E. coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides into target cells. To thoroughly exploit the biotechnological potential of EtxB-based fusion proteins, a simple method has to be worked out for their expression and purification. Production of these chimeric toxins faces problems with regard to both their low yield and stability. In this study we describe a protocol for the optimal production and secretion of undegraded EtxB hybrids into the extracellular medium of cultures of non-toxinogenic Vibrio strains. The highest level of expression of two chimeric toxins, EtxB-R2 and EtxB-pol, was obtained by using Vibrio sp.60 cultures, reaching 52 mg/l and 8 mg/l, respectively. The presence of 0.3 mM EDTA in the culture medium totally preserved both chimeric toxins from proteolysis, also during a prolonged expression. This system may be useful for the preparation of other EtxB-based fusion proteins.
EDTA protects E. coli heat-labile enterotoxin B subunit-based fusion proteins from proteolytic degradation during their production by Vibrio spp.
LOREGIAN, ARIANNA;PALU', GIORGIO
1997
Abstract
E. coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides into target cells. To thoroughly exploit the biotechnological potential of EtxB-based fusion proteins, a simple method has to be worked out for their expression and purification. Production of these chimeric toxins faces problems with regard to both their low yield and stability. In this study we describe a protocol for the optimal production and secretion of undegraded EtxB hybrids into the extracellular medium of cultures of non-toxinogenic Vibrio strains. The highest level of expression of two chimeric toxins, EtxB-R2 and EtxB-pol, was obtained by using Vibrio sp.60 cultures, reaching 52 mg/l and 8 mg/l, respectively. The presence of 0.3 mM EDTA in the culture medium totally preserved both chimeric toxins from proteolysis, also during a prolonged expression. This system may be useful for the preparation of other EtxB-based fusion proteins.Pubblicazioni consigliate
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