Recombinant protein production in a variety of Nicotiana hosts: a ...

Plant Biotechnology Journal (2011) 9, pp. 434–444

doi: 10.1111/j.1467-7652.2010.00563.x

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis Andrew J. Conley 1,2,†, Hong Zhu2, Linda C. Le2, Anthony M. Jevnikar3, Byong H. Lee4, Jim E. Brandle2,5 and Rima Menassa2,* 1

Department of Biology, University of Western Ontario, London, ON, Canada

2

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, Canada

3

Transplantation Immunology Group, Lawson Health Research Institute, London, ON, Canada

4

Food Research and Development Centre, Agriculture and Agri-Food Canada, Sainte-Hyacinthe, QC, Canada

5

Vineland Research and Innovation Centre, Vineland Station, ON, Canada

Summary

Received 28 January 2010; revised 27 May 2010; accepted 8 July 2010. *Correspondence (fax 519 457 3997; email [email protected]) † Present address: VTT Technical Research Centre of Finland, Tietotie 2, 02040 VTT, Finland.

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable a-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana

Keywords: molecular farming,

varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient con-

recombinant protein production, transgenic plants, transient expres-

centrations of recombinant proteins, in addition to producing a large amount of bio-

sion, erythropoietin, interleukin-10,

mass and a relatively low quantity of alkaloids, probably making it the most effective

a-amylase, Pseudomonas aeruginosa.

plant host for recombinant protein production.

production of recombinant proteins because it is readily amenable to genetic engineering and it has a high biomass

Introduction For the past 20 years, plants have been successfully investigated as an alternative means of producing a wide variety of therapeutically important recombinant proteins, such as antibodies, vaccines and biopharmaceuticals (Ma et al., 2005). When compared to conventional expression

yield (more than 100 000 kg per hectare) (Sheen, 1983). Furthermore, the tobacco expression platform is based on leaves, which removes the need for flowering, significantly reducing the potential for gene leakage into the environment through pollen or seed dispersal. Most importantly,

systems, such as microbial fermentation and mammalian cell cultures, plant production systems are inexpensive, easily scalable, free from human pathogens and offer the potential for direct oral administration (Fischer et al.,

tobacco is a nonfood, nonfeed crop, which minimizes regulatory barriers by eliminating the risk of plant-made recombinant proteins entering the food chain (Rymerson et al., 2002; Twyman et al., 2003). Although tobacco is inherently

2004). Although a wide range of plant hosts have been developed, tobacco has the most established history for the

biosafe, the most important factor limiting the commercial success of tobacco bioreactor technologies is the low production yields obtained for certain recombinant proteins

434

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Protein production in various Nicotiana hosts 435

(Doran, 2006). To address this particular issue, certain highyielding agronomic lines could be selected from a wide range of available tobacco germplasm to significantly boost heterologous protein expression (Streatfield, 2007). The presence of toxic alkaloids in tobacco may limit its therapeutic applica-

1997). Because of its thermostability, a-amylase is a valuable enzyme in food processing and textile industries. To increase the economic feasibility of the tobacco expression platform, we evaluated the transient and stable expression of these four recombinant proteins in numer-

tions; however, many low-alkaloid varieties of tobacco have been developed for the production of recombinant proteins that are more suitable for direct oral administration of plant tissue or crude protein extracts (Joensuu et al., 2008).

ous species and cultivars of Nicotiana. This study aims to determine the ideal Nicotiana variety that possesses both desirable agronomic attributes, such as fast growth rates, high biomass yields, high soluble protein levels and low-

Recombinant human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in many models of neuronal, retinal, cardiac and renal injury, in addition to its well-established role in red blood cell produc-

alkaloid content, and the ability to accumulate recombinant proteins at high levels.

tion for the treatment of anaemia (Maiese et al., 2005). Recently, plant-derived EPO was shown to prevent the death of kidney epithelial cells following inflammatory injury (Conley et al., 2009c). Human interleukin-10 (IL-10) is a

