**7. Conclusion**

bFGF protein accumulated to levels of ~2.3% of seed TSP and biological activity of the trans‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Recombinant expression of Insulin was first reported using *E*. *coli* [59] and has since been commercialized. Like many pharmaceuticals derived from other expression systems, the po‐ tential for contamination along with high costs associated with production remain consider‐ able for this hormone. To show that a soybean expression system could address some of these issues Cunha et al., used a sorghum γ-kafirin seed storage protein promoter and αcoixin PSV signal peptide to target recombinant proinsulin expression to soybean seeds [65]. Transgenic protein was stably expressed in seeds though accumulation levels were not re‐ ported. Transgenic seeds containing proinsulin were stable for up to seven years when stor‐

The soybean platform has also been used to produce a therapeutic for reducing systolic blood pressure. Novokinin is a hypotensive peptide that has vasorelaxing activity [73]. No‐ vakinin was expressed in soybean seeds under the control of a modified β-conglycinin pro‐ moter and accumulated to 0.5% of seed TSP. A purified soy-derived formulation, as well as a less pure defatted flour formulation, was orally administered to groups of spontaneously hypertensive rats. Both the purified and partially purified formulations successfully reduced

Haemophilia B is a bleeding disorder that results from a deficiency of human coagulation factor IX (hFIX). The current treatment for this disease is intravenous infusion of plasma-de‐ rived or recombinant hFIX protein. While this treatment is effective at preventing and ar‐ resting hemorrhage, it is very costly and the protein is difficult to produce in large quantities. Using a biolistic transformation approach, hFIX expression was targeted to soy‐ bean seeds using the soy 7S promoter and coixin signal peptide [75]. Recombinant hFIX pro‐ tein accumulated to 0.23% of seed TSP, and purified protein exhibited blood-clotting activity up to 1. 4% of normal plasma demonstrating functionality and efficacy of the soy-derived protein. The recombinant protein was stable for 6 years when stored at room temperature. Soybeans are also capable of supporting expression and stable accumulation of large and complex proteins that can be difficult or impossible to express using current expression sys‐ tems. Human thyroglobulin (hTG) is a 660 kDa homodimeric protein that is used as a pro‐ tein standard and diagnostic for the detection of thyroid disease. To date, no expression system has been capable of producing a recombinant form of hTG which is likely due to strict requirements for correct post-translational modification and proper folding during protein synthesis. As a result, commercial hTG supplied to manufacturers for their assay kits is derived from cadaver and surgically removed thyroid tissue. The heterogeneity and lack of uniformity of commercially-purified hTG preparations is a major factor of variation between kits of different manufacturers. To explore the potential of soybean as a platform for production of large and complex proteins, Powell et al., used the 7S promoter and en‐ dogenous hTG signal peptide to target recombinant expression of hTG to soybean seeds [76]. Transgenic lines showed stable expression of full length hTG dimeric protein over mul‐ tiple generations, and accumulated the protein to levels approaching 1.5% of seed TSP. Functionality of soy-derived hTG was demonstrated with commercial ELISA kits developed

genic protein was confirmed by its mitogenic activity in mice.

ed under ambient storage conditions.

Relationships

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systolic blood pressure after a single dose [74].

Over the past 25 years, soybean production in the United States has grown by nearly 57% while during that same period the number of acres used to grow soybeans has increased by only 24%. It is predicted that the worldwide requirement for grain will rise by 40-70% by the year 2050, driven in large part by the growing world population and the increase in demand for protein-rich diets. Clearly the demand for soybean protein and soybean oil is outpacing grain production, which in turn is outpacing available land for growing soybeans. Over the next 50 years, farmers will need to produce as much food as they did in the previous 10,000 years combined, and with fewer resources. The identification of various agricultural im‐ provements such as herbicide, insect, and disease resistance which will allow farmers to ob‐ tain increased yields with reduced environmental inputs will be crucial. Traits that not only increase grain yield, but also improve the absolute levels of soy protein and soy oil within the seed will also be important for producers and consumers worldwide. While soybean is recognized for its high protein content, it is also the most widely grown oil-seed crop in the United States. Enhancing nutritional value of soybean oil will greatly increase the effective‐ ness and value of soy as a food crop, help meet the needs of a growing population, and im‐ prove human health.

Over the past decade, soybean has emerged as an ideal expression platform with potential to address current healthcare needs. These unmet needs include cost-effective alternatives to existing protein-based therapeutics, simplified methods for the administration of therapeu‐ tics, and the development of reagents that could lead to better diagnostic assays and novel medical devices. Soybeans are unique with respect to protein expression platforms. They are safe to consume, cost-effective to grow, rich in protein content, and stable for years under

ambient storage conditions. They have been engineered to express a variety of potential therapeutics, including mAbs, vaccine antigens and adjuvants, hormones, growth factors, and blood-clotting factors. Seed-based expression of 660 kDa homodimerich TG underscores the potential of the soybean system to produce large and complex proteins that cannot be produced in yeast, insect, and mammalian cell cultures. The efficacy of engineered thera‐ peutics in crude soymilk formulations could lead to oral vaccines and therapies that require little, if any, purification from other seed proteins. Reports demonstrating long-term stabili‐ ty of seed-derived therapeutics in the absence of climate control directly address cold chain issues associated with vaccines and other therapeutics. With seed protein levels of ~40% and transgene expression levels approaching 3% of TSP, a single soybean plant yielding 300 seeds can produce >500 mgs of transgenic protein. To put this in perspective, a single soy‐ bean plant can produce 500 doses of a vaccine antigen administered at 1 mg/ml, or alterna‐ tively, \$50,000 [USD] of a therapeutic valued at \$100/mg [USD].

The use of soybean as a platform for the production of therapeutics represents a technology with the potential to revolutionize our current approaches to healthcare. Harnessing the full potential of the soybean platform will depend on further increasing stable transgene expres‐ sion levels, developing efficient purification methods, obtaining interest from pharmaceuti‐ cal partners, and overcoming issues associated with commercialization. The production of vaccines, antibodies, and other therapeutic proteins will undoubtedly continue to develop over the next decade. As biotechnology evolves, so does the role of soybean - from the field to the bedside.
