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.

Abshire KZ, Neidhardt FC (1993): Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage. J Bacteriol 175:3734-43.
Abu Kwaik Y (1998): Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection. Infect Immun 66:203-12.
Ang S, Lee CZ, Peck K, Sindici M, Matrubutham U, Gleeson MA, Wang JT (2001): Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray. Infect Immun 69:1679-86.
Aoki M, Uchiumi T, Tsuji E, Hachimori A (1998): Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3. Biochem J 331 ( Pt 1):143-8.
Avaeva SM (2000): Active site interactions in oligomeric structures of inorganic pyrophosphatases. Biochemistry (Mosc) 65:361-72.
Avaeva SM, Rodina EV, Vorobyeva NN, Kurilova SA, Nazarova TI, Sklyankina VA, Oganessyan VY, Harutyunyan EH (1998): Changes in E. coli inorganic pyrophosphatase structure induced by binding of metal activators. Biochemistry (Mosc) 63:592-9.
Baykov AA, Hyytia T, Volk SE, Kasho VN, Vener AV, Goldman A, Lahti R, Cooperman BS (1996): Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence. Biochemistry 35:4655-61.
Bumann D, Aksu S, Wendland M, Janek K, Zimny-Arndt U, Sabarth N, Meyer TF, Jungblut PR (2002): Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori. Infect Immun 70:3396-403.
Carystinos GD, MacDonald HR, Monroy AF, Dhindsa RS, Poole RJ (1995): Vacuolar H(+)-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiol 108:641-9.
Celis H, Franco B, Escobedo S, Romero I (2003): Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria. Arch Microbiol 179:368-76.
Celis H, Romero I (1987): The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase of Rhodospirillum rubrum: effects of pH and divalent cations. J Bioenerg Biomembr 19:255-72.
Chen J, Brevet A, Fromant M, Leveque F, Schmitter JM, Blanquet S, Plateau P (1990): Pyrophosphatase is essential for growth of Escherichia coli. J Bacteriol 172:5686-9.
Dubnova EB, Baikov AA (1991): [Catalytic properties of inorganic pyrophosphatase in rat liver mitochondria]. Biokhimiia 56:2181-7.
Fraichard A, Trossat C, Perotti E, Pugin A (1996): Allosteric regulation by Mg2+ of the vacuolar H(+)-PPase from Acer pseudoplatanus cells. Ca2+/Mg2+ interactions. Biochimie 78:259-66.
Gomez-Garcia MR, Losada M, Serrano A (2003): Concurrent transcriptional activation of ppa and ppx genes by phosphate deprivation in the cyanobacterium Synechocystis sp. strain PCC 6803. Biochem Biophys Res Commun 302:601-9.
Hachimori A, Shiroya Y, Hirato A, Miyahara T, Samejima T (1979): Effects of divalent cations on thermophilic inorganic pyrophosphatase. J Biochem (Tokyo) 86:121-30.
Hachimori A, Takeda A, Kaibuchi M, Ohkawara N, Samejima T (1975): Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus. J Biochem (Tokyo) 77:1177-83.
Hoe HS, Kim HK, Kwon ST (2002): Cloning, analysis, and expression of the gene for inorganic pyrophosphatase of Aquifex pyrophilus and properties of the enzyme. Mol Cells 13:296-301.
Hornick RB (1987): Peptic ulcer disease: a bacterial infection? N Engl J Med 316:1598-600.
Howard A, Lundgren DG (1970): Inorganic pyrophosphatase from Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans). Can J Biochem 48:1302-7.
Islam KM, Miyoshi T, Isobe T, Kasuga-Aoki H, Arakawa T, Matsumoto Y, Yokomizo Y, Tsuji N (2004): Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum. J Vet Med Sci 66:221-3.
Islam MK, Miyoshi T, Kasuga-Aoki H, Isobe T, Arakawa T, Matsumoto Y, Tsuji N (2003): Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites. Eur J Biochem 270:2814-26.
