Plant Cell Rep (2014) 33:1801–1814 DOI 10.1007/s00299-014-1658-8

ORIGINAL PAPER

Improved expression of recombinant plant-made hEGF David Rhys Thomas • Amanda Maree Walmsley

Received: 7 June 2014 / Revised: 9 July 2014 / Accepted: 14 July 2014 / Published online: 22 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Abstract Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by *34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-

Communicated by Jim Register. D. R. Thomas (&)  A. M. Walmsley School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia e-mail: [email protected]

made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields. Keywords Human epidermal growth factor  Transient expression  Codon optimisation  Unfolded protein response  Nicotiana benthamiana  Sub-cellular targeting

Background Native human EGF (hEGF) is a 53 amino acid protein containing three disulphide bonds that promotes the proliferation and differentiation of epithelial cells (Jorissen 2003; Nanba et al. 2013). Based on its ability to increase the growth of epidermal cells, hEGF has been tested as a treatment for a variety of injuries (Berlanga-Acosta et al. 2009). Human epidermal growth factor is able to enhance the healing of burns, cuts, corneal damage, diabetic ulcers, and gastric ulcers (Andree et al. 1994; Yao and Eriksson 2000; Alemdaroglu et al. 2006). The application of hEGF either pre- or post- wounding can also mitigate and heal damage to internal organs caused by ischemia (Berlanga et al. 2002; Tomaszewska and Dembinski 2002). Further, there is evidence that hEGF can promote recovery from mucosal injuries resulting from radiotherapy (Lee et al. 2008; Oh et al. 2010; Wu et al. 2009). However, despite its potential widespread applications hEGF is only approved for the treatment of diabetic foot ulcers (Berlanga et al. 2013; Yera-Alos et al. 2013). One of the reasons for this limited use is the high cost of hEGF, and that it may require multiple doses at high

123

1802

concentrations (Wong et al. 2001). As hEGF is a small peptide it diffuses away from the site of application rapidly, and is rapidly eliminated from plasma (Kuo et al. 1992). hEGF is also degraded in chronic wounds and burns, due to high proteolytic activity (Tarnuzzer and Schultz 1996; Yang et al. 2005). For the large-scale production of inexpensive recombinant proteins, Escherichia coli is the dominant production platform. However, disulphide bond containing proteins, such as hEGF, can be difficult for E. coli to produce. For example, the production of recombinant tissue plasminogen activator, which also contains multiple disulphide bonds, was found to be more expensive to produce in E. coli than in mammalian cell culture due to the need to correctly refold the recombinant protein (Datar et al. 1993). As alternative protein production platforms, plants are able to produce natively folded proteins from relatively inexpensive inputs (Rybicki 2009). Human epidermal growth factor has been expressed in various plant systems, including Solanum tuberosum, Nicotiana benthamiana, Nicotiana tabacum, Oryza sativa, and hairy root cultures (Bai et al. 2007; Higo et al. 1993; Parsons et al. 2010; Salmanian et al. 1996; Torrent and Llompart 2009; Wirth et al. 2004, 2006; Wu et al. 2014). In these previous studies hEGF was targeted to multiple subcellular compartments with varying degrees of success. When targeted to the chloroplast, hEGF was only detectable when fused to the first 186 amino acids of bacterial bglucuronidase, while targeting hEGF to the cytoplasm reached only 0.001 % of total soluble protein (TSP) (Wirth et al. 2004, 2006). When expressed with a Zera tag to induce protein body formation, hEGF accumulation was increased 100-fold compared to the cytoplasm (Torrent and Llompart 2009), while targeting to the apoplast resulted in a 1,000-fold increase over the cytoplasm (Wirth et al. 2004). Bai et al. (2007) targeted hEGF to the ER and recorded yields of up to 0.1 % of TSP, similar to that found in the apoplast by Wirth et al. (2004) (Bai et al. 2007). The addition of codon optimization and matrix attachment regions improved the accumulation of ER targeted hEGF to 0.3 % of TSP (Bai et al. 2007). In a recent study, Wu et al. (2014) were able to produce hEGF at 1.8 % of total soluble protein when targeted to the ER of stably transformed rice cells in culture, and up to 7.4 % of TSP in stably transformed rice seedling using three copies of codon optimised ER targeted hEGF; the highest yield of hEGF as a percent of TSP in plants to date (Wu et al. 2014). In this study, hEGF was transiently expressed in N. benthamiana and targeted to the vacuole, ER and apoplast. In addition, the accumulation of hEGF was also improved by codon optimisation, co-expression of the P19 silencing inhibitor, and optimising plant growth stage. Despite significant improvements in yield through these methods, the

123

Plant Cell Rep (2014) 33:1801–1814

highest yield achieved in this study of 0.269 mg/g of fresh weight was lower than previous studies. However, when measured as a percent of TSP hEGF accumulated to 6.24 % of TSP; approximately 20-fold higher than previously recorded in Nicotiana species in the literature. Further, the highest yielding samples produced hEGF at 9.05 % TSP, matching the concentrations reached by Wu et al. (2014). The recombinant hEGF increased the cellular proliferation of mouse keratinocyte cells, and its bioactivity was found to match that of commercially available E. coli derived hEGF over a range of concentrations. This study not only shows that plants are able to produce recombinant hEGF with the same dose-dependant activity as commercial E. coli derived hEGF without required additional in vitro refolding, but also that the use of codon optimization and vacuole targeting in a transient expression system can lead to greatly improved yields of hEGF. It was also found that hEGF accumulation in the ER appeared to be limited by endogenous mechanisms, which coincided with a decrease in total protein content in leaf tissue. Taken together, these results strongly suggest that plants can provide a feasible production platform for the commercial production of recombinant hEGF.

