Transgenic Res (2010) 19:291–298 DOI 10.1007/s11248-009-9306-8 BRIEF COMMUNICATION Temporal and spatial distribution of erythropoietin in transgenic tobacco plants Andrew J. Conley Æ Anthony M. Jevnikar Æ Rima Menassa Æ Jim E. Brandle Received: 2 February 2009 / Accepted: 4 July 2009 / Published online: 18 July 2009 Ó Her Majesty the Queen in Right of Canada 2009 Abstract Plants have shown promise as bioreactors for the large-scale production of a wide variety of recombinant proteins. To increase the economic feasibility of this technology, numerous molecular approaches have been developed to enhance the production yield of these valuable proteins in plants. Alternatively, we chose to examine the temporal and spatial distribution of erythropoietin (EPO) accumulation during tobacco plant development, in order to establish the optimal harvesting time to further maximize heterologous protein recovery. EPO is used extensively worldwide for the treatment of anaemia and is currently the most commercially valuable biopharmaceutical on the market. Our results indicate that the concentration of recombinant EPO and endogenous total soluble protein (TSP) A. J. Conley Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada A. J. Conley
R. Menassa (&)
J. E. Brandle Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON N5V 4T3, Canada e-mail: Rima.Menassa@agr.gc.ca A. M. Jevnikar Transplantation Immunology Group, Lawson Health Research Institute, London, ON N6A 5A5, Canada J. E. Brandle Vineland Research and Innovation Centre, Vineland Station, ON L0R 2E0, Canada declined significantly for every leaf of the plant during maturation, although the rate of these declines was strongly dependent on the leaf’s position on the plant. As a result, the amount of EPO produced in leaves relative to TSP content remained essentially unchanged over the course of the plant’s life. Decreasing levels of recombinant protein in leaves was attributed to proteolytic degradation associated with tissue senescence since transgene silencing was not detected. We found that significantly higher concentrations of EPO within younger leaves more than compensated for their smaller size, when compared to their low-expressing, fully-grown counterparts. This suggests that fast-growing, young leaves should be periodically harvested from the plants as they continue to grow in order to maximize recombinant protein yield. These findings demonstrate that EPO accumulation is highly influenced by the plant’s physiology and development. Keywords Human erythropoietin
EPO
Transgenic tobacco
Molecular farming
Recombinant protein production Introduction In recent years, transgenic plants have shown great potential for providing a safe, efficient and inexpensive means of producing large amounts of recombinant pharmaceutical proteins. Plants also offer further 123 292 benefits over conventional expression systems, including rapid scalability, the ability to correctly fold, assemble and process complex proteins, and the potential for direct oral administration of unprocessed or partially processed plant material (Ma et al. 2003; Fischer et al. 2004). Although a wide range of plant host systems have been proposed, tobacco has the most established history as a bioreactor for recombinant protein production. Tobacco has a high biomass yield (more than 100,000 kg per hectare) and the platform is based on leaves (Sheen 1983), eliminating the need for flowering and thus minimizing the possibility of gene leakage into the environment through pollen or seed dispersal. Furthermore, because tobacco is a non-food, non-feed crop, there is little risk of transgenic material contaminating the food chain (Rymerson et al. 2002; Twyman et al. 2003). Recombinant human erythropoietin (EPO) is a glycoprotein hormone that controls red blood cell production and is used extensively around the world for the treatment of anaemia caused by renal failure, chemotherapy and acquired immunodeficiency syndrome (AIDS). Recently, EPO has also been shown to act as a pleiotropic cytokine with remarkable tissue-protective activities in many models of neuronal, retinal, cardiac, and renal injury (Ghezzi and Brines 2004; Maiese et al. 2005). With this in mind, we recently demonstrated that plant-derived EPO possesses cytoprotective activity in a model of kidney epithelial cell death (Conley et al. 2009). The low production yield of many recombinant proteins continues to be the most challenging problem limiting the commercial exploitation of transgenic plant systems (Doran 2006). The effective use of transgenic plants as biofactories requires