Stability and structural insights of U-Omp19, a Brucella abortus broad spectrum protease inhibitor with immune adjuvant activity.

DARRIBA, MARÍA L.; CERRUTI, MARÍA L.; CARABAJAL, MARIANELA V.; BRUNO, LAURA.; KLINKE, SEBASTIÁN.; CASSATARO, JULIANA; PASQUEVICH, KARINA A.

Tucuman

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS). IX IberoAmerican Congress of Biophysics. XLV Reunion Anual SAB 2016; 2016

Latin American Federation of Biophysical Societies ? Sociedad Argentina de Biofísica

In our laboratory we study U-Omp19, a Brucella abortus protein with immune adjuvant properties. Bioinformatic analysis indicated that U-Omp19 has significant identity (30%) with bacterial protease inhibitors of the I38 family. Enzymatic kinetic assays demonstrated that it is broad spectrum protease inhibitor, which may play a role in its adjuvant activity by increasing the half-life of co-delivered antigens [1, 2].In order to further characterize this new protein adjuvant, we performed stability studies of U-Omp19 samples stored for different periods at different temperatures and/or subjected to stress conditions. SEC, CD, DLS, SDS-PAGE and enzymatic assays demonstrated that after 9 months of storage at -20 and -80 ºC both the physicochemical and protease inhibitory properties of U-Omp19 remained unaltered. Furthermore, the inhibitory activity and protein integrity of U-Omp19 were stable after lyophilizing and repeated freeze-thaw cycles. Interestingly, although a progressive degradation of the N-terminal of the protein is observed after 1 month of storage at 20 or 4 ºC, its CD and UV spectra together with its protease inhibitory activity were similar to those of the -20 and -80 ºC samples. Initial NMR studies (PLABEM service) showed that U-Omp19 bears a disordered N-terminal region (residues 1-64) and a C-terminal compact β-barrel (residues 70-158), which suggests that the U-Omp19 inhibitory activity is located at the C-terminal β-barrel. Ongoing X-ray crystallographic studies with N-terminally truncated forms of U-Omp19 will shed light on the protease inhibitory properties of this oral adjuvant vaccine candidate.Referencias1.Coria, L.M., et al., J Immunol, 2016. 196(10): p. 4014-29.2.Ibanez, A.E., et al., J Control Release, 2015. 220(Pt A): p. 18-28.