Results and discussion Transient expression of EPO and IL-10 in Nicotiana leaves

contra-inflammatory regulatory cytokine that has multiple roles in the regulation of immune responses and has been proposed as a potent anti-inflammatory biological therapy for the treatment of chronic inflammatory bowel diseases (Li and He, 2004). We recently demonstrated that oral

Plant expression vectors were constructed to produce EPO, IL-10, APA and PFA fused with different C-terminal tags (Figure 1). A signal peptide directed the proteins to the secretory pathway, while retention in the endoplasmic reticulum (ER) was achieved with a KDEL motif (Gomord

administration of transgenic low-alkaloid tobacco expressing IL-10 reduced the severity of murine colitis (Menassa et al., 2007). Anti-pseudomondas aeruginosa (APA) is a novel synthetic human antibody against P. aeruginosa, a

et al., 1997). All proteins were targeted to the ER lumen because it is the most promising subcellular compartment for the proper folding and assembly of complex recombinant eukaryotic proteins in plants (Fischer and Emans,

serious human pathogen causing hospital acquired infections (Hemachandra et al., 2001). APA specifically binds to P. aeruginosa bacterial cells and has the capacity to bind the first component of human complement (C1q) in the

2000), and many reports have demonstrated higher accumulation of secreted recombinant proteins in this intracellular compartment (Conley et al., 2009b; Fiedler et al., 1997; Huang et al., 2001; Menassa et al., 2001; Ramirez

classical activation pathway (H. Zhu, unpublished data). PFA is a hyperthermostable extracellular a-amylase from Pyrococcus furiosus (PFA) that hydrolyses starch by randomly cleaving the a(1,4)-glucosidic linkages (Dong et al.,

et al., 2002; Wandelt et al., 1992). Agrobacterium-mediated transient expression was used as a convenient means of rapidly testing the level of EPO accumulation in Nicotiana benthamiana, Nicotiana rustica,

Figure 1 Schematic representation of the genetic constructs used for expression in Nicotiana plants. All transgene expression fragments were placed under the control of the enhanced cauliflower mosaic virus 35S promoter, a tCUP 5’-untranslated region and the nopaline synthase terminator. SP-EPO, endogenous human EPO signal peptide; SP-Tob, Pr1b tobacco secretory signal peptide; EPO, human erythropoietin; IL-10, human interleukin-10; APA, synthetic antibody against Pseudomonas aeruginosa; PFA, hyperthermostable a-amylase from Pyrococcus furiosus; StrepII, purification tag; HIS, (His)6 purification tag; TEV, tobacco etch virus protease recognition site; c-Myc, detection tag; KDEL, ER-retention signal; ELP, elastin-like polypeptide tag (28 · VPGVG); huFc, human heavy chain constant region; EPO, erythropoietin. ª 2010 Crown in the right of Canada Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 434–444

436 Andrew J. Conley et al.

Nicotiana sylvestris and 49 cultivars of Nicotiana tabacum (Table 1), prior to the resource-intensive process of generating transgenic plants (Huang and Mason, 2004; Wroblewski et al., 2005). Transient expression of EPO was detected in all 52 plant hosts, although the concentration

Nicotiana varieties, from highest to lowest producers of EPO, was only slightly altered in this second transient analysis (Trial 2) (r = 0.587, P = 0.05). EPO accumulated up to sevenfold higher concentration in N. tabacum (cv. I 64) than in N. tabacum (cv. Con. Havana 38), which is practi-

of EPO varied significantly amongst the various species and cultivars of Nicotiana. For example, N. tabacum (cv. TI 95) produced almost 30 times more EPO than N. rustica. To account for these differences, certain Nicotiana varie-

cally identical to the enhancement observed in the original transient analysis (Trial 1). Except for two N. tabacum cultivars (i.e. Samsun and Delfield), the transient expression data from Trial 1 correlated well with the data from Trial

ties may be more susceptible to infection via agroinfiltration or they may possess some intrinsic ability to accumulate heterologous proteins to higher levels. To establish the general utility of agroinfiltration and to