that high levels of recombinant protein accumulation be consistently obtained throughout their life cycle and in subsequent generations (De Wilde et al. 2000). The inherent instability of foreign proteins expressed in a heterologous environment and their increased susceptibility to intracellular degradation systems are probably the most important factors responsible for the low accumulation of certain recombinant proteins in plants. In particular, the developmental stage of the plant may have a significant impact on the recombinant protein yield in leaf-based production systems, as massive proteolytic degradation occurs during tissue senescence (Stevens et al. 2000; Benchabane 123 Transgenic Res (2010) 19:291–298 et al. 2008). Gene silencing is another possible mechanism responsible for decreasing transgene expression levels during later stages of development or in subsequent generations, resulting in lower levels of recombinant protein accumulation (De Wilde et al. 2000). Many strategies have been employed to increase the concentration of recombinant proteins in plant tissues (reviewed by Streatfield 2007). However, the objective of the present study was to thoroughly investigate the temporal and spatial distribution of EPO production in planta, in order to determine the ideal harvesting time during the plant’s development to further maximize recombinant protein recovery. Methods, results and discussion The construct used to express human EPO in plants (SPTob
EPONat, GenBank Accession EU746475) has been described elsewhere (Conley et al. 2009). In summary, the EPO coding sequence was placed under the control of the constitutive cauliflower mosaic virus 35S promoter (Kay et al. 1987) and targeted to the endoplasmic reticulum (ER) using a tobacco secretory signal peptide and a KDEL ERretrieval motif (Gomord et al. 1997). The expression construct was then introduced into low-alkaloid tobacco (Nicotiana tabacum cv. 81V9) by Agrobacterium-mediated transformation according to the method of Horsch et al. (1985). The primary transformant (T0) with the highest concentration of EPO and exhibiting a single integration segregation ratio was self-fertilized to generate a homozygous plant line for EPO expression. To examine the amount of recombinant EPO and total soluble protein (TSP) produced in the leaves of transgenic plants during their life cycle, 12 seedlings from the homozygous (T2) EPO plant line were potted in compost and grown to maturity in a greenhouse that was maintained at 22°C with a 16h photoperiod. The experimental variation was limited by randomly relocating the plants around the greenhouse twice a week to control for any potential local environmental effects that may influence the growth conditions of the plants. After the seedlings were transplanted into the greenhouse, the plants grew homogeneously throughout their development with their height at flowering being 120.1 ± 2.3 cm Transgenic Res (2010) 19:291–298 293 Fig. 1 Temporal and spatial expression of erythropoietin (EPO) and total soluble protein (TSP) in transgenic tobacco plants. a A graphic representation of a tobacco plant indicating the respective position and the relative size of each leaf. Every other leaf of the tobacco plant was evaluated, with the leaves numbered from the bottom (leaf 1) upwards towards the youngest leaves (leaf 17). b Size distribution of the plant leaves during their initial and final sampling for the time course analysis. Each column represents the mean value (n = 12) and the standard deviation is represented with error bars. c–e From the leaves of 12 homozygous (T2) transgenic sibling plants, samples were taken from every other leaf at 10 days time intervals for the lifetime of the plants. The average accumulation (n = 12) of plant recombinant EPO (c) in each leaf sample was determined by enzyme-linked immunosorbant assay and the TSP concentration (d) was measured according to the Bradford method. e The amount of EPO expressed per amount of TSP in all sampled leaves over the lifetime of the plant. On average, the transgenic plants began flowering 38 days after the initial sampling of the plants (i.e. time = 0 days). The initial sampling took place 15 days after the seedlings were transplanted into the greenhouse. By day 70, leaf 1 had senesced and decayed and was not available for further analysis. 