title: Plasmid vectors for constructing translational fusions to the B subunit of cholera toxin.
abstract: A family of plasmid cloning vectors has been developed for creating translational fusions to the ctxB gene encoding the B subunit of cholera toxin (CTB) in Escherichia coli. These vectors permit insertion of transcriptionally and translationally competent gene sequences upstream from ctxB. To test the utility of the system, a portion of the glucosyltransferase B (GTF) gene (gtfB) from the cariogenic bacterium Streptococcus mutans GS-5 (Bratthall serotype c), encoding the N-terminal one-third of the protein, was inserted into each vector. E. coli lysates containing the constructs were partially purified by passage over a GM1 ganglioside affinity column. Western blotting analysis of the column retentate from one of the lysates revealed the presence of a novel 58-kDa protein which cross-reacted with antisera to GTF and CTB. These vectors are of general use for making other translational fusions to ctxB. The high binding affinity of CTB can be exploited in purifying large polypeptides fused to this relatively small protein. Moreover, these vectors can be used to create neoantigens with altered immunogenicity for use in polypeptide-based vaccines.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678.
authors.forename: M T
authors.initials: MT
authors.lastname: Dertzbaugh
authors.forename: F L
authors.initials: FL
authors.lastname: Macrina
mesh_terms: D002772:Cholera Toxin / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D013295:Streptococcus mutans / Q000235:genetics; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D005964:Glucosyltransferases