Construction of plasmids useful for production of the B subunit of cholera toxin from Vibrio cholerae or a heat-labile enterotoxin from enterotoxigenic Escherichia coli

Abstract

A simple method to construct the plasmids producing the B subunit of porcine or human heat-labile enterotoxin or cholera toxin was developed, and the B subunits produced by the resulting plasmids were purified. The gene of LTp from pEWD 299 was ligated to pHSG 396 or pBluescript SK+-1 and the vector carrying one Xba1 and EcoR1 site in the LTp-A gene was constructed. The Xbal-EcoR1 fragment of LTp-A gene was exchanged for the multicloning site of pHSG 396 containing Xba1, BamH1, Cla 1, Kpn1, Sac1 and EcoR1 sites. This plasmid (pTSU28) produced the LTp-B subunit. Moreover, the fragment of the LTp-B gene of pTSU 28 was exchanged by the EcoR1-HindIII fragment of LTh-B from E. coli H10407 strain (pTSU 35) or by the Cla 1-Hind III fragment of CT-B gene amplified by the PCR procedure with the chromosomal DNA of V. cholerae 86KT25 (pTSU 32). The DNA sequence of the CT-B subunit amplified by PCR procedure was compared and found identical to that cited in the literature [11]. After these plasmids were transformed into E. coli MV 1184 strain, the toxins produced by them were purified using a Bio-Gel A 5m affinity column for both LT-Bs and an immunobilized D-galactose affinity column for CT-B. Though both columns absorbed only the B subunit, the eluates contained a single protein corresponding to the B subunit, suggesting that each mutant produces only the B subunit. © 1994 Kluwer Academic Publishers.