A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3.

Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.