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Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences.
K S Fisher1, I H Maxwell, J R Murphy
1Department of Medicine, University of Colorado Cancer Center, Denver 80262.
Infection and Immunity
|October 1, 1991
Summary
Researchers engineered new diphtheria toxin A-chain fragment (DT-A) mutants to control cell killing. These DT-A variants offer a tunable range of toxicity for gene therapy applications.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cellular toxicity can be induced by diphtheria toxin A-chain fragment (DT-A) expression plasmids.
- Modifying DT-A's enzymatic activity can alter its toxicity.
Purpose of the Study:
- To construct and evaluate DT-A mutant expression plasmids with reduced ADP-ribosylation activity.
- To assess the dose-dependent toxicity of these mutants in mammalian cells.
Main Methods:
- Generated three DT-A mutant expression plasmids by substituting glutamic acid 148 with aspartic acid, serine, or glutamine.
- Co-transfected HeLa and 293 cells with mutant DT-A plasmids and a luciferase reporter plasmid.
- Determined dose-response curves to quantify the toxicity of wild-type and mutant DT-A constructs.
Main Results:
- Mutant DT-A plasmids exhibited 100- to 300-fold reduced in vitro ADP-ribosylation activity.
- Toxicity varied significantly among mutants, with the Glu148Gln mutant being the least toxic.
- Dose required for 50% luciferase inhibition ranged from 0.01 µg (wild-type) to 1.2 µg (Glu148Gln mutant) in 293 cells.
Conclusions:
- Plasmid-based expression of DT-A mutations allows for fine-tuning of cellular toxicity.
- Engineered DT-A variants provide a spectrum of cytotoxic potential for research and therapeutic applications.