Genetic Construction, Expression, and Characterization of Diphtheria Toxin-Based Growth Factor Fusion Proteins

Abstract

The fusion protein toxins that have been described are generally composed of the catalytic and transmembrane domains of a bacterial toxin (e.g., diphtheria toxin [DT] or Pseudomonas exotoxin A) to which a polypeptide hormone, growth factor, or single-chain antibody (scFv) is genetically fused (1–3). In these constructs, the native receptor binding domain of the toxin is genetically replaced with the targeting ligand. Our laboratory has focused almost exclusively on the construction, expression, and characterization of DT-based fusion toxins (1–12). We have used these novel cytotoxic reagents as probes to study structure-function relationships of the DT catalytic and transmembrane domains, as well as to study the receptor binding components of the fusion proteins (13–19). We are currently using these novel reagents to study the underlying mechanisms involved with delivery of the fusion protein toxin’s catalytic domain across the membrane and into the cytosol of target eucaryotic cells. The catalytic domain of diphtheria toxin catalyzes the NAD⁺-dependent ADP-ribosylation of the diphthamide residue in elongation factor 2, resulting in inhibition of protein synthesis (20,21). As the delivery of a single molecule of the catalytic domain to the eucaryotic cell cytosol has been shown to result in the death of that cell, the fusion protein toxins represent a family of highly potent, receptor-specific, cytotoxic probes (22).