Results Creation of genetic constructs The plasmid p35(L)APEGF contained the sequence for the 53 amino acid hEGF peptide fused to a 50 signal peptide that facilitates its delivery into the secretory pathway, and has been shown to produce active hEGF (Wirth et al. 2004). To target the hEGF to additional sub-cellular compartments a KDEL signal peptide was used to maintain the hEGF in the ER, while the 30 signal peptide from tobacco chitinase A was used to target the vacuole. This resulted in the production of three separate constructs to be tested (Fig. 1). Comparison between sub-cellular compartments In order to determine the optimum sub-cellular location for hEGF accumulation, leaves were infiltrated with constructs targeting the apoplast, ER and vacuole simultaneously. Preliminary tests found that expression was generally highest 6–8 days post-infiltration, therefore, samples were harvested depending on when each construct peaked; at either 6 or 8 DPI. The concentration of hEGF recovered from vacuole-targeted hEGF (688.6 ng/ml) was significantly higher than from samples targeting hEGF to either the ER or apoplast (499.6 and 435.2 ng/ml, Fig. 2). However, when expression of hEGF was measured as a percent

Plant Cell Rep (2014) 33:1801–1814

1803

were changed, spread over 32 of the 53 codons. This resulted in an increase in overall GC content from 50.4 to 52.7 %, and an increase in third position GC from 56.4 to 80 %. Effect of codon optimisation on the accumulation of epidermal growth factor

Fig. 1 Expression cassettes to target recombinant hEGF to the apoplast [p35(L)APEGF], ER [p35(L)APEGF.ER], or vacuole [p35(L)APEGF.VT]. 2 9 35S denotes dual 35S promoters from CMV, X the enhancer region from tobacco mosaic virus, hEGF the coding region for mature human epidermal growth factor, AP the secretory signal peptide from N. tabacum osmotin, KDEL the KDEL ER retention signal, VT the vacuole targeting region from tobacco Chitinase A and TNos the terminator region from the Agrobacterium tumefaciens nopthaline synthase gene

When hEGF was targeted to the vacuole, the use of the codon optimised sequence significantly increased the yield of hEGF by *34 %, from an average of 623.7 to 837.1 ng/ ml (Fig. 3a). As a percentage of TSP, the codon-optimised sequence also performed better, accumulating hEGF to 0.159 % compared to 0.135 % in the non-optimised version. In contrast, when targeting the ER, the use of a codonoptimised sequence did not increase the expression of hEGF. The non-optimised and optimised constructs accumulated hEGF to 423.6 and 375.7 ng/ml at 0.070 and 0.067 % of TSP, respectively (Fig. 3b). This lack of effect suggests that there is a physiological limit on the amount of hEGF that can be retained in the ER, and that the use of codon optimisation is not sufficient to overcome this. Western blot

Fig. 2 Comparison of accumulation of hEGF when targeted to either the vacuole (VT), endoplasmic reticulum (ER) or apoplast (AP) (n = 18). Values are mean ± SE. *P \ 0.05, **P \ 0.01, ***P \ 0.001

of TSP, the ER had the highest average concentration of 0.175 % TSP, followed by the vacuole and apoplast (0.1351 and 0.0927 %, respectively). This discrepancy was caused by a decrease in the total soluble protein extracted from samples expressing ER targeted hEGF by approximately 35 % when compared to either vacuole or apoplast targeting constructs (P \ 0.001 for both). Design and creation of codon optimised constructs Based on the previous results, constructs codon optimised hEGF sequence were would target hEGF to either the vacuole designing the optimised hEGF sequence,

containing the created which or the ER. In 32 nucleotides

To confirm the size and identity of the plant-made hEGF, an SDS-PAGE and western blot was performed under reducing and denaturing conditions. The positive control was E. coli derived hEGF with a molecular weight of 6.2 kDa (Fig. 4). There was no observable difference in the size of the plant- or E. coli-derived hEGF. This suggests that there are no post-translational modifications, such as glycosylation, made to the hEGF in the plant cell, regardless of it sub-cellular targeting. Although various signal peptides were added to the hEGF constructs, both the N-terminal secretory signal peptide and C-terminal vacuole targeting peptide are removed during routine post-translational processing of the preprotein (Melchers et al. 1993; Neuhaus et al. 1991). While the KDEL retention signal is not removed, the resulting 0.5 kDa size shift would be below the detectable resolution of this experiment, and so the band sizes are consistent with expectations. Effect of P19 silencing suppressor on vacuolar accumulation of hEGF To determine whether posttranscriptional gene silencing (PTGS) influences the accumulation of hEGF, OPT.VT, which has been determined to be the highest expressing hEGF construct, was infiltrated either alone or with a construct expressing P19. P19 has been found to improve the transient expression of recombinant proteins by

123

1804

Plant Cell Rep (2014) 33:1801–1814

Fig. 3 Comparison of hEGF accumulation in the ER using either the optimised or nonoptimised sequence (a) or in the vacuole using either the optimised or non-optimised sequence (b), as a percent of TSP or as the concentration of hEGF in the extract. Values are mean ± SE (n = 20). *P \ 0.05, **P \ 0.01, ***P \ 0.001

expressing day for OPT.VT alone) as a raw concentration or as a percentage of TSP, respectively. The highest concentrations of hEGF were recovered 13 days after infiltration despite the amount of TSP recovered from samples falling by about 40 % from when the same leaves were sampled after 7 days. The optimal harvest date therefore is a balance between the accumulation of hEGF and the degradation of total plant proteins. Optimising plant age Fig. 4 Reduced and denatured western blot comparing E. coli derived recombinant hEGF (positive) to extracts from plants transformed with a YFP construct (negative) or hEGF containing constructs (as labelled)

sequestering short, double stranded RNAs that would otherwise target highly expressed mRNAs (i.e., transgenes) for degradation (Silhavy et al. 2002; Voinnet et al. 2003). The amount of hEGF recovered from samples co-infiltrated with P19 was significantly higher than samples infiltrated with OPT.VT alone at 7, 10, 13 and 16 DPI, both as a raw concentration and as a percent of TSP (Fig. 5b, c). At the same time, the amount of TSP recovered from samples did not differ between tissue infiltrated with or without P19 (Fig. 5a). The accumulation of hEGF without P19 was highest at seven DPI, when it reached an average of 4.96 lg hEGF/ml. In contrast, when hEGF expression without P19 was measured as a percent of TSP, the highest concentration of 0.87 % was after 16 days. This was due to a large decrease in the TSP recovered from samples 16 DPI as a result of increased tissue necrosis. When supplemented with the P19 silencing suppressor, the accumulation of hEGF increased up to 13 days, when samples produced an average hEGF concentration of 15.8 lg/ml at 2.54 % of TSP. This was a 3.2 and 3.9 fold increase over OPT.VT values at seven DPI (the highest

123

To further increase the amount of hEGF produced, plants were grown and infiltrated at ages that are commonly used for large scale production; 4, 5 and 6 weeks old (Buyel and Fischer 2012; Lai and Chen 2011). These plants were infiltrated with OPT.VT and P19, and harvested at 13 DPI, as these had given the highest yields so far. At the time of infiltration there was a large difference in the size of the plants, with the 6-week-old plants being much taller than the 4- and 5-week-old plants (Fig. 6a). By the time of harvest (13 DPI), however, the difference in size was negligible (Fig. 6b). The increase in size of the 5-week-old plants was largely due to an increase in stem length and leaf size, with a small amount of new leaf growth. In contrast, there was a great deal of new leaf growth from the 4-week-old plants, as evidenced by the infiltrated regions being very low on the 4-week-old plant due to new leaves growing above it (Fig. 6b, red circles). The 5-week-old plants were also just beginning to flower by the harvest day, while the 6-week-old plants contained multiple well-established flowers. The 5-week-old plants produced the greatest yield of hEGF, with an average of 0.269 mg/g of leaf tissue at 6.24 % of TSP (Fig. 7). There was no significant difference between the 4- and 6- week old plants, which produced an average of 0.196 and 0.180 mg of hEGF/g at 5.09 and 3.99 % of TSP, respectively.

