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. 2021 Nov 1;25(6):390-8.
doi: 10.52547/ibj.25.6.390.

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

Saba Karam  1   2 , Mozhgan Raigani  2 , Sahar Hassani Afshar  1   2 , Yeganeh Talebkhan  2 , Elham Bayat  2 , Samira Komijani  2 , Leila Nematollahi  2 , Farzaneh Barkhordari  2 , Mehdi Shafiee Ardestani  3 , Fatemeh Davami  2
Affiliations

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

Saba Karam et al. Iran Biomed J. .

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; Pseudomonas aeruginosa; recombinant protein; scFv.

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Figures

Fig.1
Fig.1
PCR analysis of extracted recombinant plasmids from kanamycin-resistant E. coli cells. PCR products were electrophoresed on a 1% agarose gel. Lanes 1–4, amplification of anti-PcrV scFv gene; lane 5, negative control; lane 6, 1-kb DNA markers
Fig. 2
Fig. 2
SDS-PAGE analysis of anti-PcrV scFv expression in LB medium. Lane 1, cell lysates before IPTG induction; cell lysates 4 h (lane 2), 6 h (lane 3), 8 h (lane 4), and 24 h (lane 5) after induction; lane 6, Fermentas protein marker SM0671
Fig. 3
Fig. 3
Western blotting analysis of the recombinant 6-His SUMO tag anti-PcrV scFv protein. Lane 1, cell lysates before IPTG induction; lanes 2 and 3, the 56 kDa 6-His SUMO tagged anti-PcrV scFv protein; lane 4, negative control; lane 5, a 40-kDa 6-His tagged protein as the control; lane 6, protein marker
Fig. 4
Fig. 4
SDS-PAGE analysis of expressed recombinant SUMO anti-PcrV scFv in EnBase® medium at different times. Lane 1, fermentas protein marker SM0671; lane 2, cell lysates before IPTG induction; cell lysates 4 h (lane 3), 6 h (lane 4), 8 h (lane 5), and 24 h (lane 6) after induction
Fig. 5
Fig. 5
SDS-PAGE analysis of soluble and insoluble expression of recombinant SUMO anti-PcrV scFv expression in EnBase® medium at different temperatures and times. The distribution of total cell lysates, pellet, and soluble fractions during different time intervals post IPTG induction. Coomassie stained gel under reduced condition. The distribution of total cell lysates, pellet, and soluble fractions in EnBase® medium during different time intervals post IPTG induction at (A) 25 °C and (B) at 30 °C. T, total lysate; S, supernatant or soluble fraction; P, pellet; M, Fermentas protein marker
Fig. 6
Fig. 6
The SDS-PAGE analysis of the Ni-NTA affinity chromatography of the recombinant SUMO anti-PcrV scFv protein. Cell lysates before (lane 1) and after (lane 2) IPTG induction; lane 3, flow-through; lane 4, Fermentas protein marker; lane 5, wash sample; lanes 6 and 7, the fractions of elution buffer
Fig. 7
Fig. 7
The percentage of in vitro inhibition of T3SS by anti-PcrV scFv. RBCs were incubated with log-phase P. aeruginosa in the presence of a certain amount of purified anti-PcrV scFv. Spectrophotometry was performed to understand the hemoglobin release profile in the supernatants
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