Molecular characterization and Functional Analysis of the PilQ(380-706): a Novel Secretin Domain in Pseudomonas aeruginosa

BACKGROUND: Type 4 pili (T4P) is an important virulence factor of Pseudomonas aeruginosa (P. aeruginosa). T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ(380-706)) was produced as a recombinant protein. METHODS: A 981 bp fragment of C-terminal of the pilQ secretin (pilQ(1138-2118)) from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ(1138-2118) gene in the recombinant construct (pET28a/pilQ) was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ(380-706) confirmed by Western blot analysis and twitching inhibition assay. RESULTS: The PCR and enzymatic digestion results showed the presence of the pilQ(1138-2118) gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ(380-706) is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ(380-706) protein. The PilQ(380-706) protein showed high biological activity in all of these standard assays. CONCLUSION: Since, the PilQ(380-706) protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.