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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.

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechnical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25 degrees C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon was detected at temperatures beyond 40 degrees C for specimens whose equilibrium moisture concentrations were maintained at 10 g H2 O/100 g dry protein. Differential scanning calrimetry measurements showed the presence of a molecular relaxation process which migrated from 42 degrees C at 40% relative humidity to --18 degrees C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily responsible for the observed behavior.