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Plants have been used for the experimental production of many different pharmaceutical proteins, including vaccines (Streatfield, 2007), antibodies (Twyman et al. 2007), hormones, signaling proteins, blood products and replacement enzymes (Stoger et al. 2002, 2005), as well as industrial enzymes (Fischer et al. 2003). As more recombinant proteins are developed as industrial products, the demand increases for simple and economical production platforms that allow the large-scale production of inexpensive heterologous proteins.
The advantages of plant-based production platforms have been widely acknowledged, and although no single platform has emerged as a primary candidate for process-scale manufacturing, there are several well-established transgenic crops for molecular farming including tobacco, maize and rice. Plant cell cultures offer the advantages of containment, simplicity, consistency and simpler downstream processing, making them the most likely platform in the short term to yield pharmaceutical products that comply with good manufacturing practice (GMP). However, they suffer the same limitations in terms of scalability and reliance on bioreactor-based infrastructure as other fermentation platforms. Alternatively, transient expression systems offer the prospect of rapid and highly-scalable production. Although initially used as a test platform to ensure construct validity before progressing to transgenic plants, transient expression has now emerged as a platform in its own right. The basis of transient expression is the infiltration of leaves with Agrobacterium tumefaciens, plant viruses or hybrid vectors containing elements of both, allowing high level protein expression for a short period of time. The main advantages of transient expression are the rapid scale up, making these platforms ideal for rapid response situations (e.g. pandemic vaccines), and also the flexibility in scale, making them equally suitable for patient-specific therapies. None of the plant-based production platforms can yet challenge the yields possible using more established platforms such as E. coli and mammalian cells (Ramessar et al., 2008) but their advantages in terms of safety, scalability, economy and unique delivery mechanisms make it imperative to carry out research for the improvement of productivity of plants for molecular farming.
In this thesis, we investigated the ability of wheat tissues to accumulate recombinant proteins by exploiting the natural ability of -zein to induce the formation of storage organelles in native and ectopic tissues. Seeds contain a number of unique subcellular compartments that can be used for the accumulation of recombinant proteins, including protein bodies derived from the ER, PSVs, starch granules and the surface of oil bodies. All of these destinations have been exploited to increase accumulation of recombinant proteins, to reduce proteolytic degradation, to increase the efficacy of oral delivery and/or to facilitate recombinant protein recovery and purification. There is great interest in the development of strategies for the deliberate targeting of recombinant proteins to these compartments.
Dry seeds provide an optimal environment for the stable accumulation of recombinant proteins and are considered suitable as vehicles for molecular farming (Stoger et al., 2002a). Seeds have been used for the production of many recombinant proteins, including vaccine antigens, antibodies, hormones, proteases and their inhibitors, growth factors and enzymes for industrial or medical applications (Boothe et al., 2010). Several publications have demonstrated convincingly that recombinant proteins in mature seeds show no detectable loss of stability or activity when stored for several years at ambient temperatures (Stoger et al. 2000, Nochi et al., 2007). In addition to stability, high-yielding field-grown cereal crops also provide the benefit of scalability, simply by planting more crops over a larger area. As with other platforms, the factors that must be taken into consideration when choosing a seed-based production system include biomass yield per hectare, the yield of recombinant protein per unit biomass, and the length of the breeding cycle which determines how quickly production can be scaled up (Twyman et al., 2003).
This thesis considers the potential of wheat (Triticum aestivum L. cv Bobwhite) as a production platform, by testing the expression of a model recombinant fusion protein in seed and non-seed tissues. The model protein was γ-zein-DsRed, comprising proline rich repeating domain and Pro-X domain of γ-zein to induce the formation of storage compartments, and DsRed as a fluorescent reporter. Fluorescent reporter genes have revolutionized the field of molecular biology and they provide versatile tools to screen gene expression and monitor protein localization and activity. DsRed can be expressed in plants without affecting their physiology or metabolism (Anthony and Susan 2002).
