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研究生:黃中瑜
研究生(外文):C.Y Huang
論文名稱:胃幽門螺旋桿菌之焦磷酸水解脢蛋白質的表現質體建構,純化,以及功能分析
論文名稱(外文):Cloning, Purification and Characterization of the recombinant inorganic pyrophosphatase in H. pylori
指導教授:黃海美
指導教授(外文):Hai-mei Huang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:73
中文關鍵詞:幽門螺旋桿菌焦磷酸水解酶酵素動力學酸誘導焦磷酸根蛋白質純化
外文關鍵詞:H.pyloriinorganic pyrophosphataseenzyme kineticsacid inducedpyrophosphateprotein purificaition
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在本論文中,根據TIGR以及DNA 微陣列的資料庫,挑選一些特定的胃幽門螺旋桿菌基因,做為功能性以及蛋白質結構分析. 將這些目標基因(HPXXXX)分別建構至5端含有六個Histine的表現質體pQE-30,接著將完成建構的質體轉形(transformation)至 SG13009菌株.在這些目標基因之中,菌株帶有HP0620基因的表現質體在IPTG的誘導下可以表現大量可溶性蛋白質. 所以選擇此基因做為研究的方向.
根據TIGR資料庫的註解,重組HP0620 蛋白質的功能為焦磷酸水解酶,它的機制為水解焦磷酸成為磷酸根. 此蛋白質在生化合成的步驟中扮演很重要的地位. 為了結晶和功能性分析的需要,由帶有HP0620 基因之菌株中IPTG誘導表現可溶性蛋白質.利用含螯合鎳金屬的管柱可以得到大量且純度高的重組HP0620蛋白質.
經由SDS-PAGE分析結果,HP0620蛋白質的分子量約為23千道耳吞. 分別加入焦磷酸以及磷酸根呈色的試劑 (ascorbate/ammonium molybdate),在Native Gel上可以發現焦磷酸水解酶的活性. 從動力學的結果得知 (1) 反應溶液中有2 mM Mg+2 HP0620蛋白質動力學的參數Vmax和Km分別是116 umol Pi min-1 mg-1以及593 µM.(2) ATP ,NaF ,IDP (iminodiphosphate)具有抑制活性的效果. 2mM ATP, 0.8mM NaF具有抑制80%的活性而1mM IDP則抑制50%的活性. (3)二價離子對HP0620蛋白質活性的影響. HP0620 蛋白質在具有鎂離子的條件之下, 活性較錳和鈣離子為佳. 此外,鎂離子具有活化此蛋白質活性的功能. (4) 此蛋白質最佳活性的pH範圍從8至10. 它也是個熱穩定蛋白質即使前處理60℃,15分鐘也有最佳活性.
利用蛋白質序列比對工具,HP0620蛋白質和Family I類焦磷酸酶有大於40%的同源性而且也具有相同的活性位置(conserved active sites).由此推論,HP0620蛋白質應屬於Family I類焦磷酸酶.
此外,含硫基(SH)的藥劑例如半胱胺酸(cysteine)以及還原態麩胺基硫 (reduced glutathione) 並不會對HP0620蛋白質活性有影響. HP0620蛋白質唯一的半胱胺酸胺基酸以定點突變置換成絲胺酸 (serine).利用螯合鎳金屬的管柱純化,可以得到大量突變的蛋白質. 比較野生性(wild type)和半胱胺酸突變蛋白質並沒有活性上的差異.另外,加入半胱胺酸修飾藥劑 (N-ethylmaleimide)也不會對野生性HP0620 蛋白質有影響. 由這些結果,半胱胺酸可能在HP0620 蛋白質中並沒有扮演重要的地位.