2 (Figure 2b), although the EPO concentrations were consistently higher for the second transient analysis. Therefore, this data suggest that the reproducible differences observed in recombinant protein expression levels among

evaluate the effect of differing environmental conditions on the transient expression of EPO, 16 Nicotiana varieties were chosen for further study. A new set of plants were grown in a different greenhouse and agroinfiltrated with

Nicotiana varieties can be reliably translated to other growth environments. However, the absolute amounts of recombinant protein production may vary. A change in temperature was the major difference between growth

the EPO expression vector. Again, the accumulation of EPO varied significantly amongst the transiently agroinfiltrated Nicotiana varieties (Figure 2a). The relative order of

conditions in this study, with the temperature being 5–10 C cooler for the second transient analysis. This suggests that higher temperatures are detrimental for

Nicotiana variety

EPO (ng ⁄ mg TSP)

Nicotiana variety

EPO (ng ⁄ mg TSP)

N. Rustica

1.32 ± 0.20

Delfield

Ottawa 705

2.15 ± 0.23

Coker 48

6.75 ± 0.86 8.00 ± 0.21

Labu

2.67 ± 0.30

Delhi 76

8.03 ± 1.41

TI 115

2.71 ± 0.19

Yellow Mammoth

8.11 ± 0.91

Havana 307

2.86 ± 0.18

Burley 1

8.33 ± 1.54

Xanthi

2.88 ± 0.31

Delgold

8.57 ± 1.93

TI 90

2.99 ± 0.22

Green Briar

8.90 ± 1.18

N. Sylvestris

3.00 ± 0.57

TI 161

9.39 ± 1.92

Kentucky 16

3.17 ± 0.30

Maryland 201

9.57 ± 1.21

Con. Havana 38

3.29 ± 0.64

Duquesne

9.83 ± 1.42

Burley 49

3.58 ± 0.43

CT 681

10.05 ± 0.56

81V9 MS

3.73 ± 0.43

TI 170

10.16 ± 1.76

Judy’s Pride

4.20 ± 0.29

TI 164

11.01 ± 1.74

CT 572

4.23 ± 0.42

N. Benthamiana

11.40 ± 0.58

TI 158

4.28 ± 0.23

Kentucky 10

11.43 ± 0.82

Cannelle

4.57 ± 0.40

Bell C

12.26 ± 1.98

Coker 371 Gold

4.59 ± 0.09

Samsun

12.89 ± 0.77

TI 94

4.84 ± 0.72

Bell B

14.68 ± 2.24

CT 157

4.92 ± 0.46

TI 75

16.31 ± 1.06

White Mammoth

4.96 ± 0.26

Vinica

17.31 ± 4.51

Kelly

5.13 ± 1.18

Grande Rouge

17.60 ± 2.06

Gold Dollar

5.16 ± 0.54

Belgique 3007

20.55 ± 1.28

White Gold

5.20 ± 0.38

I 64

22.12 ± 1.51

Hicks Br.

5.24 ± 0.93

Little Crittenden

22.40 ± 1.27

Bonanza

6.25 ± 0.38

TI 124

22.96 ± 2.24

Havana 425

6.30 ± 0.40

TI 95

36.05 ± 2.07

Table 1 Agrobacterium-mediated transient expression of erythropoietin (EPO) in Nicotiana benthamiana, Nicotiana rustica, Nicotiana sylvestris and 49 cultivars of Nicotiana tabacum (Trial 1)

The concentration of EPO was measured from leaf sectors harvested 4 days post-agroinfiltration by enzyme-linked immunosorbant assay. The data are expressed as the mean ± SD of six independently infiltrated leaves for each Nicotiana variety. All Nicotiana varieties presented in bold type were selected for further analysis. TSP, total soluble protein. 100 ng ⁄ mg TSP is equivalent to 0.01% of TSP.

