Recombinant Production of a Novel Fusion Protein: Listeriolysin O Fragment Fused to S1 Subunit Of Pertussis Toxin

Authors
Hossein Forghani 1
Mahin Jamshidi Makiani 1
Hossein Zarei Jaliani 2
Mina Boustanshenas 1
Seyed Mohsen Zahraei 3
1 Antimicrobial Resistance Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran
2 Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3 Infectious Disease Center for Communicable Disease Control, Ministry of Health and Medical Education, Iran
10.29252/ibj.25.1.33
Abstract
Background: Some resources have suggested that genetically inactivated pertussis toxoid (PTs) bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive pertussis toxin S1 subunit (PTS1) in a fusion form with N-terminal half of the listeriolysin O (LLO) pore-forming toxin. Methods: Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results: The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion: The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future.
Keywords
Adjuvant
Cloning
Fusion protein
Pertussis toxin