Scaling-up the production of recombinant EIT antigen by E. coli fermentation for a vaccine-preparation against EHEC for cattle
AimsThis study aims to optimize and scale up the production of the chimeric antigen EIT through E. coli fed-batch fermentations. The approach seeks for simplicity and cost-effectiveness, considering EIT as a potential vaccine candidate against EHEC for cattle. Its ability to induce a humoral immune response has been recently verified in bovines in a proof-of-concept study. Methods and ResultsAn initial screening was conducted to select the optimal medium for EIT expression, using lactose for recombinant protein induction. M9 Minimal medium supplemented with yeast extract yielded the highest relative levels of EIT, as determined by Western blot analysis. E. coli cultures were subsequently grown in a stirred-tank bioreactor, and both biomass production and EIT expression were monitored. The process was reproducible across three independent fermentations, with all parameters being improved compared with shake-flask cultivations. Antigen recovery was achieved through thermal permeabilization, as the construction includes a periplasmic signal sequence. Overall, the process resulted in an average potential output of 350 doses per liter of fermented culture, while preserving the EIT antigenic capability, as confirmed by ELISA assays. ConclusionThe production of the recombinant EIT antigen was successfully scaled up using a stirred-tank bioreactor through a quite simple and cost-effective approach, achieving increased yields for supporting further studies and interventions. Impact StatementCattle are the major reservoir and source of dissemination of enterohemorrhagic E. coli (EHEC), a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. A scalable and cost-effective preharvest vaccine for cattle could help with developing strategies aimed at reducing bacterial carriage and thus, the impact of this zoonosis.