Abstract
We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.
MeSH terms
-
ADP Ribose Transferases*
-
Animals
-
Bacillus / enzymology
-
Bacillus / genetics*
-
Bacterial Proteins
-
Bacterial Toxins / genetics*
-
Base Sequence
-
Cell Survival / drug effects
-
Chimera
-
Cloning, Molecular / methods
-
DNA Replication / drug effects
-
DNA, Bacterial / genetics
-
Escherichia coli / genetics
-
Exotoxins / genetics*
-
Exotoxins / pharmacology
-
L Cells / cytology
-
L Cells / drug effects
-
L Cells / metabolism
-
Leucine / metabolism
-
Mice
-
Molecular Sequence Data
-
Oligonucleotide Probes
-
Plasmids
-
Pseudomonas aeruginosa / genetics*
-
Pseudomonas aeruginosa Exotoxin A
-
Recombinant Fusion Proteins / metabolism
-
Recombinant Fusion Proteins / pharmacology
-
Ribonucleases / genetics*
-
Ribonucleases / metabolism
-
Ribonucleases / pharmacology
-
Thymidine / metabolism
-
Transcription, Genetic / drug effects
-
Virulence Factors*
Substances
-
Bacterial Proteins
-
Bacterial Toxins
-
DNA, Bacterial
-
Exotoxins
-
Oligonucleotide Probes
-
Recombinant Fusion Proteins
-
Virulence Factors
-
ADP Ribose Transferases
-
Ribonucleases
-
Bacillus amyloliquefaciens ribonuclease
-
Leucine
-
Thymidine