Expression of active, processed ricin in transgenic tobacco

Paul C. Sehnke, Luis Pedrosa, Anna Lisa Paul, Arthur E. Frankel, Robert J. Ferl

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

The cDNA encoding the plant toxin precursor preproricin was introduced into tobacco via Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants were assayed for type II ribosome-inactivating protein expression and activity. Western blot analysis of soluble leaf extracts using anti-ricin a- chain (RTA) antibodies identified 34- and 32-kDa proteins, which were electrophoretically indistinguishable from castor seed RTA. Analysis with anti-ricin b-chain (RTB) antibodies identified both a 34-kDa protein major band, which co-migrated with castor seed RTB, and a 30-kDa protein minor band. Enzyme-linked immunoassay of the transgenic leaf extracts with anti- RTA and anti-RTB indicated microgram per gram production on a fresh weight basis of soluble extractable recombinant ricin. Sugar binding enzyme-linked immunoassay employing an immobilized glycoprotein, asialofetuin, and anti- RTB antibodies confirmed the characteristic type II ribosome-inactivating protein galactose binding lectin activity of the recombinant ricin. The enzymatic activity of recombinant ricin was characterized for cell-free translation inhibition, as well as for overall cytotoxicity. A 50% inhibitory dose of 3 x 10-11 M was observed for the immunoreactive leaf extract material using a rabbit reticulocyte translation inhibition assay, while a 50% lethal dose of 1 x 10-12 M was calculated with human T-lymphotropic virus-1 infected leukemic T-cells.

Original languageEnglish (US)
Pages (from-to)22473-22476
Number of pages4
JournalJournal of Biological Chemistry
Volume269
Issue number36
StatePublished - Sep 9 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Sehnke, P. C., Pedrosa, L., Paul, A. L., Frankel, A. E., & Ferl, R. J. (1994). Expression of active, processed ricin in transgenic tobacco. Journal of Biological Chemistry, 269(36), 22473-22476.