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
1.Salmond, G. P., and P. J. Reeves. 1993. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci. 18:7-12.
2.Pugsley, A.P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57:50-108.
3.Filloux, A., G. Michel, and M. Bally. 1998. GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol Rev. 22:177-198.
4.Wandersman, C. 1992. Secretion across the bacteria outer membrane. Trends Genet. 8:317-321
5.Fath, M. J., and R. Kolter. 1993. ABC transporters : bacterial exporters. Microbial. Rev. 57:995-1017
6.Binet, R., S. Letoffe, J. M. Ghigo, P. Delepelaire, and C.Wandersman. 1997. Protein secretion by Gram-negative bacterial ABC exporters-a review. Gene 192:7-11
7.Lee, V.T., and Schneewind, O. 1999 Type III machines of pathogenic Yersiniae secrete virulence factors into the extracellular milieu. Mol Microbiol 31:1619-1629.
8.Cornelis, G. R. and Wolf-Watz, H. 1997. The Yersinia Yop virulon : a bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23:861-867
9.Christie, P. J., Ward, J. E., Gordon, M. P. and Nester, E. W. 1989. Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. Proc. Natl. Acad. Sci. USA 86, 9677-9681 J. Bacteriol. 179: 3085 - 3094.
10.Christie, P. J., and Vogel, J,P. 2000. Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8(8): 354-60.
11.Burns, D. L. 1999. Biochemistry of typeIV secretion. Curr Opin Microbiol. 2 : 25-29.
12.Lory, S. 1998. Secretion of protein and assembly of bacterial surface organelles: shared pathways of extracellular protein targeting. Curr Opin Microbiol. 1:27-35
13.Chen, L. Y., D. Y. Chen, J. Miaw, and N. T. Hu. 1996. XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem. 271:2703-2708.
14.Lauer, P., Albertson, N.H., and Koomey, M. 1993. Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrhoeae. Mol Microbiol 8:357-368.
15.Sauvonet, N., Vignon, G., Pugsley, A.P., and Gounon, P. 2000. Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J.19,2221-2228
16.Hu, N.T., Leu, W.M., Lee, M. S., Chen, A., Chen, S. C., Song, Y. L., and Chen, L. Y. 2002. XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J. 365, 205-11
17.Sandkvist, M., M. Bagdasarian, S. P. Howard, and V. J. DiRita. 1995. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J. 14:1664-1673.
18.Py, B., L. Loiseau, and F. Barras. 2001. An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep. 2:244-248.
19.Turner, L. R., Lara, J.C., Nunn, D.N., and Lory, S. 1993.Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J. Bacteriol. 175: 4962 - 4969
20.Planet, P. J., Kachlany, S. C., DeSalle, R., and Figurski , D. H. 2001. Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci USA. 98: 2503 - 2508.
21.Yeo, H. J., Savvides, S.N., Herr, A. B., Lanka, E., and Waksman , G., 2000. Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell. 6(6): 1461-72.
22.Scott, M. E., Dossani, Z. Y., and Sandkvist, Maria. 2001. Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway. Proc Natl Acad Sci USA. 98: 13978-13983.
23.Sabine Krause, Montserrat Bárcena, Werner Pansegrau, Rudi Lurz, José María Carazo, and Erich Lanka. 2000. Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc Natl Acad Sci USA. 97: 3067 - 3072.