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

Karam, Saba; Raigani, Mozhgan; Hassani Afshar, Sahar; Talebkhan, Yeganeh; Bayat, Elham; Komijani, Samira; Nematollahi, Leila; Barkhordari, Farzaneh; Shafiee Ardestani, Mehdi; Davami, Fatemeh

doi:10.52547/ibj.25.6.390

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

Authors

Saba Karam ¹

Mozhgan Raigani ²

Sahar Hassani Afshar ¹

Yeganeh Talebkhan ²

Elham Bayat ²

Samira Komijani ²

Leila Nematollahi ²

Farzaneh Barkhordari ²

Mehdi Shafiee Ardestani ³

Fatemeh Davami ²

¹ 1International Campus, School Of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Avenue, 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

10.52547/ibj.25.6.390

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 type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: 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). Results: 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. Conclusion: Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Keywords

Fed Batch

Recombinant protein

Pseudomonas aeruginosa

scFv