title: Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system.
abstract: In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed 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 cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.
authors.affiliation: Unité des Toxines Microbiennes, CNRS URA 557, Institut Pasteur, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Guidi-Rontani
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D008727:Methotrexate; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A