title: Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences.
abstract: We previously demonstrated that cells can be killed through transfection of an expression plasmid that encodes the diphtheria toxin A-chain fragment (DT-A). This report describes the construction of expression plasmids containing three mutant DT-A-coding sequences substituting glutamic acid 148 with aspartic acid, serine, or glutamine which are known to have 100- to 300-fold-reduced ADP-ribosylation activity measured in vitro. The toxicity of these constructs was determined in cotransfection experiments using HeLa and 293 cells with a luciferase expression plasmid as the reporter. Dose responses were compared for the three new DT-A mutant plasmids and for the corresponding plasmids containing wild-type DT-A and the previously characterized tox 176 mutant. The dose required to produce 50% inhibition of control luciferase expression in 293 embryonic kidney cells for the five plasmids ranged from 0.01 micrograms for wild-type DT-A to 1.2 micrograms for the least toxic plasmid, which replaces glutamic acid 148 with glutamine. In conclusion, a wide range of DT-A toxicity can be achieved by using plasmid expression vectors that encode different DT-A mutations.
authors.affiliation: Department of Medicine, University of Colorado Cancer Center, Denver 80262.
authors.forename: K S
authors.initials: KS
authors.lastname: Fisher
authors.forename: I H
authors.initials: IH
authors.lastname: Maxwell
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
authors.forename: J
authors.initials: J
authors.lastname: Collier
authors.forename: L M
authors.initials: LM
authors.lastname: Glode
mesh_terms: D004167:Diphtheria Toxin / Q000235:genetics* / Q000633:toxicity; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000633:toxicity; D010957:Plasmids; D014162:Transfection
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A