Plant Cell Rep (2014) 33:1801–1814

1805

Fig. 5 OPT.VT was infiltrated with (?P19) or without (-) the P19 silencing suppressor and harvested 7, 10, 13 and 16 DPI. a is the total soluble protein extracted from samples, b the raw amount of hEGF extracted and c the amount of hEGF as a percentage of the total soluble protein. Values are mean ± SE (n = 20). *P \ 0.05, **P \ 0.01, ***P \ 0.001

Biological activity of plant-made hEGF To confirm that the recombinant hEGF is biologically active, the plant-made protein was tested in an MTT cell proliferation assay with 3T3-J2 cells (Fig. 8). The MTT assay is a colorimetric assay that measures cell numbers via the rate at which MTT is converted to insoluble purple formazan crystals by cellular enzymes. Cells were grown for 48 h in the presence of either plant-made (OPT.VT) or commercial E. coli derived hEGF, or a negative plant control. Cell growth was standardised to a negative control containing only cells and growth media, and were adjusted with negative plant extract so that each sample contained the same amount of total soluble proteins. When added to mouse keratinocytes, plant-made hEGF had a similar effect on cell proliferation as commercial

E. coli derived hEGF over a range of concentrations from 0.01 to 10 ng/ml (P [ 0.05). Both recombinant hEGF proteins increased cell growth up to 5 ng/ml, but had a decreased influence at a higher concentration. Both produced concentrations of cells significantly higher than the plant control when added at 5 ng/ml, and both had a similar maximal effect on cell growth (21 and 27 % more cells from plant and E. coli produced hEGF, respectively).

Discussion In this report, the transient expression of hEGF in N. benthamiana was enhanced using a variety of methods, including sub-cellular targeting, codon optimisation and the use of a silencing inhibitor. While the increase in hEGF

123

1806

Plant Cell Rep (2014) 33:1801–1814

Fig. 6 Representative samples of 4-, 5- and 6-week-old plants (left to right) before (a) and 13 days after (b) infiltration. Red circles in b show the location of an infiltrated leaf

Fig. 7 Accumulation of hEGF in 4-, 5- and 6-week-old plants co-infiltrated with OPT.VT and the P19 silencing suppressor. Results compared as mg of hEGF per gramme of leaf tissue (a) or as a percent of total soluble proteins (b). Values are mean ± SE (n = 20). *P \ 0.05, **P \ 0.01, ***P \ 0.001

production achieved through any single method was relatively modest, through the combined application of multiple enhancements, an overall improvement of approximately tenfold was achieved compared to the original apoplast targeted construct. This resulted in an average concentration of hEGF of 6.24 % of TSP (range of 3.88–9.05 %), which is approximately 20-fold above the highest recorded

123

concentration as a percentage of TSP in Nicotiana tabacum of 0.3 %, and similar to that achieved in rice seedlings (Bai et al. 2007; Wu et al. 2014). The hEGF produced was the expected size when separated by SDS-PAGE. Further, the plant-made hEGF was found to have biological activity equivalent to that of commercially available hEGF, without needing any additional refolding steps.

Plant Cell Rep (2014) 33:1801–1814

Fig. 8 Increase in 3t3-j2 cell number compared to control of cell treated with plant-made hEGF (green), E. coli made hEGF (blue), or negative plant extract from cells expressing YFP (grey). Values are mean ± SE (n = 12). Asterisks denote a significant difference in cell number from the plant control, and follow the same colour code at the graph *P \ 0.05, **P \ 0.01, ***P \ 0.001

Effect of sub-cellular targeting on hEGF accumulation When comparing sub-cellular compartments for hEGF accumulation, the vacuole produced concentrations approximately 38 % higher than in the ER and 58 % higher than in the apoplast. Interestingly, while there was no significant difference in the raw amount of hEGF accumulated in the ER or apoplast, when accumulation was measured as a percent of TSP, the ER was the best performer, with concentrations significantly higher than in the apoplast, and similar to that of the vacuole. This disparity was caused by a decrease in TSP associated with the accumulation of hEGF in the ER. The effect of codon optimisation on hEGF expression When compared to the non-optimised constructs, the codon-optimised sequence increased hEGF accumulation by 34 % when targeted to the vacuole. In contrast, there was no significant difference detected between the constructs targeted to the ER. As the accumulation of hEGF in the ER had a negative affect on the protein content of the cell, it is likely that this represents a physiological limitation that could not be overcome by the codon-optimised sequence. It is possible that this limitation could be caused by the influx of hEGF proteins into the ER saturating protein folding chaperones, activating the unfolded protein response (UPR) (Kudo et al. 2013; Liu and Howell 2010; De Wilde et al. 2013). The effect of the P19 silencing suppressor on hEGF expression The use of the P19 silencing suppressor significantly increased hEGF accumulation in the vacuole at 7, 10, 13

