RECOMBINANT CHOLERA-TOXIN B-SUBUNIT IN ESCHERICHIA-COLI - HIGH-LEVEL SECRETION, PURIFICATION, AND CHARACTERIZATION

The gene coding for cholera toxin subunit B (CT-B) was fused to a modi fied ompA signal sequence and subsequently cloned into a high expressi on vector based on the regulatory signals of the arabinose operon of S almonella typhimurium. Upon induction of gene expression in Escherichi a coli, a product of the expected size for CT-B monomer was detected a t a level of approximately 60% of total periplasmic protein. At pilot scale, batch cultivation in a 20-liter bioreactor allowed a production level of 1 g/liter of recombinant CT-B (rCT-B), the majority of which was released into the culture medium. The latter phenomenon was depen dent on the medium selected for cultivation. A simple and inexpensive purification scheme was developed which enabled the recovery of 81% of rCT-B from the culture supernatant. Comparing amino acid composition, amino-terminal sequence, mass spectrum, pentamerisation, and G(M1)-bi nding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an inter esting alternative to overexpressing systems developed in V. cholerae. (C) 1994 Academic Press, Inc.