Abstract
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.