Mutagenesis of Pseudomonas exotoxin in identification of sequences responsible for the animal toxicity.

Pseudomonas exotoxin (PE) is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The deletion of the cell recognition domain (domain Ia) of PE results in a molecule that does not bind to target cells and has low toxicity in mice (Hwang, J., FitzGerald, D.J.P., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To determine the specific sequences required for cell binding as well as cell and animal toxicity, a series of domain I mutants was constructed. Using a T7 promoter-based expression system and an OmpA signal sequence, large amounts of the various mutant toxins were secreted into the periplasm from which they were easily purified in milligram quantities. The data indicate that amino acids at positions 246, 247, and 249 have an important role in the toxicity of PE. Conversion of these amino acids to glutamic acid or glycine but not to lysine or deletion of amino acids 241-250 diminishes the toxicity of PE. When combined with a mutation at position 57 a molecule is created that has very low toxicity against cultured cells or in mice.