Biological activity of Proteus mirabilis urease subunities

BROLL, V.; FRUTTERO, L.L.; FRANZ, L.; MOYETTA, N.R.; DEMARTINI, D.R.; ZAMBELLI, B.; CIURLI, S.L.; CARLINI, C.R.

Porto Alegre

Encuentro; XVI Encontro do Programa de Pós-Graduação em Biologia Celular e Molecular (PPGBCM) da Universidade Federal do Rio Grande do Sul; 2015

Universidade Federal do Rio Grande do Sul

Ureases are widely spread nickel-dependent enzymes. Their main function is to catalyze the hydrolysis of urea producing ammonium and carbon dioxide. However this protein also produces effects independently of its enzymatic activity, such as neurotoxicity to mammals, exocytose induction, and pro-inflammatory activity with production of oxygen reactive species and cytokines. The mapping of structure-activity relationships of these non-enzymatic properties of ureases is still poorly understood. The bacterium Proteus mirabilis is an opportunistic pathogen that causes severe urinary infections, and produces large amounts of urease, which acts as an important virulence factor. Urease from Proteus mirabilis (PMU) is a trimeric protein in which each monomer is composed by three structural subunits, PmUre alpha (66.0 kDa), PmUre beta (12.2 kDa) and PmUre gamma (11 kDa) expressed by the structural genes ureC, ureB and ureA, respectively. In order to evaluate which subunit of PMU could be related to the non-enzymatic biological activities of PMU, different plasmids were produced to obtain each subunit separately. All constructs where produced and each protein expressed with a Strep-tag at the N-terminal portion. PmUre gamma is a soluble protein purified with high purity in two chromatographic steps, namely an affinity chromatography with the StrepTactin Sepharose HP column and a size exclusion chromatography. PmUre beta and PmUre alpha were instead purified to high purity from inclusion bodies, following similar chromatographic protocols. After the refolding procedure, the same steps to purify PmUre gamma were performed. Having established the purification protocols for all the structural PMU subunits, experiments were carried out to verify the spectrum of biological activities displayed by the isolated subunits. The Pmure beta appear to be the most active among the three structural subunits.