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Analysis of interactive relationship between XpsE and XpsLN by co-expressing them in E. coli
|關鍵字:||Type II secretion system;第二型分泌機制||出版社:||生物化學研究所||引用:||參考文獻 J. Abendroth, P. Murphy, M. Sandkvist, M. Bagdasarian and W. G. J. 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. 348: 845-855 C. Anderson, C. Hughes and V. Koronakis (2000) Channel vision, EMBO Rep. 1: 313-318 W. Bitter, M. Koster, M. Latijnhouwers, H. de Cock, and J. Tommassen (1998) Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol. Microbiol. 27: 209-219 N. F. Brown, C.-A. Logue, J. A. Boddey, R. Scott, R. G. Hirst, I. R. Beacham (2004) Identiﬁcation of a novel two-partner secretion system from Burkholderia pseudomallei. Mol Gen Genomics. 272: 204-215 J. L. Camberg, and M. Sandkvist (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol. 187: 249-256 L. Y. Chen, 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 I. L. Chien (2002) Purification and characterization of the XpsE protein of the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. P. J. Christie and J. P. Vogel (2000) Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8: 354-360 N. Dautin and H. D. Bernstein (2007) Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu. Rev. Microbiol. 61: 89-112 F. Dums, J. M. Dow, and M. J. Daniels (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet. 299: 357-3564 J.E. Galan and A. Collmer (1999) Type III secretion machines: bacterial devices for protein delivery into host cells. Science. 284: 1322-1328 N. T. Hu, M. N. Hung, S. J. Chiou, F Tang, D. C. Chiang, H. Y. Huang, and C. Y. Wu (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol. 174: 2679-2687 N.T. Hu, P. F. Lee, and C. Chen (1995) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. Mol. Microbiol. 18: 769-777 N. T. Hu, W. M. Leu, M. S. Lee, A. Chen, S. C. Chen, Y. L. Song, and L. Y. Chen (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J. 365: 205-211 T. L. Johnson, J. Abendroth. W. G. .J. Hol and M Sandkvist (2006) Type II secretion: from structure to function. FEMS Microbiol Lett. 255: 175-186 K. M. Kao (2005) Type II secretion apparatus of Xanthomonas campestris: Determination of ATPase activity of XpsE and mutant analysis of XpsE with mutations at nucleotide-binding motifs. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. H. M. Lee, S. W. Tyan, W. M. Leu, L. Y. Chen, D. C. Chen, and N. T. Hu (2001) Involvement of the XpsN protein in formation of the XpsL-XpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol. 183: 528-535 M. S. Lee, L. Y. Chen, W. M. Leu, R. J. Shiau, and N. T. Hu (2005) Association of the major pseudopilin XpsG with XpsN (GspC) and secretion XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J Biol Chem. 280: 4585-4591 N. A. Linderoth, M. N. Simon, and M. Russel (1997) The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science. 278: 1635-1638 D. Nunn (1999) Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol. 9: 402-408 N. Nouwen, N. Ranson, H. Saibil, B. Wolpensinger, A. Engel, A. Ghazi, and A. P. Pugsley (1999) Secretion PulD: association with pilot PulS, structure, and ion–conducting channel formation. Proc Natl Acad Sci U S A. 96: 8173-8177 A.P. Pugsley, C d’Enfert, I. Reyss, and M. G. Kornacker (1990) Genetics of extracellular protein secretion by gram-negative bacteria. Annu Rev Genet. 24: 67-90 B. Py, 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 M. Sandkvist (2001) Type II secretion and pathogenesis. Infect Immun. 69: 3523-3525 S. J. Shiue, 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 S. J. Shiue, I. L. Chien, N. L. Chan, W. M. Leu, and N. T. Hu (2007) Mutation of a key residue in the type II secretion systm ATPase uncouples ATP hydrolysis from protein translocation. Mol. Microbiol. 65: 401-412 R. T. Tsai, 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 A. Yamagata 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||摘要:||
