title: Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli.
abstract: A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment. A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser. Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A. Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148. The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells. Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells. This combination of approaches provides a means of applying recombinant DNA methods in E. coli to study structure-function relationships in whole diphtheria toxin.
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000235:genetics* / Q000276:immunology / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics; D007158:Immunologic Techniques; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins
chemical_list: D004167:Diphtheria Toxin; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases