Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor.
Fusion toxins are hybrid proteins consisting of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure and the lack of prior immunological exposure in patients. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL-2 (interleukin-2), another ADP tripeptide and RTB (ricin toxin B chain) with lectin-site mutations W37S and Y248H. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL-2-RTB[W37S/Y248H] protein (fusion protein of IL-2 with modifications W37S and Y248H) was recovered at high yields from day 6 insect cell supernatants, partially purified by affinity chromatography and reassociated with RTA (ricin toxin A chain). The fusion toxin was soluble, immunoreactive with antibodies to RTB, IL-2 and RTA and had a molecular weight of 80 kDa by SDS-PAGE. The molecule reacted poorly with asialofetuin, but bound strongly to IL-2 receptor based on selective cytotoxicity to IL-2 receptor bearing cells. The specific cytotoxicity could be blocked with IL-2 but not lactose. Thus, we report a novel targeted fusion toxin protein with full biological activity.
Frankel
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AE
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Burbage
C
C
Fu
T
T
Tagge
E
E
Chandler
J
J
Willingham
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007785:Lactose / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D015375:Receptors, Interleukin-2 / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D012276:Ricin / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D012276:Ricin; D007785:Lactose