Josse J (1966): Constitutive inorganic pyrophosphatase of Escherichia coli. 1. Purification and catalytic properties. J Biol Chem 241:1938-47.
Kim EJ, Zhen RG, Rea PA (1995): Site-directed mutagenesis of vacuolar H(+)-pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J Biol Chem 270:2630-5.
Klemme JH, Gest H (1971a): Regulation of the cytoplasmic inorganic pyrophosphatase of Rhodospirillum rubrum. Eur J Biochem 22:529-37.
Klemme JH, Gest H (1971b): Regulatory properties of an inorganic pyrophosphatase from the photosynthic bacterium Rhodospirillum rubrum. Proc Natl Acad Sci U S A 68:721-5.
Klemme JH, Klemme B, Gest H (1971): Catalytic properties and regulatory diversity of inorganic pyrophosphatases from photosynthetic bacteria. J Bacteriol 108:1122-8.
Kondrashin AA, Remennikov VG, Samuilov VD, Skulachev VP (1980): Reconstitution of biological molecular generators of electric current. Inorganic pyrophosphatase. Eur J Biochem 113:219-22.
Konturek PC, Pierzchalski P, Konturek SJ, Meixner H, Faller G, Kirchner T, Hahn EG (1999): Helicobacter pylori induces apoptosis in gastric mucosa through an upregulation of Bax expression in humans. Scand J Gastroenterol 34:375-83.
Kuhn NJ, Wadeson A, Ward S, Young TW (2000): Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co(2+) or Mn(2+) ions against fluoride inhibition. Arch Biochem Biophys 379:292-8.
Kuhn NJ, Ward S (1998): Purification, properties, and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis. Arch Biochem Biophys 354:47-56.
Lahti R (1983): Microbial inorganic pyrophosphatases. Microbiol Rev 47:169-78.
Lahti R, Hannukainen R, Lonnberg H (1989): Effects of spermine and spermidine on the inorganic pyrophosphatase of Streptococcus faecalis. Interactions between polyamines and inorganic pyrophosphate. Biochem J 259:55-9.
Lahti R, Heinonen J (1981): Reversible changes in the activity of inorganic pyrophosphatase of Streptococcus faecalis. The effect of compounds containing SH-groups. Acta Chem Scand B 35:33-8.
Lahti R, Lonnberg H (1985): Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase. Biochem J 231:485-8.
Lahti R, Niemi T (1981): Purification and some properties of inorganic pyrophosphatase from Streptococcus faecalis. J Biochem (Tokyo) 90:79-85.
Liu B, Bartlam M, Gao R, Zhou W, Pang H, Liu Y, Feng Y, Rao Z (2004): Crystal structure of the hyperthermophilic inorganic pyrophosphatase from the archaeon Pyrococcus horikoshii. Biophys J 86:420-7.
Logan RP (1994): Helicobacter pylori and gastric cancer. Lancet 344:1078-9.
Marchesini N, Luo S, Rodrigues CO, Moreno SN, Docampo R (2000): Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites. Biochem J 347 Pt 1:243-53.
Marshall BJ, Warren JR (1984): Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1:1311-5.
Mendall MA, Goggin PM, Molineaux N, Levy J, Toosy T, Strachan D, Camm AJ, Northfield TC (1994): Relation of Helicobacter pylori infection and coronary heart disease. Br Heart J 71:437-9.
Merrell DS, Goodrich ML, Otto G, Tompkins LS, Falkow S (2003): pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun 71:3529-39.
Mitchell SJ, Minnick MF (1997): Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase from Bartonella bacilliformis. Can J Microbiol 43:734-43.
Morita JI, Yasui T (1978): Purification and some properties of a neutral muscle pyrophosphatase. J Biochem (Tokyo) 83:719-26.