1807

and 16 DPI. The greatest yield of hEGF was achieved after 13 days with P19, at which point the average concentration of hEGF was 3.2-fold higher than the highest expression from OPT.VT alone, which occurred when harvested at 7 DPI. This increase is smaller than some recorded in the literature. For example, the expression of yellow fluorescent protein (YFP) and the antibody trastuzumab were increased *38- and 15-fold, respectively. These proteins were expressed transiently in N. benthamiana from constructs containing the same promoter and terminator, but different enhancer sequence to this study (Garabagi et al. 2012; Voinnet et al. 2003). However, the results in this study are consistent with results seen for the transient expression of the HIV-1 Nef protein in N. benthamiana. This protein was expressed from a construct containing the same promoter, terminator and enhancer region as this study, and only realised a threefold increase with the use of the P19 silencing inhibitor (Lombardi et al. 2009). This suggests that, while still valuable, the effect of P19 is heavily influenced by the choice of untranslated regions controlling the gene of interest. Leaf tissue had begun to bleach and small patches were necrotic 13 days after infiltration, while by day 16 much of the leaf tissue was necrotic. This is reflected in a sharp drop in the amount of TSP recovered from samples between day 10 and 13, with a further small decrease by day 16. Harvesting leaves that have been co-infiltrated with P19 at 13 DPI offered two advantages; the higher yields of hEGF produced, and a reduction in TSP by approximately 40 %. The reduction in TSP would be beneficial for downstream applications and purification, as lower amounts of contaminating protein could increase the efficiency of purification and reduce downstream costs. The effect of plant age on hEGF expression The use of P19 and OPT.VT was then carried forward to test expression in younger plants, which can offer greater yields of recombinant proteins. The concentrations of hEGF recovered from the 5-week-old plants were significantly higher than those from either 4- or 6-week-old plants. The differences in hEGF expression could be explained by the different resource allocation in the plants; the 4-week-old plants were growing new leaves while the 6-week-old plants were flowering, and both of these activities could divert resources away from protein accumulation. Another advantage of using 5-week-old plants is that due to the lack of new leaf growth, at harvest time the majority of leaves present would be producing recombinant hEGF. This would not be the case for the 4-week-old plants, as most of the leaves developed after infiltration, and so would not have been exposed to the Agrobacterium.

123

1808

Comparisons to the literature While the average concentration of hEGF from 5-week-old plants of 6.24 % of TSP was well above the previous recorded maximum in Nicotiana tabacum plants of 0.3 %, the average yield of 0.269 mg/g recovered in this study was well below the values recorded in other studies; Bai et al. (2007) reported an average yield of 0.31 mg/g fresh weight (individual values ranged from 0.1 to 0.4 mg/g) in N. tabacum, while Torrent and Llompart (2009) reported 0.5 mg/g, also in stably transformed N. tabacum. One of the reasons for the large difference in the %TSP concentration and raw concentration of hEGF in our data is that by the time of harvesting, the infiltration sites were becoming necrotic, and the amount of total protein had declined sharply, increasing the amount of hEGF as a proportion. However, there are potentially additional factors at play. Understandably different research groups not only measure protein concentration in different ways but at different stages in the life of a plant that has been grown under different conditions. Unfortunately, this affects key variables considerably and thereby makes direct comparison between studies difficult. By demonstration, in the present study, an average of 4.4 mg of protein was recovered per gramme of tissue harvested from the 5-week-old plants. This is relatively low, and so could have been a limiting factor on the amount of hEGF produced. Total protein values in the literature appear to be largely influenced by the growth temperature, with higher temperatures producing lower TSP. Using N. tabacum grown at 15 °C under high light conditions, Stevens et al. (2000) recovered approximately 28 mg of TSP per gramme of tissue from the top leaves, 15 mg/g from middle leaves, and 8 mg/g in low leaves. However, when grown at 25 °C in high light conditions, recovery dropped to approximately 24, 9 and 3 mg/g for high, middle and low leaves, respectively (Stevens et al. 2000). Similarly, Hassan et al. (2012) recovered 5–7 mg/g from N. tabacum grown with a 28/21 °C day/night temperature (Hassan et al. 2012). Fu et al. (2010) grew N. tabacum during summer, and, after considerable optimisation, were able to recover 12.85 mg of TSP per gramme of leaf tissue (Fu et al. 2010). Plesha et al. (2009) harvested N. benthamiana that was grown in a greenhouse where temperatures ranged up to 30 °C, and recovered 3–7 mg of TSP per gramme of tissue (Plesha et al. 2009). While plants in this study were grown in a greenhouse where air temperature was maintained at 18–23 °C, over summer (when the 4, 5 and 6-week-old plants were grown) the surface temperature of leaves may have been increased beyond this by an increase in sunlight. Anecdotally, an increased rate of water evaporation from trays was observed during this time compared to cooler months, and supports the notion that an

123

Plant Cell Rep (2014) 33:1801–1814

increase in plant temperature could have influenced TSP concentrations. The highest concentration of hEGF produced in plants to date is 0.5 mg/g. This was achieved using a Zera tag to accumulate hEGF in protein bodies in N. tabacum (Torrent and Llompart 2009). However, this was only 100-fold higher than the untargeted hEGF, while Wirth et al. (2004) increased hEGF expression 1,000-fold by targeting hEGF to the apoplast, which was the least effective sub-cellular target in the present study. It is likely, therefore, that the strong expression from Zera-tagged hEGF was enhanced by conditions conducive to recombinant expression. This is further evidenced by the fact that Torrent and Llompart (2009) were able to produce hEGF in the cytoplasm at 5,000 ng/g, while Wirth et al. 2004 could only produce hEGF in the cytoplasm to 1 ng/g (Torrent and Llompart 2009). In preliminary studies in our lab, the highest concentration of hEGF in the cytoplasm achieved was 354 ng/ g (data not shown), also well below that of Torrent and Llompart (2009). Stable and transient expression of hEGF Another likely cause of differences in results is the platforms used. The expressions of hEGF in the apoplast, ER, protein bodies, and cytoplasm have all used stably transformed N. tabacum (Bai et al. 2007; Higo et al. 1993; Torrent and Llompart 2009; Wirth et al. 2004). It is possible that the accumulation of recombinant proteins varies between species and infiltration methods. While transient expression is often cited as producing greater yields than stably transformed plants, this may not always be the case (Daniell et al. 2009; Medrano et al. 2009; Paul and Ma 2011; Plesha et al. 2009; Sharma and Sharma 2009). For example, the production of a mutant human glutamic acid decarboxylase (hGAD65) was found to be higher in stably transformed plants than when the same construct, or a MagnICON based construct, was transiently expressed in N. benthamiana (Avesani et al. 2013). The authors suggest the stable transformation can be advantageous due to the potential to screen and cross plants to produce hosts with genomic backgrounds suitable for recombinant protein production. They have also indicated that the protein may be activating a feedback loop, whereby the protein inhibits its own production, as they were also unable to increase yields by targeting it to the ER (Avesani et al. 2010). Other groups have also found that the ER targeted recombinant proteins can result in negative impacts on the plant such as a decrease in total soluble protein and increase in necrosis (Badri et al. 2009; Gils et al. 2005). Further, ER-targeted recombinant proteins have been shown to induce changes characteristic of the UPR,

Plant Cell Rep (2014) 33:1801–1814

including the up-regulation of BiP (Kudo et al. 2013; Nuttall et al. 2002; De Wilde et al. 2013). As disulphide bond formation is necessary for the correct folding of hEGF, access to folding chaperones in the ER presents a feasible bottleneck that could inhibit protein synthesis and/ or activate the UPR. The burst of hEGF produced from transient expression may overload the ER machinery, while stably transformed plants may produce hEGF at a slower rate that the ER is able to process. Broader comparisons between stable and transient systems are beyond the scope of this study. However, they have been previously discussed in depth by Daniell et al. (2009) and Tiwari et al. (2009), and should be referred to if of interest.

