Expression of active, processed ricin in transgenic tobacco.
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.
Sehnke
P C
PC
Department of Horticultural Sciences, University of Florida, Gainesville 32611.
Pedrosa
L
L
Paul
A L
AL
Frankel
A E
AE
Ferl
R J
RJ
D016960:Agrobacterium tumefaciens; D000818:Animals; D001483:Base Sequence; D002367:Ricinus communis / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D015368:Human T-lymphotropic virus 1 / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D037121:Plant Lectins; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012156:Reticulocytes / Q000187:drug effects; D012276:Ricin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D014026:Nicotiana / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D037121:Plant Lectins; D011498:Protein Precursors; D011994:Recombinant Proteins; C045247:preproricin; D012276:Ricin