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

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.

Fig. 1

Design of the mutant PTS1 cloning method. (A) For introducing the PTS1 gene into pPSG-IBA35 expression vector, two fragments of the multiple cloning site (blue extension) of the vector were added to the inserted gene by a simple PCR. (B) The product of the first PCR was used as a megaprimer in a second quick-change PCR to replace a fragment of the vector by the PTS1 gene

Fig. 2

Design of the fusion LLO-PTS1 cloning method. (A) Two gene fragments were amplified in separate PCRs and ligated together in a SOEing PCR protocol. (B) For introducing the LLO-PTS1 gene into pPSG-IBA35 expression vector, the product of the SOEing PCR was used as a megaprimer in a second quick-change PCR to replace a fragment of the vector by the LLO-PTS1 gene

Fig. 3

Modeling of the new disulfide bond in the mutant PTS1 gene. Two amino acids, Phe53 and Asn197, were chosen to convert to Cys to form a new disulfide bond. Native disulfide bond of the PTS1 molecule is also shown. The numbering of the amino acids was performed concerning the Met of the signal sequence as the first amino acid

Fig. 4

Quick-change PCR and purity of the mutant PTS1 protein. Quick-change PCR of the mutant PTS1 gene was performed using the whole gene as megaprimer to introduce the gene into the expression vector pPSG-IBA35. Lane A, PTS1 gene; lane B, 1 kb DNA size marker; lane C, quick-change PCR product; lane D, mutant PTS1 protein purified from BL21 DE3 strain; lane E, pre-stained protein size marker; lane F, mutant PTS1 protein purified from SHuffle T7 strain

Fig. 5

The SOEing PCR and purity of the LLO-PTS1 protein. SOEing PCR of the mutant PTS1 gene and LLO gene fragment was performed to produce the fusion gene for using as a megaprimer to produce the expression construct of LLO-PTS1 in pPSG-IBA35 vector. Lanes A and B, PCR product of the amplification of the PTS1 and LLO genes; lane C, 1 kb DNA size marker; lane D, SOEing PCR product; lane E, pre-stained protein size marker; lane F, mutant PTS1 protein purified from SHuffle T7 strain

Fig. 6

MCF-7 cell viability in the presence of LLO-PTS1. MCF-7 breast cancer cells were cultured and exposed to increasing concentrations of purified PTS1 and LLO-PTS1 proteins. Independently increasing concentrations of 5-fluorouracil was also tested as the positive control