Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

• Published in: Iranian biomedical journal Vol. 25; no. 6; pp. 390 - 398
• Main Authors: Karam, Saba, Raigani, Mozhgan, Hassani Afshar, Sahar, Talebkhan, Yeganeh, Bayat, Elham, Komijani, Samira, Nematollahi, Leila, Barkhordari, Farzaneh, Shafiee Ardestani, Mehdi, Davami, Fatemeh
• Format: Journal Article
• Language: English
• Published: Iran Pasteur Institute of Iran 01.10.2021
• Subjects: Antibodies; Antibodies, Bacterial - analysis; Antigens, Bacterial - immunology; Bacterial Toxins - immunology; Batch culture; Cultivation; E coli; Fed batch; Full Length; Lysis; Monoclonal antibodies; Opportunist infection; Pore Forming Cytotoxic Proteins - immunology; Proteins; Pseudomonas aeruginosa; Single-Chain Antibodies - analysis; Solubility; Temperature; Time Factors; Ventilation; Virulence; Virulence factors; Fed Batch; Pseudomonas aeruginosa; scFv; recombinant protein
• ISSN: 1028-852X; 2008-823X
• DOI: 10.52547/IBJ.25.6.390

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 type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. In the current investigation, a recombinant single-chain fragment variable (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). : Increased efficiency was achieved by EnBase® compared to Luria–Bertani 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. : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).