ª 2010 Crown in the right of Canada Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 434–444


(a)

(b)

(c)

(d)

Figure 2 Transient expression of erythropoietin (EPO) and interleukin-10 (IL-10) in 16 different species and cultivars of Nicotiana (Trial 2). Nicotiana leaves were agroinfiltrated and the accumulation of EPO (a) and IL-10 (c) were quantified by enzyme-linked immunosorbant assay. Each column represents the mean value of 12 agroinfiltrated leaves, and the standard deviation is represented by error bars. For both EPO and IL-10, the five lowest recombinant protein expressing Nicotiana varieties were colored green, the six median expressers were coloured orange and the five highest expressers were colored blue. (b) A comparison of the EPO concentration obtained in Trial 1 (Table 1) and Trial 2 (a), demonstrating the correlation between separate transient analyses when utilizing two groups of plants grown under differing greenhouse conditions. (d) A comparison of the EPO (a) and IL-10 (c) concentration, demonstrating the correlation between two recombinant proteins that were transiently expressed in the same set of plants grown under the same greenhouse conditions. TSP, total soluble protein.

recombinant protein accumulation, which has also been previously observed by Stevens et al. (2000). An IL-10 expression vector was also simultaneously agroinfiltrated into the leaves of the Trial 2 plants (Figure 2c) to determine the effect of Nicotiana variety on the tran-

commercial production; however, it still remains the model plant system for transient expression because it grows quickly and is readily susceptible to agroinfiltration (Marillonnet et al., 2005; McCormick et al., 1999). Our results demonstrated that high biomass–yielding tobacco

sient accumulation of another pharmaceutically important recombinant protein. Similar to the EPO findings, N. tabacum (cv. I 64) also produced the highest concentration of IL-10. For both EPO and IL-10, the five lowest, the six

varieties, such as ‘I 64’, ‘TI 95’ or ‘TI 124’, also transiently produced much higher concentrations of recombinant proteins. Therefore, these varieties may be considered more efficient plant hosts for the large-scale

median and the five highest heterologous protein expressing Nicotiana varieties remained the same, although minor rearrangements within each grouping were observed between the two proteins (r = 0.845, P = 0.01). As a

transient expression of heterologous proteins when compared to smaller Nicotiana varieties producing lower concentrations of recombinant protein, such as N. benthamiana. A similar conclusion was reached by Sheludko

result, a strong correlation existed between the accumulation of EPO and IL-10 amongst the Nicotiana varieties when grown under the same conditions (Figure 2d), suggesting that certain Nicotiana hosts may be consistently

et al. (2007), after comparing the transient expression of a GFP reporter protein in six Australian species of Nicotiana. Although agroinfiltration was used in the present study,

superior for high-level transient expression, irrespective of the recombinant protein being produced. Nicotiana benthamiana has a very small biomass relative to the larger tobacco varieties more suitable for

adaptations of this plant transient expression system have been established over the past few years. For example, expression vectors are now routinely co-agroinfiltrated with suppressors of gene silencing, which have been shown to



significantly enhance the production levels of recombinant proteins in plant leaves (Silhavy et al., 2002; Ve´zina et al., 2009; Voinnet et al., 2003). Icon Genetics has developed a technology called magnifection, which combines the high transformation efficiency of Agrobacterium with the high

the mean of the five transformants from each population with the highest concentration of EPO, IL-10, APA and PFA (Figure 3e–h) was determined to allow for comparison amongst the Nicotiana varieties. Notably, our in-house lowalkaloid N. tabacum variety (cv. 81V9) was among the top

expression yield of viral vectors, leading to accumulation levels exceeding 40% of total soluble protein (TSP) and allowing for gram-sized quantities to be attained in as little as 5 days (Giritch et al., 2006; Gleba et al., 2005; Marillonnet

three producing cultivars for EPO, IL-10 and APA. Although a twofold difference in the concentration of EPO, IL-10 and PFA was observed between the highest and lowest expressing Nicotiana varieties, no significant difference could be

et al., 2005). In addition, a suite of Cowpea mosaic virusbased vectors have been developed that are capable of expressing heterologous proteins to 20% of TSP without relying on viral replication (Sainsbury and Lomonossoff,

observed between the majority of Nicotiana varieties when examining the average of the top five expressing lines for each plant host. However, larger 11-fold differences in recombinant protein accumulation levels were observed

