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

DARRIBA, MARIA LAURA; CERRUTI, MARIA LAURA; CARAVAJAL MARIANELA; LAURA BRUNO; KLINKE S; CASSATARO JULIANA; KARINA A. PASQUEVICH

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

In our laboratory we studyU-Omp19, a Brucella abortus protein with immune adjuvantproperties. Bioinformatic analysis indicated that U-Omp19 has significant identity(30%) with bacterial protease inhibitors of the I38 family. Enzymatic kineticassays demonstrated that it is broad spectrum protease inhibitor, which may play a role in its adjuvantactivity by increasing the half-life of co-delivered antigens [1, 2].In order to furthercharacterize this new protein adjuvant, we performed stability studies ofU-Omp19 samples stored for different periods at different temperatures and/orsubjected to stress conditions. SEC, CD, DLS, SDS-PAGE and enzymatic assaysdemonstrated that after 9 months of storage at -20 and -80 ºC both thephysicochemical and protease inhibitory properties of U-Omp19 remainedunaltered. Furthermore, the inhibitory activity and protein integrity ofU-Omp19 were stable after lyophilizing and repeatedfreeze-thaw cycles. Interestingly, although aprogressive degradation of the N-terminal of the protein is observed after 1month of storage at 20 or 4 ºC, its CD and UV spectra together with itsprotease inhibitory activity were similar to those of the -20 and -80 ºCsamples. Initial NMR studies (PLABEM service) showedthat U-Omp19 bears a disordered N-terminal region (residues 1-64) and aC-terminal compact β-barrel (residues 70-158), which suggests that theU-Omp19 inhibitory activity is located at the C-terminal β-barrel. Ongoing X-ray crystallographicstudies with N-terminally truncated forms of U-Omp19 will shed light on theprotease inhibitory properties of this oral adjuvant vaccine candidate.