Abstract
Background: Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such as bacterial or mammalian cell culture. In order to explore transgenic plants for large-scale production and storage of recombinant antibodies we tried to optimize the accumulation and stability of functionally active single chain Fv (scFv) antibodies in transgenic tobacco plants. Objectives: Two different scFv antibodies which were expressed in different plant organs and plant cell compartments have been used for the study. Accumulation levels and antibody properties such as stability and antigen-binding activity were investigated. Study design: For ubiquitous expression in tobacco plants, transcription of the scFv genes was controlled by the strong cauliflower mosaic virus (CaMV) 35S promoter. We used seed specific legumin B4 (LeB4) and the unknown seed protein (USP) promoters from Vicia faba for storage organ specific expression. Results: High accumulation of the two different scFv proteins in transgenic tobacco plants was only achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 4–6.8% of total soluble proteins (TSP) in leaves and up to 3–4% in ripe tobacco seeds. Transgenic tobacco seeds as well as tobacco leaves facilitated stable storage of ER-accumulated scFvs over an extended (seeds) or a short (leaves) period of time. Functionally active scFv proteins could be extracted after harvesting of the leaf material—drying and storage for 1 week at room temperature. Both the amount and the binding activity of the scFv proteins remained unchanged. Conclusion: A plant expression system where the scFv-proteins are targeted in the ER provides not only the highest accumulation level of active single chain Fv antibodies ever reported but also a short- or long-term storage of the foreign protein in the harvested plant material.
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