2008; Sainsbury et al., 2009). It would be interesting to test whether the Nicotiana host effect on Agrobacterium-mediated transient expression would also hold when utilizing these other novel transient expression platforms.

amongst the cultivars expressing APA. No correlation was observed between the accumulation levels of the four recombinant proteins amongst the transgenic Nicotiana varieties, possibly because a much larger sample size would

Many novel production systems rely on the transient

be required to reduce the high intrinsic variability associated with positional effects and RNA silencing. In general, the variety of Nicotiana had little practical impact on the concentration of EPO, IL-10, APA and PFA in planta, suggesting that their agronomic properties, such as growth rate and

expression of recombinant proteins in plants (Marillonnet et al., 2005; McCormick et al., 1999; Santi et al., 2008; Ve´zina et al., 2009); however, our goal typically involves the generation of stable transgenic plants for field produc-

biomass yield, may be more important when determining the optimal transgenic plant host for recombinant protein production. Previous studies have demonstrated that comparisons of

tion (Brandle, 2004). Thus, to complement our transient expression analyses, 1600 independent transgenic Nicotiana plant lines were regenerated from the EPO, IL-10, APA and PFA expression constructs (25 positively express-

different expression constructs within a single plant host using transient analyses were predictive of those conducted using stable transgenic plants (Conley et al., 2009c; Huang and Mason, 2004; Patel et al., 2007; Spiegel et al., 1999).

ing plants for each of the 16 selected Nicotiana varieties). All plants were sampled at an equivalent age because the developmental stage of a transgenic plant has been shown to affect its capacity for recombinant protein pro-

For our study, which evaluated the effect of various Nicotiana hosts on the production of four different recombinant proteins, the transient analyses were not indicative of the stable transgenic plant analyses. In transient assays, hun-

duction (Conley et al., 2010; Farran et al., 2008; Stevens et al., 2000). As expected, the concentration of the recombinant proteins (Figure 3a–d) varied considerably amongst the populations of primary transformants, which can be attributed to the chromosomal position effects

dreds of nonintegrated copies of the heterologous transcript are expressed for a few days within the plant cells. In effect, this reduces the gene expression variability normally associated with stable transgenic plants (Kapila et al., 1997). Therefore, transient analysis is a good indicator of

associated with random gene insertion (Hobbs et al., 1990; Krysan et al., 2002) or to RNA silencing (Schubert et al., 2004) and possibly to transgene copy number. The concentration of EPO, IL-10, APA and PFA approached

expression construct performance for the accumulation of recombinant proteins in plants. On the other hand, only a single copy of the transgene is most often integrated into the genome of transgenic Nicotiana plants, with the level of

200, 1600, 25000 and 5000 ng ⁄ mg TSP, respectively, in the highest accumulating plant lines. All transgenic plants exhibited normal growth and morphology compared with nontransgenic plants.

foreign protein accumulation being the net result of longterm transcription, translation and proteolytic degradation (Wroblewski et al., 2005). These inherent differences between transient and stable expression systems may be

Only plant lines that yield high levels of recombinant protein are sought for large-scale production schemes. Thus,

responsible for the lack of correlation observed in this study when utilizing a variety of plant hosts. On the other hand,

Generation of transgenic Nicotiana plants expressing EPO, IL-10, APA and PFA



(a)

(e)

(b)

(f)

(c)

(g)

(d)

(h)

