High cell density fermentation of recombinant Vibrio cholerae for the production of B subunit of Escherichia coli enterotoxin

Abstract

High cell density fermentation studies were performed to produce the B subunit of Escherichia coli heat-labile enterotoxin (LTB) from a Vibrio cholerae culture that carries a recombinant plasmid with an ampicillin resistance gene, tac promoter, and the gene encoding LTB. Upon induction with isopropyl-β-D-thiogalactopyranoside (IPTG) the culture secreted the protein into the extracellular milieu. Fed-batch fermentation with stepwise addition of a total of 5 mM of IPTG during the active growth phase of the organism resulted in the production of 400 mg/L of LTB in 9 h and a cell optical density (OD) of 24. The LTB was purified to homogeneity with 70% recovery from the fermentation broth and was found to be chemically and biologically identical to the native protein by N-terminal amino acid sequencing and receptor binding assay.