100 ng/mg TSP is equivalent to 0.01% of TSP and the time to flowering being 53.1 ± 1.4 days. For the purposes of this paper, flowering was defined as the point in time where the first emergence of a flower petal was visible. The transgenic plants were moderately fertilized biweekly allowing the plants to naturally age, which could be observed by progressive yellowing of the leaves, followed by necrosis and death of the oldest tissues (Bleecker and Patterson 1997). Every other leaf of the tobacco plants was analyzed, with the leaves numbered from the bottom (leaf 1, oldest) upwards to the top (leaf 17, youngest) (Fig. 1a). The size distribution of the plant leaves at the initial and final sampling time point are illustrated in Fig. 1b. The initial sampling of leaf 1 (i.e. time = 0 days) occurred 15 days after transplantation of the seedlings into the greenhouse. For practical purposes, the initial sampling of every other 123 294 subsequent leaf commenced 5 days from the previously sampled leaf. After the initial sampling, each particular leaf was successively sampled at 10 days time intervals for the lifetime of the plant. From the leaves of the 12 independent transgenic plants, protein extracts were prepared and the concentration of EPO was determined by enzyme-linked immunosorbant assay (ELISA) as described by Conley et al. (2009). The amount of EPO (Fig. 1c) and TSP (Fig. 1d) decreased significantly in every leaf of the plant during its maturation, although at differing rates. Over the lifetime of the plant, the EPO and TSP concentration decreased to 15 and 22% of their original value for leaf 1, while only decreasing to 60 and 86% of their original value for leaf 17. These parallel trends represent a fourfold faster rate of decreasing EPO and TSP concentration for leaf 1 compared to leaf 17. On average, the accumulation of EPO and TSP decreased to 38 and 48% of their original value for all plant leaves. For every leaf, the EPO and TSP concentration declined quickly during the short period of time following the initial sampling and then leveled off for the remainder of the plant’s life. A large proportion of the total decrease (i.e. 71 and 75% on average) in EPO and TSP concentration occurring for all leaves over the lifetime of the plant was attributed to the first 20 days after their initial sampling. At any stage of the plant’s life, a higher concentration of EPO and TSP was obtained in the upper, younger leaves compared to the lower, older leaves. For example, leaf 17 produced seven times more EPO and TSP than leaf 1 at day 40. Therefore, the upper, younger leaves produced significantly more EPO than the lower, older leaves at any particular moment of the plant’s development, but they also produced significantly more TSP as well. As a result, the amount of EPO produced in the plant leaves relative to the TSP content remained essentially unchanged throughout the life of the plant (Fig. 1e), which has been observed for other recombinant proteins expressed in plant systems (Stevens et al. 2000; Molina et al. 2004; Farran et al. 2008). For the vast majority of leaves across numerous time points, the recombinant protein accumulated in the narrow range of 60–100 ng of EPO per mg of TSP. Upon closer inspection, the production rate of EPO relative to TSP generally declined over the lifetime of the plant, 123 Transgenic Res (2010) 19:291–298 suggesting that the concentration of EPO relative to TSP decreases slightly as the leaves mature (Fig. 1e). The onset of flowering had no apparent impact on the production rate of EPO or TSP for the transgenic plants. The accumulation of recombinant protein was dependent on the leaf’s age and its position on the plant. In the case of a low-accumulating, unstable protein, such as EPO, the highest level of accumulation was achieved in the fast-growing, young leaves, whereas the older leaves contained very low concentrations of the target protein. This tendency has been reported with other proteins particularly susceptible to proteolytic degradation when expressed in plant systems (Birch-Machin et al. 2004; Molina et al. 2004; Herz et al. 2005; Farran et al. 2008). However, in the case of more stable proteins, expressed in chloroplasts, the highest yields are often obtained in mature leaves of fully grown plants (Staub et al. 2000; Maclean et al. 2007), since the recombinant proteins can occasionally form stable inclusion bodies (Fernandez-San Millan et al. 2003), aggregates or crystalline structures (De Cosa et al. 2001) that are protected from degradation. As the transgenic plants matured, a general trend of decreasing TSP content from young to old leaves was observed as a result of senescence. The programmed nature of leaf senescence ensures that essential nutrients are salvaged from older tissues and remobilized for transport to younger tissues, in order to