The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins
