Takeshi Arakawa

Expression of cholera toxin B subunit oligomers in transgenic potato plants

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr ∼ 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr ∼ 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein β-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas1′2′) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human β-casein cDNA. The presence of human β-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human β-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human β- casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human β-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as β-casein. The above experiments demonstrate the expression of human milk β- casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children

Synthesis of a cholera toxin B subunit-rotavirus NSP4 fusion protein in potato

Abstract. To increase the efficacy of the small amounts of therapeutic protein generally synthesized in transformed plants, we investigated the feasibility of producing a fusion protein in potato capable of targeting a therapeutic protein to a specific organ in the body. An enterocyte-targeted rotavirus fusion gene was constructed by linking the gene encoding the cholera toxin B subunit (CTB) to a DNA fragment encoding an epitope of the rotavirus enterotoxin protein (NSP4). Solanum tuberosum plants carrying a plant expression vector harboring the fusion gene were generated by Agrobacterium tumefaciens-mediated in vivo transformation methods. Immunoblot analysis of transformed tubers indicated the presence of the CTB-NSP4 fusion protein oligomers that retained enterocyte receptor GM1 ganglioside binding affinity. The CTB-NSP4 fusion protein multimers were synthesized in the range of 0.01–0.1% of the total soluble tuber protein.

Food plant-delivered cholera toxin B subunit for vaccination and immunotolerization.

Developments in recombinant DNA technology have enabled molecular biologists to introduce a variety of novel genes into plant species for specific purposes. From crop improvement to vaccine antigen and antibody production, plants are attractive bioreactors for production of recombinant proteins, as their eukaryotic nature often permits appropriate post-translational modification of recombinant proteins to retain native biological activity. The autotrophic growth of plants requires only soil minerals, water, nitrogen, sunlight energy and carbon dioxide for the synthesis of constituent proteins. Furthermore, production of biologically active proteins in food plants provides the advantage of direct delivery through consumption of edible transformed plant tissues. The production of cholera toxin B subunit in potato plants and applications for prevention of infectious and autoimmune disease are explained in this contribution.

Improvements in human health through production of human milk proteins in transgenic food plants.

Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.