Dissertation/ Thesis
Προβολή σε EDS

Helicobacter pylori urease: Purification, molecular cloning, and enzymatic activation in Escherichia coli

Λεπτομέρειες βιβλιογραφικής εγγραφής
Τίτλος: Helicobacter pylori urease: Purification, molecular cloning, and enzymatic activation in Escherichia coli
Συγγραφείς: Hu, Li-Tai
Συνεισφορές: Mobley, Harry L. T.
Έτος έκδοσης: 1992
Συλλογή: UMB Digital Archive (University of Maryland, Baltimore)
Θεματικοί όροι: Biology, Molecular, Microbiology, Chemistry, Biochemistry, Cloning, Escherichia coli, Helicobacter pylori--enzymology, Urease--isolation & purification
Περιγραφή: University of Maryland, Baltimore. Microbiology. Ph.D. 1992 ; Helicobacter pylori (formerly Campylobacter pylori), a gram-negative microaerophilic spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, a nickel metalloenzyme of H. pylori represents a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea protects the acid-sensitive bacterium as it colonizes human gastric mucosa. H. pylori urease was purified by chromatography on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kDa, and was composed of two distinct subunits with apparent molecular size of 66 kDa and 29.5 kDa. A stoichiometry of (29.5 kDa-66 kDa){dollar}\sb6{dollar} is predicted for the structure of the native enzyme. The {dollar}K\sb{lcub}\rm M{rcub}{dollar} for urea was estimated at 0.2 mM. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.7 kb fragment carrying ureC,D,A,B (pHP402). An enzymatically inactive recombinant urease was purified from the soluble protein of French press lysates of Escherichia coli DH5{dollar}\alpha{dollar} (pHP402) according to the same protocol used for wild-type H. pylori urease. Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column. Although urease itself is encoded by two subunit genes, ureA and ureB, accessory genes are required for enzymatic activity. Clones were isolated that expressed enzymatically active recombinant H. pylori urease in E. coli. Conditions were developed under which near wild-type urease activity was achieved. E. coli SE5000 containing recombinant H. pylori urease genes (pHP808) was grown in minimal medium containing no amino acids; NiCl{dollar}\sb2{dollar} was added to 0.75 {dollar}\mu{dollar}M. Structural genes ureA and ureB (pHP902) were overexpressed in ...
Τύπος εγγράφου: doctoral or postdoctoral thesis
Γλώσσα: English
Relation: http://hdl.handle.net/10713/2541; Yes
Διαθεσιμότητα: http://hdl.handle.net/10713/2541
Αριθμός Καταχώρησης: edsbas.F8CB9963
Βάση Δεδομένων: BASE
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PubTypeId: dissertation
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Items – Name: Title
  Label: Title
  Group: Ti
  Data: Helicobacter pylori urease: Purification, molecular cloning, and enzymatic activation in Escherichia coli
– Name: Author
  Label: Authors
  Group: Au
  Data: <searchLink fieldCode="AR" term="%22Hu%2C+Li-Tai%22">Hu, Li-Tai</searchLink>
– Name: Author
  Label: Contributors
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  Data: Mobley, Harry L. T.
– Name: DatePubCY
  Label: Publication Year
  Group: Date
  Data: 1992
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  Group: HoldingsInfo
  Data: UMB Digital Archive (University of Maryland, Baltimore)
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  Data: <searchLink fieldCode="DE" term="%22Biology%22">Biology</searchLink><br /><searchLink fieldCode="DE" term="%22Molecular%22">Molecular</searchLink><br /><searchLink fieldCode="DE" term="%22Microbiology%22">Microbiology</searchLink><br /><searchLink fieldCode="DE" term="%22Chemistry%22">Chemistry</searchLink><br /><searchLink fieldCode="DE" term="%22Biochemistry%22">Biochemistry</searchLink><br /><searchLink fieldCode="DE" term="%22Cloning%22">Cloning</searchLink><br /><searchLink fieldCode="DE" term="%22Escherichia+coli%22">Escherichia coli</searchLink><br /><searchLink fieldCode="DE" term="%22Helicobacter+pylori--enzymology%22">Helicobacter pylori--enzymology</searchLink><br /><searchLink fieldCode="DE" term="%22Urease--isolation+%26+purification%22">Urease--isolation & purification</searchLink>
– Name: Abstract
  Label: Description
  Group: Ab
  Data: University of Maryland, Baltimore. Microbiology. Ph.D. 1992 ; Helicobacter pylori (formerly Campylobacter pylori), a gram-negative microaerophilic spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, a nickel metalloenzyme of H. pylori represents a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea protects the acid-sensitive bacterium as it colonizes human gastric mucosa. H. pylori urease was purified by chromatography on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kDa, and was composed of two distinct subunits with apparent molecular size of 66 kDa and 29.5 kDa. A stoichiometry of (29.5 kDa-66 kDa){dollar}\sb6{dollar} is predicted for the structure of the native enzyme. The {dollar}K\sb{lcub}\rm M{rcub}{dollar} for urea was estimated at 0.2 mM. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.7 kb fragment carrying ureC,D,A,B (pHP402). An enzymatically inactive recombinant urease was purified from the soluble protein of French press lysates of Escherichia coli DH5{dollar}\alpha{dollar} (pHP402) according to the same protocol used for wild-type H. pylori urease. Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column. Although urease itself is encoded by two subunit genes, ureA and ureB, accessory genes are required for enzymatic activity. Clones were isolated that expressed enzymatically active recombinant H. pylori urease in E. coli. Conditions were developed under which near wild-type urease activity was achieved. E. coli SE5000 containing recombinant H. pylori urease genes (pHP808) was grown in minimal medium containing no amino acids; NiCl{dollar}\sb2{dollar} was added to 0.75 {dollar}\mu{dollar}M. Structural genes ureA and ureB (pHP902) were overexpressed in ...
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  Data: doctoral or postdoctoral thesis
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RecordInfo BibRecord:
  BibEntity:
    Languages:
      – Text: English
    Subjects:
      – SubjectFull: Biology
        Type: general
      – SubjectFull: Molecular
        Type: general
      – SubjectFull: Microbiology
        Type: general
      – SubjectFull: Chemistry
        Type: general
      – SubjectFull: Biochemistry
        Type: general
      – SubjectFull: Cloning
        Type: general
      – SubjectFull: Escherichia coli
        Type: general
      – SubjectFull: Helicobacter pylori--enzymology
        Type: general
      – SubjectFull: Urease--isolation & purification
        Type: general
    Titles:
      – TitleFull: Helicobacter pylori urease: Purification, molecular cloning, and enzymatic activation in Escherichia coli
        Type: main
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            NameFull: Hu, Li-Tai
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            NameFull: Mobley, Harry L. T.
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          Dates:
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              M: 01
              Type: published
              Y: 1992
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