本研究針對十字花科黑腐病菌中唯一的胞內蛋白XpsE與內膜蛋白XpsL之間的交互作用進行探討。希望嘗試利用在大腸桿菌中，同時送入表現XpsE與MBP-XpsLN的載體，共同表現XpsE與MBP-XpsLN，再以連續通過兩種不同resin純化XpsE/MBP-XpsLN複合體的方式及SDS-polyacrylamide電泳來分析複合體中XpsE與MBP-XpsLN的比例。並期望利用這樣的測試方法檢查四個在XpsE N2區域保留性高的胺基酸D202、H204、R217與R271定點突變成alanine之後，對於XpsE與MBP-XpsLN之間交互作用的影響。實驗結果發現，作為負面對照組的XpsEKMRA雙突變蛋白，過去在MBP-XpsLN pull down實驗結果顯示其不與MBP-XpsLN結合，然而在共表現系統中卻發現其與MBP-XpsLN的結合能力與XpsEwt相似。利用另外兩個在MBP-XpsLN pull down實驗結果同樣不與MBP-XpsLN結合的XpsER286A與XpsEC突變蛋白進行檢查發現，兩者也能夠與MBP-XpsLN結合。針對這樣的矛盾結果，推測可能是因為共表現系統的蛋白表現量過高所致，已知獨立的 XpsE N1區域在高蛋白濃度情況下具有試管內與XpsLN結合的能力。然而，在不加入IPTG，僅利用質體本身基礎蛋白表現量的情況下，降低細胞內蛋白含量，XpsEKMRA仍然會與MBP-XpsLN進行結合。因此，目前實驗條件下，共表現系統並不適合用來分析XpsE與XpsLN的交互作用關係。此外，在構築XpsER271A與MBP-XpsLN共表現菌株時，發現共表現的轉形菌株長成微小菌落後便不再生長，而單獨表現XpsER271A的轉形菌株仍可順利成長，暗示即使只是基礎表現量，XpsER271A與MBP-XpsLN的共表現就不利於大腸菌的生長。相較之下，單獨表現的正常XpsE或包含XpsER271A的其它突變XpsE蛋白只有在IPTG誘導蛋白大量合成情況下，生長才受到明顯抑制。
The focus of this study is the interactive relationship between XpsE, the only cytoplasmic protein in the Type II secretion system of Xanthomonas campestris pv. campestris, and the N-terminal cytoplasmic domain of the inner membrane protein XpsLN. Attempts were made to analyze the interactive relationship by co-expressing Strep-tagged XpsE and MBP-XpsLN in Escherichia coli. Subsequent to purification of the XpsE-Strep/MBP-XpsLN complex through two consecutive affinity columns, the ratio of each protein in the complex was estimated from signal intensity of each appearing on Comassie blue-stained SDS-polyacrylamide gel. Effect of mutating four conserved residues in the N2 domain, D202, H204, R217 and R271, of XpsE on their interactive relationship with XpsLN were also analyzed. The double mutant XpsEKMRA that has been demonstrated to be defective in XpsLN-binding in the MBP-XpsLN pull down assay was chosen as the negative control. Unexpectedly, the double mutant is similar to the wild type XpsE by forming stable complex with MBP-XpsLN in the coexpression system employed here. Likewise, two other mutated XpsE, XpsER286A and N-terminally truncated XpsEC, both of which were defective in XpsLN-binding in the MBP-XpsLN pull down assay, were also capable of forming stable complex with MBP-XpsLN in the coexpression system. We speculated that the inconsistency may arise from too high protein expression levels in the coexpression system. It is known that at high protein concentrations, the N1 domain is capable of binding to XpsLN in vitro. However, by omitting IPTG from the coexpression system, which allows a low basal level of protein production, we still observed complex formation between XpsEKMRA and MBP-XpsLN, suggesting that the coexpression system in its current state may not be suitable for analyzing interactive relationship between XpsE and XpsLN. Furthermore, unlike the strain singly expressing the mutant XpsER271A, transformants of the XpsER271A/ MBP-XpsLN coexpression strain formed very minute colonies, implicating growth defect caused by coexpressing the mutant XpsER271A and MBP-XpsLN at basal expression level. In contrast, singly expressed wild type XpsE or its variants, which include the XpsER271A, is inhibitory to E. coli growth only when overproduced.
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