1809

Methods Mach1 (Invitrogen, USA) heat competent E. coli cells were used for plasmid amplification, and hEGF expression plasmids were inserted into LBA4404 Agrobacterium tumefaciens via electroporation for plant transformation. N. benthamiana plants were germinated in Jiffy-7 pellets (Jiffy International, Norway) for 2 weeks. The Jiffy pellet was then potted into a 3:1 mix of potting mix and perlite, and plants were fertilised once a week. Unless specified, plants were used when leaves were large enough to allow multiple infiltrations; usually between 8 and 12 weeks old. Creation of new plasmids

Conclusions This study has shown that the transient expression of hEGF in N. benthamiana is able to produce high yields of recombinant protein when combined with the P19 silencing inhibitor and codon-optimised constructs. Further, the recombinant hEGF produced in plants had the same dosedependant activity on the proliferation of mouse keratinocytes as commercially available E. coli derived hEGF. During expression optimisation, it was found that the yield of protein produced in infiltrated plants varies significantly if the age of the plants differ by as little as 1 week. It was also found that of the vacuole, apoplast and ER, targeting hEGF to the vacuole resulted in the highest yield. Targeting hEGF to the ER had a negative effect on the health of the leaf tissue, resulting in a decrease in the total protein content. This is likely due to an upper limit on the amount of hEGF present in the ER being reached, as using the codon-optimised sequence was unable to increase hEGF expression as it did when targeted to the vacuole. Based on the characteristics of hEGF and the response of the leaf tissue, we hypothesise that this limit is caused by the activation of the UPR as a result of the protein processing mechanisms in the ER becoming saturated. Plants provide an attractive system for the production of high value recombinant proteins. The high yields of a biologically active protein without the need for further downstream folding steps support the notion that N. benthamiana is a viable option for the commercial production of hEGF. While recombinant protein yields are consistently improving, it is becoming apparent that there are biological limitations in plant systems that may ultimately cap the amount of recombinant protein that can be produced. For the continued improvement of plant-based protein production systems, it will become increasingly important to modify the host plants in order to address the impact of these native limiting factors.

The plasmid p35(L)APEGF was provided by Dr. Sonia Wirth (2004). For the creation of constructs targeting hEGF to either the vacuole or ER, the fragment containing hEGF and the osmotin 50 signal peptide was amplified from p35(L)APEGF using primers NgoMIV (AAA GCC GGC GCG CAG TTC CCA CCA C) and Xho-EGF (AAA CTC GAG ATG GGC AAC TTG AGA TGT T) which added a XhoI and NgoMIV restriction sites at the 50 and 30 ends, respectively. This fragment was digested with XhoI and NgoMIV, and then ligated into both pIBT210.VT and pIBT210.ERR which contain either a vacuole targeting signal or KDEL ER retention signal, respectively (Fig. 9). From these vectors, fragments containing the hEGF with the 50 signal peptide and either the vacuole or KDEL signal peptides were amplified using the primers BamHI (AAA GGA TCC ATG GGC AAC TTG AGA TCT TC) and VT3 (CTA CAT AGT ATC AAC GAG AAG) or ERR3 (CTA AAG TTC ATC CTT TTC AGA T), respectively, before being digested with BamHI. The p35(L)APEGF plasmid was digested with BamHI and SmaI and the fragments containing hEGF with the additional 30 signal peptides were ligated into this backbone, creating the binary constructs p35(L)APEGF.VT and p35(L)APEGF.ER, which target hEGF to the vacuole or ER, respectively (Fig. 1). These constructs were transformed into Mach1 E. coli cells following manufacturer’s instructions. Plasmids were then purified and sequenced before being introduced into A. tumefaciens. The coding region of the EGF gene was optimised by using codons most commonly used in N. benthamiana (http://www.kazusa.or.jp/codon). When two or more codons for the same amino acid were used at similar rates, a preference was given to codons with high GC content, especially in the third position, as similar characteristics are found in highly expressed native proteins (Chiapello et al. 1998; Wang and Roossinck 2006). Along with the codon optimised hEGF, additional residues were included in the sequence to facilitate future cloning (Fig. 10). The optimised sequence with the 50 signal peptide was then synthesised by GeneArt (USA) in the construct p.optEGF.

123

1810

From p.optEGF, the region containing the 50 signal peptide and the codon optimised hEGF sequence was amplified with the primers NgoMIV-opt (AAA GCC GGC TCA CCT CAA CTC CCA CCA CT) and BamHI ? (AAA GGA TCC CGC CGA TCA CCA CCC G). This fragment was then digested with BamHI and NgoMIV and purified before being ligated into pIBT210.ER and pIBT210.VT that had been similarly digested, to create the plasmids pIBT210.OPT.ER and pIBT210.OPT.VT. Fragments containing the 50 signal peptide, hEGF gene and either the ER or vacuole signal peptide were amplified using primers BamHI and either OPT.ER (CTA AAG TTC ATC CTT CCT CAA CTC CCA CCA CTT CA) or VT3 using templates of pIBT210.OPT.ER or pIBT210.OPT.VT. These fragments were digested with BamHI and ligated into p35(L)APEGF backbone to give the plasmids p35(L).OPT.ER and p35(L)OPT.VT. Transient expression For plant infiltrations, A. tumefaciens cultures were seeded fresh from a glycerol stock, and grown in YM media supplemented with 50 mg/ml rifampicin. Cultures were grown at 28 °C shaking at 175 rpm until cultures reached an OD of *1. Cells were then harvested by centrifuging at 3,000g for 10 min and resuspended to an OD of 0.5–0.6 in 10 mM MES and 200 lM acetosyringone in distilled water. Infiltrations were performed by gently nicking the underside of N. benthamiana leaves with a needle and using a 1 ml syringe to force the A. tumefaciens into the leaf through the wound. Sampling and protein extraction Sampling was performed by pressing a 2 cm round metal ring through the leaf of interest. The circle of tissue was transferred to a 2 ml centrifuge tube with 500 ll of extraction buffer (20 % glycerol, 0.05 % tween-20 in PBS (8 g/l NaCl, 0.05 g/l KCl, 7.2 g/l Na2HPO4, 1.2 g/l KH2HPO4) with 19 EDTA free protease inhibitor (Roche, Switzerland) and stored at -20 °C. Samples were disrupted by adding two 5 mm tungsten beads and shaking in a tissuelyser for 90 s at 24 Hz. The beads were then removed and the samples spun at 14,000 g for 60 min. The supernatant was transferred to fresh tubes and stored at 20 °C for use.

