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 language | English (US) |
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Pages (from-to) | 22473-22476 |
Number of pages | 4 |
Journal | Journal of Biological Chemistry |
Volume | 269 |
Issue number | 36 |
State | Published - Sep 9 1994 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology