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Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.
This is the peer reviewed version of the following article: Limaye, A., Koya, V., Samsam, M., & Daniell, H. (2006). Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 20(7), 959–961. http://doi.org/10.1096/fj.05-5134fje, which has been published in final form at http://doi.org/10.1096/fj.05-5134fje. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving http://olabout.wiley.com/WileyCDA/Section/id-820227.html#terms.
transmucosal carrier, furin, ganglioside receptors, genetically modified crops
Limaye, A., Koya, V., Samsam, M., & Daniell, H. (2006). Receptor-Mediated Oral Delivery of a Bioencapsulated Green Fluorescent Protein Expressed in Transgenic Chloroplasts Into the Mouse Circulatory System. FASEB Journal, 20 (7), 959-961. http://dx.doi.org/10.1096/fj.05-5134fje
Date Posted: 01 March 2022