臺灣博碩士論文加值系統

XpsM蛋白為革蘭氏陰性菌第二類型胞外蛋白分泌途徑中必要的成員之一，此蛋白由213個氨基酸組成，其N-端位於第10-28個氨基酸序列為一疏水性強的區域，推測XpsM可能利用此序列以N-端朝向胞內的方式插在內膜中。本研究首先構築一系列XpsM::PhoA融合蛋白，以鎳離子管柱層析分析XpsM與XpsM之間的交互作用，選擇含XpsM N-端較短片段的兩株XpsM::PhoA融合蛋白74::PhoA與99::PhoA，分析它們各別與全長 (His)6-tagged XpsMh的結合能力，發現與全長XpsM結合可能只須XpsM N-端74個氨基酸，此序列包含transmembrane region (TM)。為了分析TM在XpsM與XpsM交互作用中的相對位置，本研究進一步利用定點突變 (site-directed mutagenesis) 的方法，將XpsM疏水性區域的氨基酸逐一改為cysteine，經starch plate檢查突變蛋白分泌澱粉水解酶的功能，發現僅V19C突變蛋白失去分泌功能，其它均維持正常，說明XpsM TM中除了第19個氨基酸，其它位置均可以cysteine取代；經西方墨點法在不含β-mercaptoethanol (β-ME) 的情況下分析各突變蛋白，發現 A12C、A23C、Y24C、L25C、V26C、L27C、V28C 七個突變株出現雙倍體，這些雙倍體在加入β-ME 後即行消失，說明這些雙倍體確實為cysteine和cysteine之間形成disulfide bond的結果。由於 XpsM 的TM序列中靠近胞質週緣區的連續六個及靠近細胞質的一個氨基酸各自與一個XpsM相鄰，因此推論XpsM在細胞膜中形成的複合體，可能是大於雙倍體的多倍體。進而利用cross-linking實驗分析單獨表現正常的XpsMh蛋白，結果也顯示XpsM蛋白在細胞膜中可能形成多倍體形式的複合體。

The XpsM protein is one of the major components required for the type II secretion in gram-negative bacteria. It is composed of 213 amino acid residues. A hydrophobic region located between the 10th and the 28th residues at the N-terminus serves as the membrane anchoring sequence. The monotopic XpsM protein is localized in cytoplasmic membrane with its N-terminus facing the cytoplasm. In this study, I constructed a series of XpsM::PhoA protein. Using metal chelating chromatography, I analyzed two of the PhoA fusion proteins, 74::PhoA and 99::PhoA, for their interaction with the full-length, (His)6-tagged XpsMh, The N-terminal 74 amino acid residues of the XpsM are probably sufficient for its self-association. To analyze the relative position of the neighboring XpsM TM region, I substituted each of the 19 hydrophobic residues of the XpsM with cysteine residue by performing site-directed mutagenesis. All but the V19C mutant protein remained functional in α-amylase secretion. Treating intact cells with oxidizing agent caused, in 7 mutant proteins, the appearance of an extra band on immunoblot from SDS-PAGE in absence of β-mercaptoethanol (β-ME). The extra band disappeared in presence of β-ME, suggesting that it has arisen from disulfide bond formation. Since six of the mutants giving rise to XpsM dimer were located in succession between the 23rd and 28th residue, we speculated that in the functional apparatus each XpsM is probably surrounded by more than one neighboring XpsM proteins. Results from crosslinking experiment also imply the formation of XpsM homo-multimer in cytoplasmic membrane.

縮寫字對照表 ………………………………………1
中文摘要 ……………………………………… ……2
英文摘要 …………………………………………….3
前言 ………………………………………………….4
材料與方法 ………………………………………….13
結果 ………………………………………………….26
討論 ………………………………………………….34
參考文獻 …………………………………………….38
圖表 ………………………………………………….44
附錄 ………………………………………………….55

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