INTERNALIZATION AND TRANSLOCATION OF A NEW CHIMERIC PROTEIN COMPOSED OF PSEUDOMONAS-AERUGINOSA EXOTOXIN-A AND MOUSE DIHYDROFOLATE-REDUCTASEAS A MODEL SYSTEM

In an attempt to introduce a large peptide that is not normally transl ocated across membranes into the cytosol of eukaryotic cells, we creat ed a new chimeric protein termed CEDH between Pseudomonas aeruginosa e xotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate red uctase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1 -187).ETA(609-613). We have defined, genetically constructed and expre ssed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary c ells. Furthermore, the chimeric protein allowed the growth of dihydrof olate reductase-deficient Chinese hamster ovary cells in the absence o f hypoxanthine and thymidine, These results demonstrated that the chim eric protein exhibited enzyme activity and possessed the tightly folde d native structure, and that the DHFR protein can be selectively inter nalized and translocated via domains of exotoxin A. These data show th at the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target c ells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.