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dc.contributor.authorPang, Yin-Yuinen_US
dc.identifier.citation1. 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.zh_TW
dc.description.abstractT2SS 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.en_US
dc.description.tableofcontentsIntroduction 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 46en_US
dc.subjectprotein complexen_US
dc.subjectATPase activityen_US
dc.titleThe Biochemical Analysis of Coexpressed and Copurified XpsE/MBP-XpsLN Complexen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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