title: Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active.
abstract: Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactose-binding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens-mediated gene transfer. Yields of ricin in soluble cell extracts were 1 microg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.
authors.affiliation: Department of Surgery, Medical University of South Carolina, Charleston, South Carolina, 29425, USA.
authors.forename: E P
authors.initials: EP
authors.lastname: Tagge
authors.forename: J
authors.initials: J
authors.lastname: Chandler
authors.forename: B
authors.initials: B
authors.lastname: Harris
authors.forename: M
authors.initials: M
authors.lastname: Czako
authors.forename: L
authors.initials: L
authors.lastname: Marton
authors.forename: M C
authors.initials: MC
authors.lastname: Willingham
authors.forename: C
authors.initials: C
authors.lastname: Burbage
authors.forename: L
authors.initials: L
authors.lastname: Afrin
authors.forename: A E
authors.initials: AE
authors.lastname: Frankel
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001212:Asialoglycoproteins / Q000378:metabolism; D002367:Ricinus communis / Q000737:chemistry; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D060748:Fetuins; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D015151:Immunoblotting; D007624:KB Cells / Q000187:drug effects / Q000378:metabolism; D007612:Kanamycin / Q000494:pharmacology; D007785:Lactose / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D037121:Plant Lectins; D010947:Plants, Toxic; D014176:Protein Biosynthesis / Q000187:drug effects; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012276:Ricin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D013601:T-Lymphocytes / Q000187:drug effects; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D060748:Fetuins; D037121:Plant Lectins; D011498:Protein Precursors; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; C045247:preproricin; D007612:Kanamycin; D012276:Ricin; D007785:Lactose