The expression of biologically active cholera toxin in Escherichia coli

Chromosomal DNA from Vibrio cholerae E1 Tor strain 1621 was digested with Hind III and the fragments obtained fractionated by centrifugation through a sucrose gradient. A 15Kb fragment which contained the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterized by restriction endonuclease digestion. Sequences homologous to the LT gene were identified by hybridization and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.