PREPRORICIN EXPRESSED IN NICOTIANA-TABACUM CELLS IN-VITRO IS FULLY PROCESSED AND BIOLOGICALLY-ACTIVE

Ricin, the highly toxic glycoprotein expressed in the endosperm of cas tor seeds, is composed of a galactose-binding lectin B chain (RTB) dis ulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modifie d ricin has been conjugated to monoclonal antibodies and used for targ eted therapy of cancer and autoimmune diseases. Replacement of chemica lly coupled molecules with a genetically engineered targeted ricin wou ld improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed muta genesis. 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 pres ent study, we introduced the cDNA encoding preproricin into cultured t obacco cells via Agrobacterium tumefaciens-mediated gene transfer, Yie lds of ricin in soluble cell extracts were 1 mu g/g in cells or, appro ximately, 0.1% of the total soluble protein, The ricin was partially p urified by P2 monoclonal antibody anti-RTB affinity chromatography. Th e RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa u nder nonreducing conditions and at 32-35 kDa under reducing conditions . The tobacco ricin bound to immobilized asialofetuin as avidly as cas tor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibit ed rabbit reticulocyte lysate protein translation similar to castor be an ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M fo r castor bean ricin), The human cutaneous T cell lymphoma cell line HU T102 showed similar sensitivity to tobacco ricin when compared to cast or bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively).