Polyclonal Antibody against the Active Recombinant Helicobacter Pylori Urease Expressed in Escherichia coli

다국어 초록 (Multilingual Abstract)

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. In order to express the recombinant urease at 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) column chromatography and the purified enzyme possessed the specific activity of 1,255 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 a H pylori ATCC strain and the recombinant E coli strains expressing various bacterial ureases were examined for the cross-reactivity.
Helicobacter pylori is a microaerophilic, spiral-shaped, Gram-negative bacterium which colonized gastric mucosa of humans, non-human primates and pigs. h pylori produces bacterial urease of high activity up to 6% of the soluble cell protein(Hu and Mobley, 1990) and this surface-presented, two-subunit enzyme(Dunn et al., 1990) is distinct from other bacterial ureases which are maed of three subunits and localized in the cytoplasm h pylori urease server as a major surface immunogen(Newell, 1987, Perez-Perez and Blaser, 1987) and as an important survival factor for the bacterium in the acidic environment of the gastric lumen(Eaton et al., 1991). As in the cases of other bacterial enzymes, H pylori urease requires nickel ions as an essential cofactor for the enzyme activity. Because of the fastidious culture conditions of H pylori and its slow growth rate, growing large number of cells for various biochemical characterization and analyses is difficult. There have been many attempts to express recombinant H pylori urease in the recombinant E coli strains which harbors a recombinant plasmid containing the entire urease gene cluster. However, recombinant E coli cells grown in rich media such as LB broth used to produce functionally inactive apourease instead of the active holoenzyme(Cussac et al., 1992, Lee et al., 1995). This seemed to be duc to chelation of the minute amount of nickel ions by certain components present in the medium since it was shown that catalytically active recombinant urease could be expressed only when the host cells were grown in minimal medium that contains no Ni-chelating agents(Hu and Mobley, 1993). Recently, Mobley et al cloned and sequenced the nixA gene which encodes a specific nickel-transport membrane protein and found that when the nixA was co-expressed in E coli with H pylori urease gene cluster, it potentiated expression of catalytically active urease even when cultured in complex medium (Mobley et al., 1995). In this report, we subcloned a minimal urease gene cluster into a high copy-number plasmid vector and used it to express the recombinant H pylori urease at high level by supplementing nickel ions in milimolar quantity in the complex medium without co-expressing the nickel transporter gene, nixA, of Helicobacter pylori. Nearly homogeneous urease was purified and was used to raise polyclonal antibody specific H pylori urease.

목차 (Table of Contents)

• Abstract
• Materials and Methods
• Results and Discussion
• References