Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode |
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| Authors: | Saba Karam Mozhgan Raigani Sahar Hassani Afshar Yeganeh Talebkhan Elham Bayat Samira Komijani Leila Nematollahi Farzaneh Barkhordari Mehdi Shafiee Ardestani Fatemeh Davami |
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| Institution: | 1.International Campus, School Of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2.Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Avenue, Tehran, Iran; 3.Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran |
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| Abstract: | 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+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv |
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