Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein.
A synthetic peptide, encoding amino acid residues 345 to 359 of the glucosyltransferase B enzyme of Streptococcus mutans GS-5, was genetically fused to the N-terminal end of the B-subunit gene of cholera toxin. The protein was overexpressed in Escherichia coli and retained the antigenicity associated with cholera toxin B subunit (CTB) as well as that associated with glucosyltransferase B. The addition of 15 amino acids to the N-terminal end of CTB did not appear to affect the gross structure of the protein significantly. The chimeric protein monomers assembled into a functional oligomer which exhibited only minor conformational differences from native CTB as measured by circular dichroism. The chimera bound to GM1 ganglioside and thus retained the biological activity of CTB. These results demonstrate that genetic fusion of small peptides to the N terminus of CTB has only a minimal effect on the structure and function of the protein. Furthermore, the chimera was shown to be immunogenic when fed to mice. This work has important implications in the construction of CTB chimeras for use as oral vaccines.
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298.
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Dertzbaugh
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Peterson
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Macrina
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D000942:Antigens, Bacterial / Q000235:genetics; D001428:Bacterial Vaccines / Q000235:genetics*; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology; D002942:Circular Dichroism; D004220:Disulfides; D000939:Epitopes; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012995:Solubility; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics; D014614:Vaccines, Synthetic
D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D004220:Disulfides; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D002772:Cholera Toxin; D005964:Glucosyltransferases