• BMB Reports
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• v.31 no.3
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• pp.240-244
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• 1998

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and in a potent immunogen. In order to express the recombinant urease at a higher level, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a high copy-number vector. The recombinant H. pylori urease expressed in an E. coli strain that was grown in a rich medium supplemented with added nickel was purified to near homogeneity by using DEAE-Sepharose, Superdex HR200, and Mono-Q (FPLC) columns and the purified enzyme possessed the specific activity of 1255 U/mg. Polyclonal antibodies raised against the purified recombinant H. pylori urease were shown to be very specific when subjected to Western blot analysis, in which crude extracts from the H. pylori ATCC strain and the recombinant E. coli strains expressing various bacterial ureases were exnmined for cross-reactivity.

• Journal of Life Science
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• v.13 no.1
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• pp.67-72
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Recombinant H. pylori urease expressed in E. coli was purified by simple purification procedures utilizing (CNBr-activated Sepharose-anti-urease IgG immunoaffinity chromatography or epoxy- activated Sepharose-urea affinity chromatography. Urease was apparently bound so tightly to the anti-urease IgG resin that it could not be eluted at various elution conditions except at certain extreme pH 1, including 100 mM carbonate (pH 10.5) buffer solution, which was shown to elute slightly inactivated but relatively pure enzyme. Urease eluted from the epoxy-activated Sepharose-urea affinity column showed higher activity, but the smaller UreA subunit of the enzyme appeared as a Fainter band of diminished intensity when subjected to SDS-polyamide gel electrophoresis.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.317-325
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• 2001

For heuristic purposes, the relative ratio of urease contents inside and outside cells was surveyed using nine ureB+ strains of Helicobacter pylori. the ratio of the enzyme specific activity appeared to vary greatly between the various H. pylori strains, ranging from 0.5 to 2.5. Besides the above compartment, urease was also richly found in the membrane fraction, especially in either peripheral or integral form. The urease distribution across the H. pylori membrane was significantly influenced by the ambient pH; the specific activity of external urease was highest at pH 5.5 with a narrow plateau, whereas the internal specific activity was highest within a pH range of 4.5 to 6.5 with a broad plateau. These finding strongly suggest that H. pylori urease is secretory and responded to the external pH. However, at pH 4.0 or below, no urease activity was detected in either the internal or external compartment, although an increase in the color development with 2,4,6-trinitrobenzene sulfonate (TNBS) was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these phenomena may be related to a specific proteolysis in certain proteins, including urease or ${\gamma}$-glutamyl transpeptidase. Interestingly, the effect of ammonium ions n alleviating the enzyme inactivation inside the H. pylori cells was remarkably similar to that of D-glucose. In addition, it would appear that the cation acted as a surrogate of not only $Na^+$ but also $K^+$ thereby increasing the H. pylori P-type ATPase activity. This is of great interest, as it implies that the urease action in H. pylori is indispensible at any locus.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.239-243
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• 2000

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration, produces urease which elicits a powerful immunoglobulin response in H. pylori-infected individuals. To establish a model plant vaccine agains H. pylori, 750 bp -ureA DNA amplified by polymerase chain reaction from pH 808 plasmid harboring urease gene cluster was cloned and manipulated to be expressed in tobacco plants. From the regenerated transgenic tobacco plants, ureA DNA integration,m its mRNA expression and protein synthesis were analyzed and confirmed by standard molecular techniques. The CaMV 35S promoter-driving ureA construct was expressed to produce a 30 kDa protein which was identical with bacterial UreA in size when detected on immunoblot of SDS polyacrylamide gel electrophoresis.

