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研究生:簡伊翎
研究生(外文):I-Ling Chien
論文名稱:十字花科黑腐病菌XpsE蛋白之純化及其特性分析
論文名稱(外文):Purification and Characterization of theXpsE Protein of the Type II Secretion Apparatus of Xanthomonas campestris pv. campestris
指導教授:胡念台
指導教授(外文):Nien-Tai Hu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:75
中文關鍵詞:十字花科黑腐病菌
外文關鍵詞:XpsE
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中文摘要
革蘭氏陰性菌之第二類型胞外蛋白分泌途徑須由二段步驟,將胞外蛋白由胞內送至胞外。先由Sec蛋白系統,將胞外蛋白送至胞質週緣區後,再由另一群12-14個蛋白所組成的分泌系統,將胞外蛋白從胞質週緣區送達胞外。十字花科黑腐病菌中,外膜分泌系統由xpsD~O十二個基因的蛋白產物所組成,其中XpsE 蛋白是唯一沒有疏水性區域的蛋白,因此被認為是胞內蛋白。XpsE胺基酸序列中含有nucletide-binding motif,預測有ATPase或 kinase的活性,推測此蛋白可能為提供此分泌機器的能量來源。本論文主要進行XpsE蛋白之純化及其生化特性分析,由蛋白質N端定序得知,XpsE蛋白的起始密碼為ATT,首先構築以ATT為起始密碼的全長xpsEh基因,在大腸桿菌中大量表現,再利用鎳離子親和性管柱純化此蛋白,隨後將經部分純化之XpsEh蛋白作有限的蛋白酶切割分析(limited proteolysis),發現利用三種酵素 (trypsin、endoproteinase Glu-C及endoproteinase Asp-N) 皆會切出50 kDa的蛋白片段,且N端少36個胺基酸的XpsE(△1-36)也會切割出此50 kDa的片段,經由N端定序得知,此切割位於第152-153個胺基酸之間,顯示在第153個胺基酸鄰近有一較易被蛋白酶所切割的區域,推測XpsE蛋白自153胺基酸為界的C端胺基酸序列可能形成一獨立的區域。將C端帶Strep-tag,但具有功能的XpsE-Strep蛋白加入不同核苷酸進行蛋白酶實驗得知,XpsE-Strep蛋白在加入ATPgS的情形下比較不易被蛋白酶切割,說明XpsE蛋白似乎有ATP結合態(ATP-binding state),且會造成蛋白本身構形改變。由分子篩管柱層析得知,加入了ATPgS會使部分XpsE-Strep蛋白呈現大於440 kDa的大分子,推測ATP的結合可能會引起XpsE蛋白形成多倍體。

Abstract
In the type II secretion pathway, extracellular proteins are secreated in two steps. In the first step, the proteins are exported from cytoplasm to periplasm through the Sec apparatus. Subsequeatly, they are secreted through a secretion apparatus composed of twelve to fourteen proteins to reach the extracellular milieu. In Xanthomonas campestris, the secretion apparatus are constituted of the protein products of twelve genes, xpsD~O. Among them, XpsE protein is predicted to be a cytoplasmic protein for its lack of membrane spanning sequence. It possesses a nucleotide-binding motif, and has been suggested to have either ATPase or kinase activity. Therefore it was hypothesized to serve as energy generation for the secretion apparatus. N terminal amino acid sequence of the the functional XpsE protein revealed that the start codon of XpsE protein is ATT, which is located 36 codons upstream of the originally assigned start codon ATG. In this study, recombinant XpsE protein was partially purified from Escherichia coli and analyzed. The C-terminally (His)6-tagged XpsE protein purified from nickel affinity chromatography was analyzed with limited proteolysis. A 50 kDa protein band was detected, when the XpsE protein was digested with trypsin, Endoproteinase Glu-C or Endoproteinase Asp-N. Its N terminal amino acid sequence matches that in full-length XpsE beginning from the 153rd residue, suggesting that the region C-terminal to the 153rd residue may form an independent domain. EDTA treatment appeared efficient in removing the nucleotide remaining associated with the XpsE. Incubation of EDTA-treated XpsE with ATPgS made the protein less susceptible to trypsin digestion. Furthermore, incubation of EDTA-treated, dimer-sized XpsE also made approximately half of the protein fractionate as molecules of sizes greater thrn 440 kDa upon gel filtration chromatography. This result suggests that ATP-binding may cause XpsE to multimerize.

目 錄
中文摘要 ……………..……….…………...…….…... 1
英文摘要 ………..……..………..……………..…..… 3
前言 ……………………..…..………………..……… 5
材料與方法 ……………..…..………………….……. 12
結果 .…………….……..…..………………………… 24
討論 ……………………..…..……………………..… 35
參考文獻 ………………..…..…………………..…… 39
圖表 ……………………..…..……………………..… 43
附錄 ……………………..…..……………………….. 63

參考文獻
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