Oliva G, Romero I, Ayala G, Barrios-Jacobo I, Celis H (2000): Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori. Arch Microbiol 174:104-10.
Parfenyev AN, Salminen A, Halonen P, Hachimori A, Baykov AA, Lahti R (2001): Quaternary structure and metal ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii, and Streptococcus mutans. J Biol Chem 276:24511-8.
Plaxton WC (1996): The Organization and Regulation of Plant Glycolysis. Annu Rev Plant Physiol Plant Mol Biol 47:185-214.
Rapoport S, Scheuch D (1960): Glutathione stability and pyrophosphatase activity in reticulocytes; direct evidence for the importance of glutathione for the enzyme status in intact cells. Nature 186:967-8.
Rodrigues CO, Scott DA, Bailey BN, De Souza W, Benchimol M, Moreno B, Urbina JA, Oldfield E, Moreno SN (2000): Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets. Biochem J 349 Pt 3:737-45.
Romero I, Garcia-Contreras R, Celis H (2003): Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing. Arch Microbiol 179:377-80.
Routzahn KM, Waugh DS (2002): Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J Struct Funct Genomics 2:83-92.
Salminen T, Kapyla J, Heikinheimo P, Kankare J, Goldman A, Heinonen J, Baykov AA, Cooperman BS, Lahti R (1995): Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis? Biochemistry 34:782-91.
Samygina VR, Popov AN, Rodina EV, Vorobyeva NN, Lamzin VS, Polyakov KM, Kurilova SA, Nazarova TI, Avaeva SM (2001): The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications. J Mol Biol 314:633-45.
Schwarm HM, Vigenschow H, Knobloch K (1986): Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris. Biol Chem Hoppe Seyler 367:127-33.
Scott DR, Marcus EA, Weeks DL, Lee A, Melchers K, Sachs G (2000): Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease. Infect Immun 68:470-7.
Scott DR, Weeks D, Hong C, Postius S, Melchers K, Sachs G (1998): The role of internal urease in acid resistance of Helicobacter pylori. Gastroenterology 114:58-70.
Seufferheld M, Vieira MC, Ruiz FA, Rodrigues CO, Moreno SN, Docampo R (2003): Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes. J Biol Chem 278:29971-8.
Sosa A, Ordaz H, Romero I, Celis H (1992): Mg2+ is an essential activator of hydrolytic activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum. Biochem J 283 ( Pt 2):561-6.
Tominaga N, Mori T (1977): Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans. J Biochem (Tokyo) 81:477-83.
Triccas JA, Gicquel B (2001): Analysis of stress- and host cell-induced expression of the Mycobacterium tuberculosis inorganic pyrophosphatase. BMC Microbiol 1:3.
Urbina JA, Moreno B, Vierkotter S, Oldfield E, Payares G, Sanoja C, Bailey BN, Yan W, Scott DA, Moreno SN, Docampo R (1999): Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs. J Biol Chem 274:33609-15.
Verhoeven JA, Schenck KM, Meyer RR, Trela JM (1986): Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus. J Bacteriol 168:318-21.
Ware D, Postgate JR (1970): Reductant-activation of inorganic pyrophosphatase: an ATP-conserving mechanism in anaerobic bacteria. Nature 226:1250-1.
Ware DA, Postgate JR (1971): Physiological and chemical properties of a reductant-activated inorganic pyrophosphatase from Desulfovibrio desulfuricans. J Gen Microbiol 67:145-60.
Wen Y, Marcus EA, Matrubutham U, Gleeson MA, Scott DR, Sachs G (2003): Acid-adaptive genes of Helicobacter pylori. Infect Immun 71:5921-39.
Wood HG (1977): Some reactions in which inorganic pyrophosphate replaces ATP and serves as a source of energy. Fed Proc 36:2197-206.
Young TW, Kuhn NJ, Wadeson A, Ward S, Burges D, Cooke GD (1998): Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase? Microbiology 144 ( Pt 9):2563-71.