Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation
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Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, γ-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera® (γ-Zein ER-accumulating domain) is the N-terminal proline-rich domain of γ-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera® domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera® domain. We demonstrated that Zera®-F1-V protein accumulation was at least 3× higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera® technology to induce protein body formation in non-seed tissues. Zera® expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera® technology to substantially increase the accumulation of value-added proteins in plants.
KeywordsProtein body Zera Plant-derived vaccines Plant bioreactors Plague Transgenic plants
Nicotiana tabacum NT1 cells
Total soluble protein
Unfolded protein response
γ-Zein ER-accumulating domain
The authors sincerely acknowledge Joyce Van Eke (Boyce Thompson Institute, Ithaca, NY) for providing the tobacco NT1 calli; Stephen Temple and Forage Genetics International (Nampa, ID) for alfalfa clone R2236 and alfalfa transformation protocol; and the research team at ERA biotech (Barcelona, Spain) for plasmid pUC18:Zera® and Zera® antibody. The authors are particularly grateful to Dow Agrosciences (Indianapolis, IN), ERA Biotech (Barcelona, Spain), and the ASU/ITESM Collaborative on Biotechnology Research for the partial support of this project. The authors are also very grateful with Paul Arnold for his help with the editing of the paper, and with the undergraduate students Amber Gustin, Deborah Pauley and Julliane Miller for their collaboration with plant transformation experiments and plant tissue culture.
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