Plant Cell Rep (2014) 33:1801–1814

Systems, USA) diluted to 2.5 lg/ml in PBS and incubated overnight at 4 °C. Plates were then washed three times with 0.005 % PBST (PBS with 0.05 ml/l tween-20), and blocked for 2 h with 200 ll of 5 % skim milk in PBS at 37 °C. Plates were washed and 100 ll of samples was added to wells. As a standard, E. coli derived recombinant hEGF (liquid rhEGF, Life Technologies) was used. Samples were incubated at 37 °C for 1 h, and then plates were washed with 0.005 % PBST. 50 ll of polyclonal rabbit anti-hEGF antibody (Life Technologies) diluted to 2 lg/ml with 1 % skim milk in PBS was added to each well, and plates were incubated for 1 h at 37 °C then washed three times. 50 ll of goat anti-rabbit HRP (Pierce Antibodies, USA) diluted to 133 lg/ml in 1 % skim milk in PBS buffer was then added to each well and incubated at 37 °C for 1 h. The plate was then washed four times, and 50 ll of room temperature TMB peroxidase (Bio-Rad, USA) was added to the wells. The colour was left to develop for 10–15 min, and the reaction stopped by the addition of 50 ll 1 N H2SO4. Plates were then read at 450 nm using a Multiskan Ascent plate reader (ThermoFisher Scientific, USA). A standard curve of commercial hEGF was derived from serial dilutions of the positive control, and this was used to infer the concentration of hEGF in the other samples. Only OD values that fell within the linear section of the curve were used, and only if the R2 value was above 0.95. Results were only considered real (i.e., not background noise) if the OD of the sample was greater than three times the OD of a negative control. Bradford’s assay Measures of total soluble protein (TSP) were carried out on each sample by Bradford’s assay. A standard of bovine serum albumin (BSA) (Sigma-Aldrich, USA) ranging from 0.5 to 0.05 mg/ml was used. 10 ll of samples were added in duplicate to a 96 well U-bottomed plate (ThermoFisher Scientific), and 200 ll of Bradford reagent (Bio-Rad) was added to each sample. The reaction was left to develop for 5 min, and then plates were read at 595 nm. The BSA standard curve was used to infer the concentration of TSP in samples, but was not used if the BSA samples’ readings were not linear or if the R2 value was below 0.95. OD values of samples were only used if they fell within the linear range of the standard curve. SDS page and western blot

ELISA ELISAs were used to quantify the amount of hEGF in samples. 96 well plates (CostarÒ, USA) were coated with 50 ll mouse monoclonal anti-EGF antibody (R&D

123

Samples were mixed with loading dye (69 concentration: 375 mM Tris, 10 % SDS, 50 % glycerol and bromophenol blue to colour, in water) to a final concentration of 29 and 2-mercaptoethanol to a final

Plant Cell Rep (2014) 33:1801–1814

1811

concentration of 10 %. Samples were then heated at 95 °C in a heating block for 5 min. Samples were run on precast polyacrylamide gels (AnyKd, bio-rad) in running buffer (10 % Tris:glycine buffer (25 mM Tris, 192 mM glycine), 0.1 % SDS) alongside a standard (Dual Xtra, Bio-Rad). The gel was then equilibrated in transfer buffer (10 % Tris:glycine buffer, 20 % methanol) for 10 min. Protein samples where then transferred to Immobilon-P PVDF membrane(EMD Millipore) in transfer buffer for 45 min at 75 V and 4 °C. After the protein had been transferred, the membrane was removed from the cassette and placed in a pre-wetted snapID cassette (EMD Millipore, Germany). For the western blot, a blocking buffer was prepared by passing 0.1 % skim milk in PBS with 0.05 % tween-20 through Whatman grade 1 filter paper, and probing was performed according to manufacturer’s instruction. As a primary antibody, polyclonal anti-hEGF antibody diluted to 1 lg/ml (Abcam, England) in blocking buffer was used, while goat antirabbit IgG secondary antibody (0.02 lg/ml) in blocking buffer was used as secondary. The probed membrane was then transferred to a container where it was incubated with 1 ml of ECL Prime western blot detecting reagent (GE Healthcare, England) for 1 min. Kodak Biomax lightFilm (Eastman Kodak, USA) was then exposed to the membrane in a darkroom for 5 min, and developed using a CP1000 X-ray developer (Agfa-Gevaert, Belgium).

To determine the optimal age of plants for the expression of hEGF, 20 seeds were sowed into Jiffy pots every week for 3 weeks. After 2 weeks the Jiffies were planted into potting mix. When the groups were 4, 5 and 6 weeks old the two uppermost leaves with diameters greater than 5 cm were co-infiltrated with OPT.VT and P19 constructs as described above.

Experimental design

MTT assay of cell growth

For the comparison of two or more constructs, 15–20 leaves were infiltrated with all constructs being tested in discrete patches, being careful to ensure infiltrated regions stayed separate. The infiltration positions of each construct were rotated around the leaf to prevent positional affects influencing expression. Samples were taken as previously described. To test the effect of the tomato bushy stunt virus P19 silencing protein on hEGF expression, leaves were infiltrated with a hEGF construct on one side of the leaf midrib and the hEGF construct co-infiltrated with a construct containing the P19 silencing inhibitor under the control of dual 35S promoters on the other side. Plants were sampled either at 7 and 13 days post infiltration (DPI), or at 10 and 16 DPI. The experiment was separated like this because leaves were not large enough for more than four samples each. For infiltration, P19 was prepared in the same manner as the hEGF constructs, but was resuspended to a final OD of 1 in the same medium as the hEGF constructs (the final OD of the infiltration buffer would be 1.5–1.6).

