Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24089
標題: 志賀氏痢疾桿菌colicin Ib之純化與結晶
Purification and preliminary crystallization of colicin Ib from Shigella flexneri
作者: 詹雅婷
Chan, Ya-Ting
關鍵字: colicin Ib;腸桿菌素
出版社: 生物化學研究所
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TolA central domain interacts with Escherichia coil porins. EMBO J. 15, 6408-6415. 27. Benedetti, H. & Geli, V. (1996). Chapter29: colicin transport, channel formation and inhibition. Handbook of Biological Physic 2, 665-691. 28. Buchanan, S. K., Lukacik, P., Grizot, S., Ghirlando, R., Ali, M. M., Barnard, T. J., Jakes, K. S., Kienker, P. K. & Esser, L. (2007). Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import. EMBO J. 26, 2594-2604. 29. Ghosh, P., Mel, S. F. & Stroud, R. M. (1993). A carboxy-terminal fragment of colicin Ia forms Ion channels. J. Membr. Biol. 134, 85-92. 30. Schramm, E., Olschlager, T., Troger, W. & Braun, V. (1988). Sequence, expression and localization of the immunity protein of colicin B. Mol. Gen. Genet. 211, 176-182. 31. Geli, V., Baty, D., Pattus, F. & Lazdunski, C. (1989). Topology and function of the integral membrane protein conferring immunity to colicin A. Mol. Microbiol. 3, 679-687. 32. Song, H. Y. & Cramer, W. A. (1991). Membrane topography of ColEl gene products: the immunity protein. J. Bacteriol. 173, 2935-2943. 33. Benedetti, H., Frenette, M., Baty, D., Knibiehler, M., Pattus, F. & Lazdunski., C. (1991). Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement. J. Mol. Biol. 217, 429-439. 34. Pilsl, H. & Braun, V. (1995). Evidence that the immunity protein inactivates colicin 5 immediately prior to the formation of the transmembrane channel. J. Bacteriol. 177, 6966-6972. 35. Ghosh, P., Mel, S. F. & Stroud, R. M. (1994). The domain structure of the ion channel-forming protein colicin Ia. Nat. Struct. Biol. 1, 597-604. 36. 洪嘉伶 (2010). 大腸桿菌中大腸桿菌素colicin Ib之分泌. 國立中興大學分子生物研究所碩士論文
摘要: 
colicin-Ia及-Ib為水溶性pore-forming (穿孔式)腸桿菌毒素,由特定具有該毒素質體DNA (pCol)的E. coli產製毒素,毒殺近親種源腸桿菌以取得生存優勢,並藉由產製抑制毒素immunity蛋白質以保護自身。由colicin Ia晶體結構得知該毒素具有3個domains: 位於中段的receptor-binding-domain負責辨認結合細菌胞外膜Cir受器,N-端的translocation-domain負責讓毒素能穿越細菌外膜,而C-端的channel-forming-domain在目標細菌內膜形成電位調控型離子通道 (voltage-dependent ion channel)以進行毒殺。由colicin-Ia及-Ib位於C-端immunity蛋白質結合區域的序列差異性(40% (176胺基酸)),以及只能辨識各自免疫蛋白質的特性,推論immunity蛋白質結合區域的結構應該會有差異。本研究主旨在探討弗式痢疾桿菌 (Shigella flexneri) colicin-Ib (638 a.a.)的晶體結構及其與colicin-Ia對免疫蛋白結合的差異性。已使用共轉殖表達質體pQE80L-cib與pACYC184-imm的DH5α菌株,分別進行6升兩批次蛋白質表達,以Ni-NTA、SP-sepharose、及HiTrap-Q等樹脂管柱層析法分批純化至>98及>99%純度及濃縮至9 mg/mL,結晶實驗以蒸氣昇華方法篩選結晶條件,已突破容易形成晶狀針叢習性及使用β-octylglucoside添加劑,在硫酸銨鹽條件下長出板柱狀單晶,於同步輻射BL13C1站測試晶體繞射X-光解析度為4.0Å.

Colicin Ia and Ib, which are produced by strains of Escherichia coli that harbor one colicinogenic plasmid, pCol, are water soluble pore-forming bacteriocin that are lethal for related strains of E. coli. These toxin-producing strains also express specific immunity protein to protect themselves against their own bacteriocin. The crystal structure of colicin-Ia has unraveled the molecular details of three distinct domains corresponding to their specific functions: The N-terminal translocation-domain mediates toxin translocation across the outer membrane, the central receptor-binding-domain binds to the outer membrane receptor Cir, and the C-terminal channel-forming-domain forms a lethal voltage gated ion channel into the inner membrane of the targeted cell. The facts that colicins Ia and Ib have different immunity specificities and the immunity binding region of the C-terminal residues differ by 40% (176 a.a.) strongly suggests different structural features in immunity specificity. The aim of this study was to determine the crystal structure of colicin Ib and to assess the structural details of the immunity binding region. We have purified colicin Ib (638 a.a.) from two batches of 6 L large scale protein expression of a transformed DH5αstrain harboring pQE80L-cib and pACYC184-imm expression plasmids. The proteins were purified by using a combination of Ni-NTA, SP-sepharose, and HiTrap-Q chromatography to homogeniety >98% and >99%, respectively. Initial screening for promising crystallization condition and further fine-tuning using 1.2 M ammonium sulfate and β-octylglucoside yielded plate-like prismatic crystals. The preliminary X-ray diffraction study of these crystals at NRRSC BL13C1 station showed resolution to 4.0Å.
URI: http://hdl.handle.net/11455/24089
其他識別: U0005-2306201116093200
Appears in Collections:生物化學研究所

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