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Key Message: Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag ( ELP) and a green fluorescent protein ( GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10- ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10- GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10- GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) represents a spectrum of diseases in which inflammation leads to acute and chronic gut injury. It is a growing health issue for which no cure exists. The pathogenesis is multifactorial with links to infectious and environmental events that trigger disease in genetically predisposed individuals. Treatment of the two major forms of IBD, Crohn's disease and ulcerative colitis, involves the reduction of inflammation with toxic immunosuppressive drugs or blocking of the pro-inflammatory effects of tumour necrosis factor-α (TNF-α) with antibodies. Here, we show that the oral administration of transgenic low-alkaloid tobacco expressing the contra-inflammatory cytokine human interleukin-10 (hIL-10) reduces the severity of colitis by down-regulating TNF-α expression directly at the sites of inflammation in IBD-susceptible IL-10−/– mice. hIL-10 expressed in plants is biologically active and displays resistance to gastrointestinal degradation. Dietary supplementation with plant tissue delivering up to 9 µg of hIL-10 daily for 4 weeks was well tolerated by treated mice. Gut histology was significantly improved relative to controls ( P = 0.002), and was correlated with a decrease in small bowel TNF-α mRNA levels and an increase in IL-2 and IL-1β mRNA levels. Transgenic plants expressing IL-10 to directly attenuate TNF-α expression at sites of inflammation in the gut may become a useful new approach in the luminal therapy of IBD. [ABSTRACT FROM AUTHOR]

Abstract: Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis, prevention or treatment. Tobacco has been and will continue to be a major crop for molecular farming and offers several practical advantages over other crops. It produces significant leaf biomass, has high soluble protein content and is a non-food crop, minimizing the risk of food-chain contamination. This, combined with its flexibility and highly-efficient genetic transformation/regeneration, has made tobacco particularly well suited for plant-based production of biopharmaceutical products. The goal of this review is to provide an update on the use of tobacco for molecular farming of biopharmaceuticals as well the technologies developed to enhance protein production/purification/efficacy. We show that tobacco is a robust biological reactor with a multitude of applications and may hold the key to success in plant molecular farming. [Copyright &y& Elsevier]