Ureases (urea aminohydrolase, EC 3.5.1.5) are nickel dependent enzymes present in plants, bacteria and fungi that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. Canatoxin, an isoform Canavalia ensiformis urease, has several biological properties unrelated to its ureolytic activity: insecticidal effect, binding to glycoconjugates, platelet aggregating and pro-inflammatory activities. Helicobacter pylori has been established as the etiologic agent of chronic gastritis and gastric and duodenal ulcers, being an important risk factor for gastric adenocarcinoma. Urease, produced in abundance by H. pylori, plays a central role in the pathogenesis of this bacterium. Considering that ureases of Bacillus pasteurii and H. pylori share some biological activities with canatoxin, like the platelet aggregating effect, this work aims the purification of recombinant urease of H. pylori and to investigate other biological properties this protein may share with canatoxin, emphasizing the pro-inflammatory effect. Recombinant H. pylori urease was produced in E. coli SE5000 transformed with plasmid pHP8080 as previously described. The purification of urease was made in four steps: 1) Ammonium sulfate precipitation; 2) Q-Sepharose ion exchange chromatography; 3) Phenyl-Sepharose hydrophobic interaction chromatography, and 4) Superose 6 HR gel filtration chromatography, the last two steps using a FPLC apparatus. The pro-inflammatory activity of the recombinant protein was tested using the mice paw edema model. The mice received intraplantar injections of urease at different concentrations, after being pretreated with different anti-inflammatory drugs or not. This study can help to establish structure versus activity relationships for urease enzymes of different sources and to elucidate some aspects involved in the physiopathology of diseases caused by urease-producing bacteria.
CAPES, CNPq, FAPERGS & FINEP