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

      Hu, Li-Tai; Mobley, Harry L. T. (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 trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 (pH808/pHP902) expressed a urease activity up to 87 {dollar}\mu{dollar}mole urea/min/mg protein (U/mg protein), a level approaching that of wild type H. pylori UMAB41 (100 U/mg protein) from which the genes were cloned.