• Journal of Life Science
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• v.18 no.2
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• pp.241-248
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• 2008

This study has been carried out to secretion antibodies for the purpose of preventing the infection of Helicobacter pylori and using them as a supplement for treatment. This experiments have been separated antigens from H. pylori and observed into antibody production and the agglutination of H. pylori for the separated antigens. As major antigenic proteins separated from H. pylori, the following could be verified: 12 kinds of band for whole cell (WC), seven kinds of band for outer membrane protein (OMP), three kinds of band for crude urease, and one kind of band for lipopolysaccharide (LPS). The IgG anti-H. pylori antibody of separated antigens showed $77.9{\pm}6.4{\mu}g/ml$ for we (L), $84.9{\pm}6.4{\mu}g/ml$ for OMP, and $123.8{\pm}2.9{\mu}g/ml$ for crude urease, at the same antigen concentration of $20{\mu}g/100ull$, which showed the most at the crude urease. And it turned out that the IgA antibodies were generated with $2.5{\pm}0.32{\mu}g/ml$ for WC (L), $2.0{\pm}0.43{\mu}g/ml$ for OMP, and $1.3{\pm}0.25{\mu}g/ml$ for crude urease, which demonstrated the most for WC (L) antigens. As a result of verifying the immunogenecity of antigenic protein through the Western blotting, major antigenic substances could be confirmed as follows: 10 kinds for WC, six kinds for OMP and three kinds for crude urease. The agglutination values on the H. pylori of the antibody were $2^5,\;2^5,\;2^6\;and\;2^7$ at the antigen serums of anti-WC (H), anti-WC (L), anti-OMP and anti-crude urease, respectively, which indicated the highest for the antigen serum of anti-crude urease. The urease activation-inhibiting absorbance of antigen serum created by each antigen was $0.14{\pm}0.01$ for WC (H), $0.16{\pm}0.01$ for WC (L), $0.18{\pm}0.03$ for OMP, and $0.18{\pm}0.04$ for urease, demonstrating a significant inhibiting effect, compared with $0.26{\pm}0.02$ of the control group.

• Archives of Pharmacal Research
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• v.20 no.3
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• pp.275-279
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• 1997

Helicobacter pylori (H. pylorl) infection is now established as the major pathogenic factor in chronic gastritis and peptic ulcer disease. in addition, there is accumulating evidence that H. pylori plays an important role in the process of gastric carcinogenesis. On the other hand, oriental traditional medicines have been used for stomach disease for thousands of years. In the present study, methanol extract from the stem bark of Magnolia sieboldii (M. sieboldii) and its components were investigated on their inhibitory effects against urease activity and growth of H. pylori in vitro. The methanol extract of M. sieboldii significantly inhibited the growth of H. pylori ATCC 43504 at 5 mg/ml. From the further fractionation, the chloroform fraction inhibited the bacterial growth dose-dependently. Among four fractions separated from the chloroform fraction by silica gel column chromatography, MS-C-2 was the most potent. Costunolide was isolated from the MS-C-2 subtraction by preparative TLC and recrystallization using n-hexane. Anti-H. pylori effect of costunolide was investigated using one commercial strain (H. pylori ATCC 43504) and three clinical strains (H. pylon 4, 43, 82548). Costunolide exhibited potent anti-H. pylori activity, and the MIC was around $100-200{\mu}g/ml$. However, costunolide had no inhibitory effect of H. pylori urease activity at the concentration used for the growth inhibition assay. From these results, we conclude that costunolide inhibits the, growth of H. pylori by the independent manner of H. pylori urease inhibition.

• Korean Journal of Food Science and Technology
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• v.38 no.1
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• pp.93-98
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• 2006

Effects of anti-Helicobacter pylori IgY powder on H. pylori infection were evaluated 3 and 7 weeks after powder feeding by urease, PCR, and histological tests, and specific IgG assay of murine gastric tissue using mouse model. To produce anti-H. pylori IgY powder, laying hens were immunized with H. pylori prior to egg yolk harvest. C57BL/6 mice showing high response to H. pylori were infected with H. pylori and fed with the anti-H. pylori IgY powder. In urease and PCR tests, urease activity and gene count of anti-H. pylori IgY powder-fed group significantly decreased in comparison with control. Histological results indicated anti-H. pylori IgY powder effectively protected mice from H. pylori.