There are different reasons why we select wheat plant as host to utilize the model fusion protein. Wheat has a well-established global infrastructure for cultivation, harvest, storage and distribution, providing the foundation for recombinant protein production in industrialized and developing countries. Like other cereals, wheat seeds maintain recombinant proteins in a stable form for years, but wheat has two major advantages over maize in that it is self-pollinating (thus reducing the likelihood of outcrossing from transgenic crops) and has a low producer price compared to other cereals. As a cereal crop, it is generally recognized as safe (GRAS) by the European Food Safety Authority (EFSA) which makes it an ideal production crop for oral vaccines and topical formulations. Potential disadvantages of wheat include the challenging transformation procedure and the generally low expression levels that have been achieved with other recombinant proteins, hence the development of novel strategies to improve protein yields in this thesis. Wheat plant has been rarely used for molecular farming because of the lower yield for recombinant proteins. The feasibility of γ-zein sequences for protein accumulation in induced protein bodies in wheat plant tissues would make it attractive host for molecular farming.
Wheat transformation was achieved by the particle bombardment of immature embryos. This type of explant was favored in early transformation protocols (Becker et al., 1994; Rasco-Gaunt et al., 2001) although other targets have been used for both particle bombardment and Agrobacterium-mediated transformation. One of the key advantages of particle bombardment is that it is a genotype-independent method, whereas Agrobacterium-mediated transformation must be optimized for individual genotypes because it is highly dependent on the biological properties of the target. Particle bombardment is also suitable for the transient expression of endosperm (Knudsen and Müller, 1991) or epidermal cells (Douchkov et al., 2005) to test the efficacy of expression constructs.
In this thesis, we achieved a low transformation frequency of up to < 1%, which is comparable to early results reported by Altpeter et al. (1996) and is regarded as an acceptable efficiency of gene transfer given the recalcitrance of wheat explants, the need to culture explants on induction medium for several months, and the challenge of regenerating explants into transgenic plantlets (Stöger et al., 2000). The transformation frequency is also dependent on the
genotype, explant type and transformation methodology, therefore, careful optimization was required to achieve this transformation frequency in the Bobwhite cultivar.
After achieving the transformation of wheat explants and the recovery of transgenic lines expressing the fusion protein, we studied the induction of artificial storage organelles and their potential exploitation for the accumulation of recombinant proteins. We found that maize prolamin sequences were able to promote the accumulation of its fusion partner within induced protein bodies that were morphologically similar to their native counterparts in maize endosperm tissue. The constitutive expression of DsRed using the γ-zein sequence as a fusion partner was demonstrated in three different tissues: leaf, endosperm and the embryo. The maize γ-zein sequence can induce the formation of protein bodies not only in maize but also in other plants such as tobacco, and in eukaryotic cells more generally (Llamport et al., 2010; Tosi et al., 2009). The main objective in this thesis was to conduct a feasibility study using a model fusion protein γ-zein-DsRed, visually confirm the formation of protein bodies and analyze their dynamic behavior in different tissues.
We demonstrated that the γ-zein-DsRed fusion protein can induce the de novo formation of protein bodies in all three tissues, resulting in the accumulation of the protein in ectopic organelles. The heterologous expression of γ-zein in leaves has previously been shown to induce the formation of spherical organelles similar to cereal protein bodies (Geli et al., 1994; Torrent et al., 2009). The underlying mechanism has not been fully characterized, but the physicochemical properties of the N-terminal γ-zein sequences are thought to promote self-assembly (Kogan et al., 2002) and are consider to favor aggregation (Mainieri et al., 2004). The mechanism of protein body induction by the N-terminal domain of γ-zein is highly conserved, and has been shown to work in the fungus Trichoderma reesei (Conley et al., 2012), mammalian CHO and COS cells, and also insect cells from Spondoptera frugiperda (Blanca et al., 2010; Torrent et al., 2009).
