIA COLI UNDER FEAST AND FAMINE CONDITIONS 6
3. PROTEIN COMPLEXES CONTAINING GLYCEROL KINASE (GLPK), L-LACTATE DEHYDROGENASE (LLDD), HYDROGENASE 2 LARGE SUBUNIT (HYBC), SUCCINATE DEHYDROGENASE FLAVOPROTEIN SUBUNIT (SDHA) 8
CHAPTER II: PROFILING OF PRIMARY AND QUATERNARY STRUCTURES OF SUCCINATE DEHYDROGENASE SUBUNIT A IN ESCHERICHIA COLI CELLS 16
1. SUMMARY 17
2. INTRODUCTION 18
3. MATERIALS AND METHODS 20
4. RESULTS 23
5. DISCUSSION 41
LIST OF REFERENCE 46
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