complete the development of the plant, which functions to favour reproduction at the expense of the soma (Bleecker and Patterson 1997; Stevens et al. 2000). Importantly, massive proteolytic degradation occurs during plant senescence, which could hinder the utility of leaf-based systems for recombinant protein production because heterologous proteins tend to be preferentially degraded since they are regarded as foreign by the cell (Enfors 1992; Benchabane et al. 2008). Therefore, it is important to harvest the leaves at the appropriate moment in time to optimize the final recombinant protein yield, since the rates of synthesis and degradation are different for all proteins over the lifetime of the transgenic plant (Stevens et al. 2000). Since recombinant protein production decreases as the tobacco leaves continue to grow and mature, we were also interested in determining the relationship between recombinant protein yield and leaf size. As a Transgenic Res (2010) 19:291–298 Fig. 2 Relationship between recombinant protein yield and leaf size throughout the plant’s life. a–c The concentration of EPO (a), the leaf area (b), and the amount of EPO per leaf (c) were determined for every leaf of the plant during their initial and final sampling. Empirical data was only collected for the odd-numbered leaves, so the even-numbered leaves were assumed to possess the average value of their two adjacent neighboring leaves to determine the complete plant-wide profile for EPO accumulation. Given that four 7.1 mm diameter leaf discs have a fresh weight (FW) of 32 ± 6 mg (n = 75, data not shown), 1 cm2 of leaf tissue weighs approximately 20 mg (FW) result, the data from Fig. 1c was alternatively represented in Fig. 2a, since each sample of protein extract was derived from a constant area of leaf material (i.e. four 7.1 mm diameter leaf discs per sample). The concentration of EPO per unit area is higher for each leaf when it is younger than when it is older, with a greater difference occurring for the bottommost leaves of the plant compared to the uppermost leaves during their sampling lifetime. From leaf 1 to leaf 17, EPO concentration per unit area increased only 38% for the initial sampling, but increased 380% for the final sampling. 295 The length and surface area of 50 variously-sized tobacco leaves were used to establish the regression between leaf length and area, resulting in a linear fit of the data [leaf area = 27.00 9 (leaf length) 458.09, (R2 = 0.95)] (data not shown). Given the actual lengths of the leaves at the initial and final sampling time point, the mathematical equation was used to calculate their corresponding leaf area (Fig. 2b). As the leaves developed, their surface area increased by approximately 80% over their sampling lifetime, on average. The EPO concentration and leaf length were only measured for the odd-numbered leaves, thus the even-numbered leaves were assumed to possess the average value of their two adjacent neighboring leaves in order to determine the entire plant-wide accumulation profile for EPO. The amount of EPO per leaf and its relative contribution to the total EPO yield per plant is demonstrated in Fig. 2c. On a per leaf basis, the bottommost leaves produced significantly more EPO when they were young even though they were much smaller than their fully-grown counterparts; however, this difference gradually diminishes as you progressed upwards from the lower leaves to the upper leaves of the plant. As a result, a total of 160 lg of EPO could be harvested from the plant if each leaf was harvested at its initial sampling point (assuming no change to the plant’s physiology as development continued), compared to 110 lg of EPO if the plant was harvested at a fully flowering stage. Although other groups have demonstrated that the large majority of a plant’s recombinant protein is present in the large, fully-expanded mature leaves of chloroplast-engineered tobacco (Fernandez-San Millan et al. 2003; Molina et al. 2004; Fernandez-San Millan et al. 2008), we have shown here that younger leaves contain the highest amount of ER-targeted EPO. This suggests a possible distinction between these two intracellular compartments for the longterm storage of recombinant proteins. Therefore, a strategy to obtain the highest possible yield of recombinant protein may be to harvest the tobacco leaves intermittently when they are relatively large and young while the plant continues to grow and produce new leaves. Moreover, the plants could be treated with additional fertilizer to reduce their rate of tissue senescence and presumably, their rate