The expression of the recombinant fusion protein in wheat was confirmed using fluorescence and confocal microscopy. In leaves, which lack protein storage vacuoles, the fluorescent protein accumulated in protein bodies 1-2 m in diameter which were dispersed throughout the cytosol (Fig.III.15). These were similar in size and appearance to the native protein bodies found in maize endosperm, and also to heterologous protein bodies induced in tobacco leaves using the N-terminal γ-zein sequences (Torrent et al., 2009). The same investigators also fused the coding sequence for enhanced cyan fluorescent protein (ECFP) to either the Zera domain or to an ER-retention signal (KDEL) and expressed the constructs in tobacco epidermis. The Zera-ECFP protein accumulated in protein bodies within the epidermal cells, the ECFP-KDEL protein accumulated in the ER lumen and a control ECFP protein lacking Zera and KDEL was secreted. Similarly, the γ-zein N-terminal sequences were also fused to the bean storage globulin phaseolin, and the recombinant fusion protein (zeolin) was expressed in tobacco leaves where it accumulated to levels in excess of 3.5% total soluble protein (Mainieri et al. 2004). The same γ-zein N-terminal domain was unable to increase yields of the recalcitrant HIV-1 Nef protein either as an N-terminal or C-terminal fusion, but the entire zeolin protein as a fusion partner increased the yield of zeolin-Nef to 1.5% total soluble protein and induced the formation of ectopic, ER-derived protein bodies (de Virgilio et al., 2008). Llompart et al. (2010) produced human growth hormone as a Zera fusion by transient expression in Nicotiana benthamiana leaves, resulting in recombinant protein yields of up to 10% total soluble protein. Joseph et al. (2012) have expressed a Zera-DsRed fusion in Nicotiana benthamiana leaves resulting in yields of up to 85% total soluble protein and the accumulation of the fluorescent proteins in ER-derived protein bodies.
In addition to leaves, we also expressed the γ-zein-DsRed protein in wheat endosperm and embryo cells to investigate the trafficking and accumulation of the protein in seed tissues and to determine the impact of recombinant protein expression in the endosperm, which forms its own endogenous compartments as a major sink for storage proteins (James et al., 2003).
We found that the γ-zein-DsRed fusion protein was stable in wheat seed tissues but that the expression profile driven by the constitutive ubiquitin promoter was potentially constrained by the specific cell type. In the endosperm, the highest expression was achieved in subaleurone layer cells. This may reflect a genuine difference in the activity of the ubiquitin promoter in different specialized cell types within the seed, but may also be explained by differential activity in terms of protein synthesis and accumulation as the cells mature, and/or the properties of the cells that affect the visibility of the fluorescence signal. Subaleurone cells are younger than the central endosperm cells and generally contain more protein bodies, whereas older cells contain more starch and the vacuole is reabsorbed. Therefore, the visualization of protein bodies within the central vacuole is easier in the subaleurone cells, where DsRed fluorescence could be detected from 10 to 25 days after anthesis, peaking during days 14-18. The protein bodies appeared clustered or aggregated in the central endosperm cells, perhaps reflecting a genuine difference in distribution compared to leaves in combination with the clearer fluorescence signal in the larger vacuoles of older cells (Fig. III.16A). These results are in accordance with the behavior of endogenous protein bodies, which also accumulate within large vacuoles (Fig. III.17A,B). The γ-zein-DsRed protein bodies in the inner endosperm cells were partially obscured large starch granules.
Zhang et al. (2003) expressed full-length maize γ-zein in barley seeds using a wheat high-molecular-weight glutenin subunit promoter, resulting in yields of up to 2% of the total grain nitrogen. Recombinant γ-zein has also been expressed in the leaves and roots of Arabidopsis thaliana, but there was no trace of the protein in seeds suggesting the protein was unstable in seeds (unlikely) or that the promoter was inactive in seeds (Geli et al., 1994). These experiments were carried out to identify the sequences responsible for protein body biogenesis and the authors concluded that the N-terminal proline-rich repeat domain and the C-terminal cysteine-rich domain were sufficient for this purpose (Geli et al., 1994).
Cell-specific differences in expression were also observed when we expressed the γ-zein-DsRed fusion protein in wheat embryos. Strong fluorescence was observed in the tissues below the scutellum, indicating that the ubiquitin promoter was not active in all of the embryo tissues or that protein synthesis and accumulation were inefficient. At the level of individual cells, the fusion protein appeared to be expressed more strongly in the embryo than the endosperm (Fig. III.13). DsRed protein bodies were observed in clusters of cells within the embryo from as early as 8 days after anthesis (Fig. III.18). The protein bodies in embryo cells were less dynamic than those in leaf cells, as determined by real-time confocal microscopy.
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The plant cell has to sort thousands of different protein to the correct subcellular compartment, and most proteins therefore contain one or more domains or motifs that provide the necessary information. Each subcellular compartment and membrane requires a different targeting sequence and key examples include N-terminal signal peptides for entry into the secretory pathway, transit peptides for entry into plastids and mitochondria, nuclear localization signals, peroxisome targeting signals and vacuolar sorting signals.
