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.
A simple method to construct the plasmids producing the B subunit of porcine or human heatlabile 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 Xbal 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 Xbal, 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.
Department of Microbiology, Fujita Health University School of Medicine, Aichi, Japan.
T
T
Tsuji
M
M
Kato
Y
Y
Kato
H
H
Kawase
S
S
Imamura
A
A
Miyama
D000818:Animals; D001483:Base Sequence; D002772:Cholera Toxin / Q000096:biosynthesis*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005784:Gene Amplification; D008040:Genetic Linkage; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014170:Transformation, Genetic; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004768:Enterotoxins; D002772:Cholera Toxin