Processing of preproricin in transgenic tobacco

The plant protein toxin ricin has found widespread application as a potenti al therapeutic agent for many human diseases and in disease-model systems s uch as those involving apoptosis, Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, sp ecifically in transgenic tobacco carrying the preproricin gene under the co ntrol the cauliflower mosaic virus 35S promoter. Production of modified ric in for altered controllable activity and/or fusion therapeutics to target d elivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgeni c tobacco was purified using lectin affinity chromatography and characteriz ed using various biochemical and biophysical techniques. Coomassie blue sta ining of an SDS-PAGE gel of lactose-agarose purified material identified pr edominant proteins of 30 and 35 kDa molecular weight. Western analysis usin g anti ricin a- and b-chain antibodies confirmed the expression and purific ation of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin, High-resolution gel filtration chr omatography characterized the lactose binding complex as a 66-kDa native mo lecular weight protein which could be separated into 30- and 35-kDa protein s upon incubation with the reducing agent dithiothreitol, N-terminal sequen cing of the recombinant ricin a-chain revealed that an equimolar ratio of t wo alternately processed peptides was present, which varied by an additiona l amino acid derived from the signal peptide. Similar analysis of ricin b-c hain again identified two forms of this polypeptide as well; however, full- length ricin b-chain and b-chain missing the first alanine residue were pre sent at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylamin opurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the me dia of the cultures, indicating that cell cultures provide a safe and simpl e means to produce properly processed recombinant ricin. (C) 1999 Acadcmic Press.