The N-terminal signal peptide ensures that a protein enters the secretory pathway before it is fully translated, because it is recognized by a signal-recognition particle that binds to receptors on the ER membrane, allowing the protein to be translocated into the ER during synthesis. In the absence of any further signals, the protein then follows the default pathway, which culminates in its secretion to the apoplast (Denecke et al., 1990). If the protein contains a Cterminal S/KDEL tetrapeptide, it is retrieved to the ER and remains in the lumen, whereas other signals allow the protein to be diverted from the secretory pathway, e.g. into the vacuole (Hadlington and Denecke, 2000).
Seeds differ from most vegetative cells with respect to protein trafficking and offer several unique compartments, including protein bodies derived from the ER, PSVs, starch granules and the surface of oil bodies, all of which have evolved as part of the specialized function of seeds as storage organs. These compartments are adapted for the storage of proteins in a stable and accessible form, and have therefore been investigated for the accumulation of recombinant proteins to increase yields and maximize stability (by preventing recombinant proteins from encountering proteases). The degradation of recombinant proteins by proteases inside the cell or after secretion is a critical determinant of overall yields (Doran 2006). We therefore targeted our model fusion protein to accumulate within induced protein bodies to avoid proteolysis and increase yields. Confocal microscopy confirmed that the recombinant fusion protein assembled within the induced protein bodies, and the aggregation (triggered by the -zein domain) and the intrinsic DsRed fluorescence suggested the protein was stable and intact (Fig.III.17A, B). This was confirmed by the absence of visible degradation products when the protein was extracted and analyzed by immunoblotting (Figure III. 14).
The morphology of the γ-zein-DsRed protein bodies in wheat leaf and embryo cells resembled that of native protein bodies in maize (Lending et al., 1988; Washida et al., 2004), rice (Muench et al., 2000; Nicholson et al., 2005; Saito et al., 2009) and soybean (Schmidt et al., 2008), induced protein bodies formed in tobacco leaves (Torrent et al., 2009), and the zeolin bodies described above (Mainieri et al., 2004; De Virgillo et al., 2008). The general morphology is spherical and approximately 2 µm in diameter.
The accumulation of the fusion protein did not appear to alter the growth or development of the transgenic wheat plants, either in terms of the general phenotype or in terms of the impact at the cellular level. For example, there was no evidence of enhanced apoptosis in the transgenic plants, which can occur if the rate of protein synthesis exceeds the rate of protein folding (Ron et al., 2007). There was also no evidence for enhanced apoptosis in experiments using the Zera domain to express ECFP in tobacco leaves (Torrent et al., 2009). These results suggest that γzein-DsRed and similar induced protein bodies do not adversely affect plant growth or development.
Torrent et al. (2009) also showed that the Zera sequences maintained the stability of the fusion partner, allowing functional recombinant protein to be recovered after 5 months at room temperature. The stability of proteins in protein bodies was also demonstrated by Nochi et al. (2007), who expressed the cholera toxin B subunit in rice protein bodies and showed they were highly resistant to proteolytic digestion in the gastrointestinal tract. Tailored protein bodies can also be used to facilitate the purification of recombinant proteins from seeds.
Molecular farming initially focused on upstream productivity by improving gene expression and protein stability, but as more products have moved towards commercial development the focus has shifted to downstream processing for process-scale manufacturing. The aggregation of the γzein-DsRed fusion protein into dense protein bodies simplifies the purification process by allowing initial recovery by crude fractionation, followed by the resolubilization of relatively pure protein aggregates which means that expensive chromatography-based separations are only needed for polishing to remove trace impurities. The accumulation of recombinant proteins in novel protein bodies will therefore lower production costs by increasing upstream productivity and by replacing expensive downstream processing steps.
The expression of recalcitrant recombinant proteins as fusions with stability-inducing partners is a generally useful strategy to improve product yields. However, the use of seed storage proteins as stabilizing partners provides additional advantages including the induction of protein body-like aggregates in cells that normally lack such organelles, offering further advantages such as protection from degradation, alternative purification strategies and post-harvest encapsulation. The advantages of seed-based systems can therefore be combined with the rapid biomass accumulation that is possible in leafy plants. Protein fusion partners that provide these enhanced benefits include natural zeins, synthetic elastin-like peptides (ELPs) and fungal hydrophobins (Table 2), all three of which can induce protein bodies and facilitate the process-scale production and purification of recombinant proteins (Conley et al., 2011).
