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  • Title: Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization.
    Author: Slos P, Speck D, Accart N, Kolbe HV, Schubnel D, Bouchon B, Bischoff R, Kieny MP.
    Journal: Protein Expr Purif; 1994 Oct; 5(5):518-26. PubMed ID: 7827509.
    Abstract:
    The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of the expected size for CT-B monomer was detected at 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 dependent 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 GM1-binding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an interesting alternative to overexpressing systems developed in V. cholerae.
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