Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor

Arthur E. Frankel, Chris Burbage, Tao Fu, Edward Tagge, John Chandler, Mark Willingham

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)913-919
Number of pages7
JournalProtein Engineering
Volume9
Issue number10
Publication statusPublished - Oct 1996

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Keywords

  • IL-2 fusion toxin
  • Ricin

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Frankel, A. E., Burbage, C., Fu, T., Tagge, E., Chandler, J., & Willingham, M. (1996). Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor. Protein Engineering, 9(10), 913-919.