將重組HP0620蛋白質做為抗原並且注射至兔子誘發免疫反應而產生多株抗體.所得到的抗體利用西方墨點法測試其效價,發現在1:5000的稀釋比例可以偵測到小於3ng的重組HP0620蛋白質. 利用所製備的抗體以西方墨點法就可以偵測到在不同的環境下,胃幽門螺旋桿菌HP0620蛋白質的表現量. 結果發現不論有無添加尿素(urea), 在pH 5.5和pH 7.2的環境中長時間或短時間培養胃幽門螺旋桿菌,都不會改變HP0620蛋白質的表現量. 此結果說明環境中的pH值改變並不會影響HP0620蛋白質的表現.
In this study, some genes from H.pylori were selected for the structural and functional analysis according to TIGR and DNA microarray databases. These target genes (HPXXXX) were cloned into expression vector, pQE-30, containing His-tag at 5’ end. Then, the constructed plasmids were transformed into E.coli SG13009 strain. Among these target genes, the clones that carried HP0620 gene in the expression plasmid produced large amount of soluble protein after IPTG induced protocol. The HP0620 gene was selected for further study.
The function of the recombinant HP0620 protein was an inorganic pyrophosphatase based on TIGR database annotation. It had the ability into catalyze the hydrolysis pyrophosphate to orthophosphate. The inorganic pyrophosphatase (PPase) plays an important role in biosynthesis processes. For the need of crystallization and functional assay, the purified HP0620 was essential. Expressed protein from 1L bacteria culture was purified through Ni-NTA superflow column in an imidazol NaCl buffer system with high purity and great quality.
The molecular mass of recombinant HP0620 was estimated to be 23KDa by SDS-PAGE. This recombinant protein in native gel showed PPase enzyme activity after addition of sodium pyrophosphate substrate and ascorbate/ammonium molybdate coupling reagent.
The kinetics results showed: (1) the enzyme had an apparent Km of 593 µM, and a Vmax estimated at 116 umol Pi min-1 mg-1 in reaction buffer containing 1 mM free Mg+2.(2) The HP0620 protein was inhibited in the presence of ATP, NaF and IDP (iminodiphosphate). 2mM ATP and 0.8mM NaF showed 80% inhibition of the enzyme. 1mM IDP showed 50% inhibition of the enzyme. (3) Mg+2 ions were preferred to Mn+2 and Ca+2 ions for optimal activity. The Mg+2 ions were allosteric activators for the HP0620 protein. (4) The optimal pH for the enzyme ranged from pH 8.0 to pH 10.0. The HP0620 protein was also a thermostability protein and performed optimal activities after the enzyme was pre-treated up to 60℃ for 15 mins.


The sequence alignment between HP0620 and Family I PPases exhibited more than 40% homologue and all species had the same conserved active sites. Based on this result, HP0620 protein could be classified into Family I PPases.
Moreover, reagents that contained SH functional group such as cysteine and reduced glutathione had no effect on the enzyme. The only cysteine residue was replaced by serine using site-directed mutagenesis methods. The cys-mt HP0620 was purified with large amount. And the mutant HP0620 protein was assayed compared with wt HP0620 protein. There was no difference in the two enzymes. Cysteine modification reagent, N-ethylmaleimide, also exerted no effect on the wt-HP0620 protein. The cysteine would not be an important residue in contrast to membrane-form PPases.
The purified recombinant HP0620 protein was an antigen and could be injected into the rabbit to boost immune response for producing polyclonal antibody. The detection limit is less than 3 ng of recombinant HP0620 protein in 1:5000 antibody dilution by Western analysis. The antibody could be used to detect the HP0620 protein expression level in H.pylori cultured in different circumstances. However, the HP0620 protein will not be induced in bacteria cultured in acid medium. There was no significant difference in HP0620 protein expression level from bacteria grown in different pH medium (pH 5.5 and pH 7.2 in the presence or absence of urea) from short time (30, 60, and 120 mins) to long time (48 hrs). It indicated that the difference of pH in the environment did not change induction of the inorganic pyrophosphatase.
中文摘要 1
Abstract 3
Introduction 6
Materials and methods 12
Results 24
Discussion 32
References 40
Figures 46
Appendix 65
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