Background:
Pseudomonas aeruginosa is one of the opportunistic pathogens
causing frequent hospital-acquired life-threatening infections in
mechanically ventilated patients. The most significant virulence factor
of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS.
Methods:
In
the current investigation, a recombinant scFv mAb against the PcrV
protein was expressed in EnBase® (fed-batch) cultivation mode. The
pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was
transformed into Escherichia coli (BL21) cells. The expression
and solubility of anti-PcrV scFv protein were investigated at two
different temperatures (25 °C and 30 °C) and at different induction
times (4, 6, 8, 12, and 24 hours).
Results:
Increased
efficiency was achieved by EnBase® compared to LB broth; owing to the
slow release of glucose, the maximum level of solubility and total
protein expression was observed in EnBase® cultivation system at 30 °C
and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 μg/mL.
Conclusion:
Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).