• The Journal of the Korean Society for Microbiology
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• v.34 no.6
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• pp.533-542
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• 1999

H. pylori produces urease abundantly amounting to 6% of total protein of bacterial mass. Urease genes are composed of a cluster of 9 genes of ureC, ureD, ureA, ureB, ureI, ureE, ureF, ureG, ureH. Production of H. pylori urease in E. coli was studied with genetic cotransformation. Structural genes ureA and ureB produce urease apoprotein in E. coli but the apoprotein has no enzymatic activity. ureC and ureD do not affect urease production nor enzyme activity ureF, ureG, and ureH are essential to produce the catalytically active H. pylori urease of structural genes (ureA and ureB) in E.coli. The kinetics of activation of H. pylori urease apoprotein were examined to understand the production of active H. pylori urease. Activation of H. pylori urease apoprotein, pH dependency, reversibility of $CO_2$ binding, irreversibility of $CO_2$ and $Ni^{2+}$ incorporation, and $CO_2$ dependency of initial rate of urease activity have been observed in vitro. The intrinsic reactivity (ko) for carbamylation of urease apoprotein co expressed with accessory genes was 17-fold greater than that of urease apoprotein expressed without accessory genes. It is concluded that accessory genes function in maximizing the carbamylating deprotonated ${\varepsilon}$-amino group of Lys 219 of urease B subunit and metallocenter of urease apoprotein is supposed to be assembled by reaction of a deprotonated protein side chain with an activating $CO_2$ molecule to generate ligands that facilitate productive nickel binding.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.865-872
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• 2000

Helocobacter phylori is the major cause of gastritis, peptic ulcer, and a principal risk factor for gastric cancer. As the firs step towards a vaccine against H. pylori infection, Hy.pylori urease was expressed and purified as a recombinant apoenzyme (rUrease) in E. coli. In order to develop an effective immunization protocol using rUrease, the host immune responses were evaluated after the oral immunization of mice with rUrease preparations plus cholera toxin relative to various conditions, such as the physical nature of the antigen, the frequency of the booster immunization, the dose of the antigen, and the route of administration. The protective efficacy was assessed using a quantitative culture following an H. pylori SS1 challenge. It was demonstrated that rUrease, due to its particulated nature, was more superior than the UreB subunit as a vaccine antigen. The oral immunization of rUrease elicited significant systemic and secretory antibody responses, and activated predominantly Th2-type cellular responses. The bacterial colonization was significantly reduced (~100-fold) in those mice immunized with three or four weekly oran doses of rUrease plus cholera toxin (p<0.05), when compared to the non-immunized/challenged controls. The protection correlated well with the elicited secretory IgA level against rUrease, and these secretory antibody responses were highly dependent on the frequency of the booster immunization, yet unaffected by the dose of the antigen (25-200$\mu\textrm{g}$). These results demonstrate the remarkable potential of rUrease as a vaccine antigen, thereby strengthening the possibility of developing an H. pylori vaccine for humans.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.264-265
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• 2000

Helicobacter pylori(H. pylori) is the causative agent of chronic gastritis and the single most important factor in peptic ulcer disease, however, the pathogenetic mechanisms underlying H, pylori infection are not well understood. Futhermore, there is a strong association between H. pylori infection and gastric cancer. Various diagnostic methods for detecting H. pylori infection are available. These can be divided into invasive methods, requiring endoscopy, and non-invasive tests, mainly 13C-urea breath tests and serologic detection of antibodies. Rapid urease test is the most recommendable endoscopic test for the diagnosis of H. pylori infection, presently. CLO test kit is the represent of rapid urease test kits. The principles of CLO test kit is that hydrolysis of urea by urease Is detected by a dye indicators showing a color change. Our device is used same principle but we improved the reaction time is more faster and positive color change is more distinctive from the color of the negative specimen. So, this kit is more reliable because it response faster and accuracy.