P. Slos et al., RECOMBINANT CHOLERA-TOXIN B-SUBUNIT IN ESCHERICHIA-COLI - HIGH-LEVEL SECRETION, PURIFICATION, AND CHARACTERIZATION, Protein expression and purification, 5(5), 1994, pp. 518-526
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.