of recombinant protein degradation. Alternatively, inducible or developmentally-regulated promoters 123 296 could be used to restrict the expression of a heterologous protein in tobacco leaves, such that it accumulates for a brief period of time prior to harvest to limit the detrimental effects of long-term protein instability (Streatfield 2007). Transgene silencing can significantly reduce the accumulation of recombinant proteins in plants, acting as another potential phenomenon hampering the commercial exploitation of plants as protein bioreactors (De Neve et al. 1999; De Wilde et al. 2000; Alvarez et al. 2008). Thus, the transgenic plant line was also utilized to evaluate the stable expression of EPO through multiple subsequent generations. Seed was germinated on selection medium and ten transgenic sibling plants from each generation were transplanted into the greenhouse and grown for ELISA analysis. For each plant, the first four expanded leaves were sampled once they reached 25 cm in length and were used to represent the concentration of recombinant EPO in the whole plant. As shown in Fig. 3, transgene silencing was not observed as EPO accumulation remained stable throughout all generations of transgenic tobacco and reached comparable levels to the parental primary transformant (T0), which produced EPO at a concentration of 73 ng/mg TSP (data not shown). Thus, the concentration of EPO was no different in the hemizygous plants relative to the homozygotes. In addition, every generation of transgenic plants exhibited normal growth and morphology. Fig. 3 Accumulation of erythropoietin (EPO) in successive generations of transgenic tobacco plants. The concentration of plant recombinant EPO was measured by enzyme-linked immunosorbant assay from the first four expanded leaves of each transgenic plant. The data is presented as the mean ± standard deviation of ten transgenic sibling plants. TSP, total soluble protein 123 Transgenic Res (2010) 19:291–298 The generational stability of EPO expression was also examined for an additional 12 genetic variants of EPO, which have been described in Conley et al. (2009). Each homozygous transgenic line was chosen based on its ability to produce relatively high levels of recombinant EPO while segregating according to a single-locus Mendelian ratio. Single integrations were chosen to minimize the occurrence of transgene silencing since the presence of multiple transgene copies generally favours more efficient transgene silencing, although single-copy transgenes can be silenced (Elmayan and Vaucheret 1996; Jorgensen et al. 1996; De Wilde et al. 2000). Of the 12 unique transgenic plant lines, only a single line demonstrated a drastic reduction of EPO yield during subsequent generations, with essentially no accumulation of EPO observed in the plant after the T3 generation (data not shown), which was probably the result of transgene silencing. In summary, this study demonstrates that recombinant protein yield is highly influenced by the tobacco plant’s age. As the plants developed and the leaves grew larger, the accumulation of both EPO and TSP decreased significantly for each leaf, although the protein production profile was distinctively different for every leaf of the plant. Consequently, two major issues must be considered when determining the optimal harvesting time to maximize recombinant protein yield. First, young leaves produce considerably higher concentrations of recombinant protein than older leaves, but their levels of TSP are also much higher, which may complicate purification of the target protein. Second, young leaves are obviously smaller than their fully-grown, mature counterparts, limiting the potential biomass yield of the plants. Therefore, we suggest that young, fully expanded tobacco leaves should be periodically harvested as the plant grows, since the highest concentration of EPO can be obtained in the smallest amount of biomass. However, this strategy should be tested on a case-by-case basis, as it probably depends on the particular recombinant protein and production host. Furthermore, all leaves should be harvested prior to flowering to save production time and to prevent accidental transmission of transgenes into the environment. Acknowledgments The authors wish to gratefully thank Laura Slade and Linda Le for technical support and Alex Transgenic Res (2010) 19:291–298 Molnar for assistance with the preparation of the figures. Thanks to Dr. Jussi Joensuu and Alex Richman for critical comments on the manuscript and helpful discussions. 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