High Cell Density Cultivation Process for the Expression of Botulinum Neurotoxin a Receptor Binding Domain

Abstract

The receptor-binding domain of botulinum neurotoxin (H_C fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale H_C 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_C 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_C fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified H_C 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_C fragment in E. coli for use in anti-botulism vaccines.

Keywords: Clostridium botulinum; fermentation; high cell density cultivation; recombinant protein expression; subunit vaccine.

Figure 1

Kinetics of bacterial growth and H_C fragment expression in shake flasks. The culture was grown in TB media at 37 °C. Samples were taken during culture growth and used to determine the optical density, the volumetric yield of H_C fragment expression, and the specific yield.

Figure 2

Kinetics of bacterial growth and H_C fragment expression using batch fermentation under growth conditions identical to those applied during shake flask experiments. The culture was grown in a 4-L fermenter in TB media at 37 °C.

Figure 3

Fed-batch fermentation for H_C fragment expression. The growth temperature was set to 37 °C from 0–12 h EFT and 18 °C from 12–30 h EFT. The addition of nutrients is indicated at the top of the graph as follows: M—magnesium sulfate, P—sodium phosphate, G—glycerol, T—trace elements, N—tryptone, and yeast extract concentrate.

Figure 4

High cell density cultivation for H_C fragment expression. The bacterium was grown in a defined medium. At 12.5 h EFT, the temperature was reduced to 28 °C, and at 13.5 h EFT, it was further reduced to 18° C. Nutrient boluses are indicated at the top of the graph as follows: M—magnesium sulfate, P—sodium phosphate, G—glycerol, T—trace elements, N—tryptone and yeast extract concentrate. From 28 h EFT until 47 h EFT, continuous feeding at a constant rate was supplied to limit the oxygen demand of the bacterium and prevent acetate accumulation caused by oxygen limitation.

Figure 5

Purification of the H_C fragment expressed during the HCDC process. Cells were disrupted by sonication, and the protein was purified from the supernatant by IMAC (A) (red and blue lines represent absorbance at 260 and 280 nm, respectively; the green line represents the elution buffer percentage). Samples of the purification process were analyzed by SDS-PAGE (B): 1. Disrupted culture supernatant; 2. Flow-through from loading supernatant to IMAC column (unbound proteins); 3. Released impurities at 40 mM imidazole; 4. Purified H_C fragment expressed during the HCDC process; 5. Purified H_C fragment expressed using shake flasks; 6. Molecular weight marker.

Figure 6

Far-UV CD spectra of H_C fragments were expressed using HCDC (black) or shake flasks (red).