To perform an MTT assay, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, Life Technologies] was dissolved in PBS to 5 mg/ml. Cells were detached from the flask by trypsin digestion and resuspended in DMEM-5 (5 % bovine serum in DMEM with 50 mg/l gentamicin). Cell counts were performed via haemocytometer, and cells were diluted in DMEM-5 to reach a final concentration of 50,000 cells/ml. 200 ll of this was added to the wells of 96 well cell culture plates (BD Bioscience, USA), excluding the outer rows, which were filled with PBS to prevent edge effects. The cells were left to adhere for 24 h in the incubator, before the medium was aspirated off and replaced with 200 ll of DMEM-5 containing the treatments to be tested. Each treatment was added to plates in at least triplicate, and each plate contained a blank control of cells with DMEM-5 alone, which was used to standardise results across multiple plates. After the addition of DMEM-5, plates were placed back into the incubator. After 48 h, 20 ll of MTT solution was added to each well, and plates were left another 2 h in the incubator to develop

Measuring hEGF accumulation For the analysis of hEGF expression, results were compared as both the crude concentration of hEGF in the extraction buffer (which approximately translates to hEGF/ 6.28 cm2 of leaf tissue), and as a percent of total soluble protein (TSP) extracted from samples. 3t3-J2 cells Mouse keratinocytes (3T3-J2 cells, kindly supplied by Dr. Shiva Akbarzadeh [Alfred Hospital, Vic, Australia]) were maintained in DMEM-10 (Dulbecco’s Modified Eagle Medium supplemented with 10 % bovine calf serum (New Zealand origin) and 50 mg/l of gentamicin (Life Technologies)) and kept in an incubator at 37 °C with 5 % CO2 concentration. Cells were passaged when they reached *80 % confluency, and were not used after 12 passages.

123

1812

Plant Cell Rep (2014) 33:1801–1814

colour. After this time, the medium was carefully aspirated from each well, being careful not to disturb the precipitate, and wells were filled with 200 ll of DMSO. Plates were placed on at shaker at 37 °C and 100 rpm for 15 min for the MTT crystals to dissolve. After this, the OD of the plates was read on a plate reader, with the OD at 630 nm being subtracted from the OD at 570 nm to control for non-specific absorbance. For each well, the OD570–630 was standardised to the average of the blank control wells. Analysis For the comparison of multiple groups, ANOVA tests with Bonferroni correction for multiple comparisons were employed, while student’s t tests were used when comparing two groups. Paired tests were employed when samples being compared were taken from the same leaf. Conflict of interest of interest.

The authors declare that they have no conflict

Appendices See Figs. 9 and 10. Fig. 9 Plasmid map of pIBT210.VT and pIBT210.ERR

Fig. 10 Comparison of the hEGF sequence acquired from Dr. Wirth (original version) and the codon optimised version from this study (modified version). Changes to the nucleotide sequence are underlined. The nucleotide sequence of the hEGF gene and (a) and the amino acid sequence of the hEGF gene with the 50 signal peptide (b) are shown. In B, the grey box represents the signal peptide, and the white box the hEGF sequence

123

Plant Cell Rep (2014) 33:1801–1814

References Alemdaroglu C, Degim Z, Celebi N et al (2006) An investigation on burn wound healing in rats with chitosan gel formulation containing epidermal growth factor. Burns 32:319–327 Andree C, Swain WF, Page CP et al (1994) In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair. Proc Natl Acad Sci 91:12188–12192. doi:10. 1073/pnas.91.25.12188 Avesani L, Vitale A, Pedrazzini E et al (2010) Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant. Plant Biotechnol J 8:862–872. doi:10.1111/j.1467-7652.2010. 00514.x Avesani L, Merlin M, Gecchele E et al (2013) Comparative analysis of different biofactories for the production of a major diabetes autoantigen. Transgenic Res. doi:10.1007/s11248-013-9749-9 Badri MA, Rivard D, Coenen K, Michaud D (2009) Unintended molecular interactions in transgenic plants expressing clinically useful proteins: the case of bovine aprotinin traveling the potato leaf cell secretory pathway. Proteomics 9:746–756. doi:10.1002/ pmic.200700234 Bai J-Y, Zeng L, Hu Y-L et al (2007) Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants. Biotechnol Lett 29:2007–2012 Berlanga J, Prats P, Remirez D et al (2002) Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am J Pathol 161:373–379. doi:10.1016/S00029440(10)64192-2 Berlanga J, Ferna´ndez JI, Lo´pez E et al (2013) Heberprot-P: a novel product for treating advanced diabetic foot ulcer. MEDICC Rev 15:11–15 Berlanga-Acosta J, Gavilondo-Cowley J, Lo´pez-Saura P et al (2009) Epidermal growth factor in clinical practice—a review of its biological actions, clinical indications and safety implications. Int Wound J 6:331–346. doi:10.1111/j.1742-481X.2009.00622.x Buyel JF, Fischer R (2012) Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield and downstream costs. Biotechnol Bioeng 109:2575–2588. doi:10.1002/bit.24523 Chiapello H, Lisacek F, Caboche M, He´naut A (1998) Codon usage and gene function are related in sequences of Arabidopsis thaliana. Gene 209:GC1–GC38 Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14:669–679. doi:10.1016/j.tplants.2009.09.009 Datar R, Cartwright T, Rosen C (1993) Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator. Nat Biotechnol 11:349–357 De Wilde K, De Buck S, Vanneste K, Depicker A (2013) Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response. Plant Physiol 161:1021–1033. doi:10.1104/pp.112.209718 Fu H, Machado Pa, Hahm TS et al (2010) Recovery of nicotine-free proteins from tobacco leaves using phosphate buffer system under controlled conditions. Bioresour Technol 101:2034–2042. doi:10.1016/j.biortech.2009.10.045 Garabagi F, Gilbert E, Loos A et al (2012) Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts. Plant Biotechnol J 10:1–11. doi:10.1111/j.1467-7652.2012.00742.x Gils M, Kandzia R, Marillonnet S et al (2005) High-yield production of authentic human growth hormone using a plant virus-based expression system. Plant Biotechnol J 3:613–620. doi:10.1111/j. 1467-7652.2005.00154.x

