幽門螺旋桿菌之腺核苷二磷酸七碳糖合成酶HP0858之表現質體建構與結構模擬

The construction of expressing plasmid and the structure stimulation of Helicobacter pylori ADP-Heptose synthase (HP0858)

葉星汎(Hsin-Fan Yeh)

指導教授 ： 殷献生

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2009年)

摘要

L-glycero-alpha -D-manno-Heptose是幽門螺旋桿菌脂多醣 核心部分的重要組成，若幽門螺旋桿菌七碳糖轉移酶Ⅰhp0279突變，可導致幽門螺旋桿菌死亡。亦即缺乏L-glycero-alpha -D-manno-Heptose可導致幽門螺旋桿菌死亡。 因此挑出L-glycero-alpha -D-manno-Heptose合成步驟的中的重要蛋白，hp0858雙功能蛋白質。建構hp0858表現細胞並希望能得到其蛋白質結晶，然無法得到結晶，原因可能是表現hp0858時的雜質以及在低離子強度下hp0858易沉澱。亦利用序列比對與結構模擬，發現hp0858在自然環境下可能非以單體存在，其兩個domain均可能進行雙體化，這可導致表現蛋白混入雜質，以及表現蛋白質的快速沉澱。無法得到結構。

關鍵字

幽門螺旋桿菌 ； 脂多醣 ； 七碳糖 ； 蛋白質結構

並列摘要

Abstract
L-glycero-alpha -D-manno-Heptose is an essential element of Helicobacter pylori lipopolysacharides (LPS) core oligosaccharides (OS), because the inactive mutation of heptosytransferase Ⅰ, hp0279 is lethel to Helicobacter pylori. It tells the lack of heptose is lethel to Helicobacter pylori. We select the bifunctional protein hp0858 , one of the protein in L-glycero-alpha -D-manno-Heptose biosynthesis pathway and construct the E coli expressing clone of hp0858 to make the protein crystal of hp0858. I fail to get the crystals, because hp0858 might be more easily precipitate in low ion strength buffer and purity may benot enough. By bioinformational tools, sequence aliment and structure stimulation, we tell that hp0858 may not be monomer in natural environment and the two domains of hp0858 might form homodimer independently This properties might makes hp0858’s impurity and rapid precipitation

並列關鍵字

Helicobacter ； pylori ； HP0858 ； heptose ； protein structure

參考文獻

Andersson, C. E. and S. L. Mowbray (2002). "Activation of ribokinase by monovalent cations." J Mol Biol 315(3): 409-19.

Bernd Kneidinger, C. M., Michael Graninger, Alla Zamyatina, Fiona McArthur, Paul Kosma, Miguel A. Valvano, and Paul Messner (2002). "Biosynthesis Pathway of ADP-L-glycero-Beta -D-manno-Heptose in Escherichia coli." JOURNAL OF BACTERIOLOGY 184(2): 7.

Bernd Kneidinger, M. G., Michael Puchberger§, Paul Kosma§, and and P. Messner (2001). "Biosynthesis of Nucleotide-activated D-glycero-D-manno-Heptose." THE JOURNAL OF BIOLOGICAL CHEMISTRY 276(24): 10.

De Leon, G. P., N. H. Elowe, et al. (2006). "An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis." Chemistry & Biology 13(4): 437-441.

Edwards, N. J., M. A. Monteiro, et al. (2000). "Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium." Molecular Microbiology 35(6): 1530-1539.