| Summary: | The receptor-binding domain of botulinum neurotoxin (H<sub>C</sub> fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale H<sub>C</sub> fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in H<sub>C</sub> fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the H<sub>C</sub> fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified H<sub>C</sub> fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the H<sub>C</sub> fragment in <i>E. coli</i> for use in anti-botulism vaccines.
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