ELPs are biochemically similar to γ-zein, and have been used to test the accumulation of GFP targeted to the cytoplasm, plastids, apoplast and ER in several different N. benthamiana tissues, with the ER variants showing the highest accumulation (Colney et al., 2009). ELPs have also been used to increase the expression of pharmaceutical proteins in transgenic tobacco leaves (Patel et al. 2007; Floss et al., 2008). The induction of protein bodies using 100 ELP repeats fused to single-chain antibody resulted in yields of 40% total soluble protein in tobacco seeds and 25% in leaves (Scheller et al., 2006).
When targeted to the ER, recombinant hydrophobin fusion proteins also induce the formation of novel protein bodies in fungi (Conley et al., 2011) and in tobacco leaves (Joensuu et al., 2010). In the latter study, GFP was expressed using the Trichoderma reesei HFBI hydrophobin sequence as a fusion partner, resulting in an unprecedented expression level equivalent to 51% total soluble protein in leaves, up to 91% of which could be recovered by aqueous two-phase extraction (Joensuu et al., 2010).
The γ-zein fusion strategy was envisaged to produce stable vaccine antigens as prolamin aggregates in wheat, but we found instead that separate fusion protein bodies were formed and deposited in the PSV, which would also be useful for the development of seed-based vaccines. Cold chains are usually required for vaccine distribution which is challenging in developing countries, but seed-based oral vaccines provide a solution by allowing the distribution of stable vaccine antigens at ambient temperatures using existing distribution infrastructures for food (Ramessar et al., 2008). Edible seeds containing oral vaccines would require minimal processing (enough to ensure dosing accuracy) and this could be done by traditional techniques in developing country settings, such as the preparation of flour paste (Lamphear et al., 2002; Takagi et al., 2005). Alvarez et al. (2010) expressed Yersinia pestis F1-V antigen as a Zera fusion in Nicotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco) NT1 cells. The yield was increased three-fold compared to the native protein without affecting plant growth and development (Alvarez et al., 2010).
Many pathogens enter the body via mucosal surfaces; therefore mucosa has been the main target both for passive and active immunization strategies with focusing on oral delivery. (Hefferon et al., 2010). In plants, the production of vaccine antigens have clear advantages because plant tissues containing vaccine antigens can directly be consumed without processing avoiding expensive purification processes. However, the potential hurdle is the digestive system that may cause destruction of the vaccine antigens before they can reach the immune cell in the ileum, clustered in regions know as Peyer's patches. The production of vaccine antigens in plants offers further advantage of extending partial protection from digestive enzymes and permits more time for the antigen to interact with immune cells. This shielding effect is improved through the targeting recombinant proteins into storage organelles such as protein bodies (Takagi et al, 2010) and starch granules (Stephen et al, 2006; Reviewed in Khan et al., 2012).
Remarkably, Takagi and colleagues (2010) compared the survival of a known oral tolerogen when administered either as a synthetic peptide, or as transgenic rice grain, where the antigen was targeted to either ER-derived protein bodies or PSVs. The rice endosperm derived antigens showed more resistance to in vitro digestion with pepsin than the soluble form. This is particularly noteworthy that targeting antigens to protein bodies were more effective provided more resistance than PSVs. Therefore, the data presented the evidence that bio-encapsulated delivery of tolerogen significantly enhanced the immunological efficacy for the suppression of allergen-specific IgE responses. Ogawa and colleagues (1987) also showed that rice prolamin bodies are only digested to a limited extent and many prolamin bodies are excreted. More recently, cholera toxin B subunit (Nochi et al., 2007) and recombinant antibodies expressed in pea seeds (Zimmermann et al., 2009). In the latter case, flour from the transgenic pea seeds was more potent than the purified antibody fragments, and additional experiments showed that the antibody was protected from degradation in the pea seeds, possibly reflecting the presence of protease inhibitors (Zimmermann et al., 2009; Reviewed in Khan et al., 2012).
To conclude, the expression of the γ-zein-DsRed fusion protein did not affect the growth and development of the wheat plants, and allowed us to study the formation and dynamic behavior of novel protein bodies in a range of different tissues. These protein bodies will help to increase protein yields and facilitate the extraction of recombinant proteins produced in wheat, and will provide encapsulation for orally administered proteins such as vaccine antigens.