1813 Hassan S, Colgan R, Paul MJ et al (2012) Recombinant monoclonal antibody yield in transgenic tobacco plants is affected by the wounding response via an ethylene dependent mechanism. Transgenic Res 21:1221–1232. doi:10.1007/s11248-012-9595-1 Higo K, Saito Y, Higo H (1993) Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco. Biosci Biotechnol 57:1477–1481 Jorissen R (2003) Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284:31–53. doi:10.1016/ S0014-4827(02)00098-8 Kudo K, Ohta M, Yang L et al (2013) ER stress response induced by the production of human IL-7 in rice endosperm cells. Plant Mol Biol 81:461–475. doi:10.1007/s11103-013-0016-5 Kuo B, Kusmik W, Poole J (1992) Pharmacokinetic evaluation of two human epidermal growth factors (hEGF51 and hEGF53) in rats. Drug Metab 20:23–30 Lai H, Chen Q (2011) Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current good manufacture practice regulations. Plant Cell Rep 31:573–584. doi:10.1007/s00299-011-1196-6 Lee KK, Jo HJ, Hong JP et al (2008) Recombinant human epidermal growth factor accelerates recovery of mouse small intestinal mucosa after radiation damage. Int J Radiat Oncol Biol Phys 71:1230–1235. doi:10.1016/j.ijrobp.2008.03.041 Liu J-X, Howell SH (2010) Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants. Plant Cell 22:2930–2942. doi:10.1105/tpc.110.078154 Lombardi R, Circelli P, Villani ME et al (2009) High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol. doi:10.1186/1472-6750-9-96 Medrano G, Reidy MJ, Liu J et al (2009) Rapid system for evaluating bioproduction capacity of complect pharmaceutical proteins in plants. Proteins 483:51–67. doi:10.1007/978-1-59745-407-0 Melchers LS, Sela-Buurlage MB, Vloemans Sa et al (1993) Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3-glucanase in transgenic plants. Plant Mol Biol 21:583–593 Nanba D, Toki F, Barrandon Y, Higashiyama S (2013) Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation. J Dermatol Sci 72:81–86. doi:10.1016/j.jdermsci.2013.05. 009 Neuhaus JM, Sticher L, Meins F, Boller T (1991) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88:10362–10366 Nuttall J, Vine N, Hadlington JL et al (2002) ER-resident chaperone interactions with recombinant antibodies in transgenic plants. Eur J Biochem 269:6042–6051. doi:10.1046/j.1432-1033.2002.03302.x Oh H, Seong J, Kim W et al (2010) Recombinant human epidermal growth factor (rhEGF) protects radiation-induced intestine injury in murine system. J Radiat Res 51:535–541. doi:10.1269/jrr.09145 Parsons J, Wirth S, Dominguez M et al (2010) Production of human epidermal growth factor (hEGF) by in vitro cultures of Nicotiana tabacum: effect of tissue differentiation and sodium nitroprusside addition. Int J Biotechnol Biochem 6:133–140 Paul M, Ma JK-C (2011) Plant-made pharmaceuticals: leading products and production platforms. Biotechnol Appl Biochem 58:58–67. doi:10.1002/bab.6 Plesha MA, Huang T, Falk BW, Mcdonald KA (2009) Optimization of the bioprocessing conditions for scale-up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system. Biotechnol Prog 25:722–734. doi:10.1021/bp.149

123

1814 Rybicki EP (2009) Plant-produced vaccines: promise and reality. Drug Discov Today 14:16–24. doi:10.1016/j.drudis.2008.10.002 Salmanian AH, Gushchin A, Medvedeva T et al (1996) Synthesis and expression of the gene for human epidermal growth factor in transgenic potato plants. Biotechnol Lett 18:1095–1098 Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832. doi:10.1016/j.biotechadv.2009.06.004 Silhavy D, Molna´r A, Lucioli A et al (2002) A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21:3070–3080. doi:10.1093/emboj/cdf312 Stevens LH, Stoopen GM, Elbers IJ et al (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182 Tarnuzzer R, Schultz G (1996) Biochemical analysis of acute and chronic wound environments. Wound Repair Regen 4:321–325 Tiwari S, Verma PC, Singh PK, Tuli R (2009) Plants as bioreactors for the production of vaccine antigens. Biotechnol Adv 27:449–467 Tomaszewska R, Dembinski A (2002) The influence of epidermal growth factor on the course of Ischemia-reperfusion induced pancreatitis in rats. J Physiol 53:183–198 Torrent M, Llompart B (2009) Eukaryotic protein production in designed storage organelles. BMC Biol 7:193–208 Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956 Wang L, Roossinck MJ (2006) Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants. Plant Mol Biol 61:699–710. doi:10.1007/s11103-0060041-8

123

Plant Cell Rep (2014) 33:1801–1814 Wirth S, Calamante G, Mentaberry A (2004) Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems. Mol Breed 13:23–35. doi:10.1023/B:MOLB.0000012329.74067.ca Wirth S, Segretin ME, Mentaberry A, Bravo-Almonacid F (2006) Accumulation of hEGF and hEGF-fusion proteins in chloroplasttransformed tobacco plants is higher in the dark than in the light. J Biotechnol 125:159–172 Wong W, Huang ER, Wong S et al (2001) Applications, and efficient large-scale production, of recombinant human epidermal growth factor. Biotechnology 18:51–71 Wu HG, Song SY, Kim YS et al (2009) Therapeutic effect of recombinant human epidermal growth factor (RhEGF) on mucositis in patients undergoing radiotherapy, with or without chemotherapy, for head and neck cancer: a double-blind placebo-controlled prospective phase 2 multi-institutional cli. Cancer 115:3699–3708. doi:10.1002/cncr.24414 Wu C, Kuo W, Chang C, Kuo J (2014) The modified rice aAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings. Plant Mol. doi:10.1007/s11103-014-0174-0 Yang C-H, Huang Y-B, Wu P-C, Tsai Y-H (2005) The evaluation of stability of recombinant human epidermal growth factor in burninjured pigs. Process Biochem 40:1661–1665. doi:10.1016/j. procbio.2004.06.038 Yao F, Eriksson E (2000) Gene therapy in wound repair and regeneration. Wound Repair Regen 8:443–451. doi:10.1046/j. 1524-475x.2000.00443.x Yera-Alos IB, Alonso-Carbonell L, Valenzuela-Silva CM et al (2013) Active post-marketing surveillance of the intralesional administration of human recombinant epidermal growth factor in diabetic foot ulcers. BMC Pharmacol Toxicol 3:44. doi:10. 1186/2050-6511-14-44