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研究生:彭穎韻
研究生(外文):Yin-Yuin Pang
論文名稱:共同表現之XpsE/MBP-XpsLN複合體之生化特性分析
論文名稱(外文):The Biochemical Analysis of Coexpressed and Copurified XpsE/MBP-XpsLN Complex
指導教授:胡念台
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:2008
畢業學年度:96
語文別:英文
論文頁數:30
中文關鍵詞:第二型分泌機器
外文關鍵詞:T2SSprotein complexATPase activitycardiolipin
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T2SS of Xanthomonas campestris pv campestris is assembled by 12 proteins. XpsE is the only cytoplasmic component and the likely energy supplier of the system, whereas XpsL is a bitopic membrane protein with a single transmembrane segment. The role of XpsL in T2SS is not so clear. It has been previously observed that the hexameric XpsE, whose formation is nucleotide-dependent, interacts in vitro directly with the cytoplasmic domain of XpsL as MBP-XpsLN. We thus speculated that XpsE may form complex with XpsLN in vivo. In this study, we attempted the complex isolation by coexpressing XpsE and MBP- XpsLN in E. coli. Copurification of MBP-XpsLN and Strep-tagged XpsE was observed on the SDS-PAGE when purified using double-affinity chromatography, indicating that a stable XpsE/MBP-XpsLN complex was formed as a consequence of their coexpression in E. coli. The molecular size of such a complex was estimated to be 800 kDa as revealed by size-exclusion chromatography. We thus postulated that the complex may be constituted of 6 molecules each component. The protein complex purified from size-exclusion chromatography exhibited an ATPase activity sixfold that of the singly expressed XpsE. In addition, the ATPase activity of the complex was stimulated by cardiolipin by threefold. The XpsE/MBP-XpsLN complex resulted from the coexpression strategy employed here might resemble an intermediate stage during secretion process in vivo, thus enabling us to study in the future the mechanistic events driven by the interaction between XpsE and XpsL.
Introduction 1
Materials and Methods 7
Plasmids and bacterial strains 7
Media, reagents and buffers 7
Mini-preparation of plasmid DNA 7
Preparation of competent cell 7
Co-transformation 8
Protein analysis and immunological techniques 8
Small-scale induction of XpsE/MBP-XpsLN and XpsE/SUMO-XpsLN 9
Purification of XpsE/MBP-XpsLN complex using amylose affinity column 10
Purification of MBP-XpsLN using amylose affinity column 11
Purification of XpsE using Strep-Tactin column 11
Purification of XpsE/SUMO-XpsLN complex using two consecutive affinity columns 12
Purification of His-tagged SUMO-XpsLN complex using nickel column 13
Size-exclusion chromatography 13
Blue native gel electrophoresis 14
ATPase activity assay 14
Results 16
Expression and purification of XpsE/MBP-XpsLN 16
Stoichiometric analysis of XpsE and MBP-XpsLN interaction 18
In vitro ATPase activity of XpsE 19
In vitro ATPase activity of copurified XpsE/MBP-XpsLN complex 20
Expression and purification of XpsE/SUMO-XpsLN 21
Stoichiometric analysis of XpsE and SUMO-XpsLN interaction 22
Discussion 24
References 28
Figures 31
Tables 44
Appendix 46
1.Abendroth, J., P. Murphy, M. Sandkvist, M. Bagdasarian and W. G. Hol. (2005). The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J. Mol. Biol. 384, 845-855.
2.Camberg, J. L., T. L. Johnson, M. Patrick, J. Abendroth, W. G. J. Hol and M. Sandkvist. (2007). Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. EMBO J. 26, 19-27.
3.Camberg, J. L. and M. Sandkvist. (2005). Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J. Bacteriol. 187, 249-256.
4.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.
5.Crowther, L. J., A. Yamagata, L. Craig, J. A. Tainer and M. S. Donnenberg. (2005). The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J. Mol. Biol. 280, 24839-24848.
6.Driessen, A. J. and N. Nauwen. (2008). Protein translocation across the bacterial cytoplasmic membrane. Annu. Rev. Biochem. 77, 643-667.
7.Filloux, A., A. Hachani and S. Bleves. (2008). The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154, 1570-1583.
8.Forest, K. T., K. A. Satyshur, G. A. Worzalla, J. K. Hansen and T. J. Herdendorf. (2004). The pilus-retraction protein PilT: ultrastructure of the biological assembly. Acta Crystallogr D Biol Crystallogr. 60, 978-982.
9.Kim, K. I., W. Cheong, S. C. Park, J. S. Ha, K. M. Woo, S. J. Choi and C. H. Chung. (2000). Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli. J. Mol. Biol. 303, 655-666.
10.Kuo, W. W., H. W. Kuo, C. C. Cheng, H. L. Lai and L. Y. Chen. (2005). Roles of the minor pseudopilins, XpsH, XpsI and XpsJ, in the formation of XpsG-containing pseudopilus in Xanthomonas campestris pv. campestris. J. Biomed Sci. 12, 587-599.
11.Lee, M. S., L. Y. Chen, W. M. Leu, R. J. Shiau and N. T. Hu. (2005). Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J. Biol. Chem. 280, 4585-4591.
12.Lee, P. A., D. Tullman Ercek and G. Georgiou. (2006). The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60, 373-395.
13.Lill, R., W. Dowhan and W. Wickner. (1990). The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 60, 271-280.
14.Py, B., L. Loiseau and F. Barras (1999). Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE the putative ATP-binding component and the membrane protein OutL. J. Mol. Biol. 289, 659-670.
15.Py, B., L. Loiseau and F. Barras. (2001). An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep. 23, 244-248.
16.Sekimizu, K., B. Y. Yung and A. Kornberg. (1988). The DnaA protein of Escherichia coli. Abundance, improved purification, and membrane binding. J. Biol. Chem. 263, 7136-7140.
17.Shiue, S. J., K. M. Kao, W. M. Leu, L. Y. Chen, N. L. Chan and N. T. Hu. (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J. 25, 1426-1435.
18.Shiue, S. J., I. Ling. Chien, N. L. Chan, W. M. Leu and N. T. Hu. (2007). Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol. Miocrobiol. 65, 401-412.
19.Souvonnet, N., G. Vignon, A. P. Pugsley and P. Gounon. (2000). Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J. 19, 2221-2228.
20.Yamagata A. and J. A. Tainer. (2007). Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J. 26, 878-890.
21.Tsai, R. T., W. M. Leu, L. Y. Chen and N. T. Hu. (2002). A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem. J. 367, 865-871.
22.Walker, J. E., M. Saraste, M. J. Runswick and N. J. Gay. (1982). Distantly related sequences in the α- and ß-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 8, 945-951.
23.Yeo, H. J., S. N. Savvides, A. B. Herr, E. Lanka and G. Waksman. (2000). Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol. Cell. 6, 1461-1472.
24.Wittig, I., H. P. Braun and H. Schägger. (2006). Blue Native PAGE. Nat. Protoc. 1, 418-428.
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