Figure 3 Accumulation of four recombinant proteins in transgenic Nicotiana plants. Quantitative analysis of plant-derived EPO (a), IL-10 (b), APA (c) and PFA (d) protein concentration in the leaf tissue of stable transgenic plants. Twenty-five primary transformants were generated for each Nicotiana variety. Each sample consisted of a single leaf disc from each of the first four expanded leaves from the plant. The average of the top five EPO (e), IL-10 (f), APA (g) and PFA (h) expressing plants is represented for each Nicotiana variety. TSP, total soluble protein; EPO, erythropoietin; PFA, hyperthermostable a-amylase from Pyrococcus furiosus.

larger transgenic populations may be necessary to better reflect the transient results, because hundreds of primary

kidney epithelial cells from cytokine-induced death in vitro (Conley et al., 2009c), whereas plant-derived IL-10 was an

transformants are generally necessary to recover very high expressing plant lines or to allow for comparison among different experimental variables. Although this study has demonstrated that EPO, IL-10, APA and PFA can be expressed in numerous varieties of

effective therapeutic treatment in a mouse model of colitis (Menassa et al., 2007). Although not evaluated in this study, we suspect that EPO and IL-10 derived from the other Nicotiana varieties would also be biologically active. Current studies in our laboratory have shown that plant-

Nicotiana, it is also important to consider the quality of the recombinant protein product. For N. tabacum (cv. 81V9), plant-derived EPO was previously shown to protect

derived APA is capable of binding P. aeruginosa bacterial cells (Zhu et al., unpublished results), but further tests are still needed to demonstrate an effective therapeutic



response for this antibody. For PFA, we can conclude that this protein is functionally active in all tested Nicotiana varieties, because its level of accumulation was actually based on its ability to degrade starch in an enzymatic assay.

N. tabacum (cv. Burley 49), which was almost 40 times higher than N. tabacum (cv. 81V9). However, for the production of medicinally useful recombinant proteins, it is theoretically possible to modify the genetic background of any Nicotiana variety to produce lower concentrations of alkaloids (Rymerson et al., 2002).

Agronomic properties of the Nicotiana varieties Although the yield of recombinant protein, represented as

Conclusions

gram per kilogram FW or as a percentage of TSP, is important, it is equally important in an industrial setting to establish the growth rate, TSP levels and leaf biomass of the various Nicotiana varieties. For each of the 16 Nicotiana vari-

In summary, this comparative study evaluated the transient and stable expression of four therapeutically or industrially important proteins (i.e. EPO, IL-10, APA and PFA) in a variety of Nicotiana plant hosts. The level of transient accu-

eties, ten wild-type individually potted plants were simultaneously grown from seed under the same greenhouse conditions and their agronomic properties evaluated. Nicotiana benthamiana plants reached the flowering

mulation for EPO and IL-10 varied significantly amongst the different Nicotiana varieties. Nevertheless, a strong correlation between recombinant protein yield and the Nicotiana varieties was observed when expressing the

stage after 55 days, whereas N. tabacum (cv. Burley 49) took 118 days to reach maturity (Figure 4a). The median flowering time for all Nicotiana varieties was 83 days. At flowering, the N. benthamiana plants were 23 cm tall, whereas N. tabacum (cv. TI 124) were 125 cm tall

same protein under two different growth conditions or when expressing two different proteins under the same growth conditions. Of the 52 Nicotiana varieties evaluated, N. tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to pro-

(Figure 4b). The median flowering height for all Nicotiana varieties was 87 cm. The amount of leaf biomass at maturity varied significantly amongst the Nicotiana varieties (Figure 4c). Although N. tabacum (cv. Burley 49) took

ducing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for large-scale transient expression. For the transgenic plant analyses, the variety of Nicotiana had little

twice as long to reach maturity, it was able to produce 26 times more leaf biomass than N. benthamiana, because a large portion of any plant’s development involves germination and seedling establishment. Once Nicotiana seedlings

practical impact on the production levels of the recombinant proteins. As well, no correlation could be observed between the transient and stable analyses. Currently, our laboratory is evaluating the agronomic characteristics of

establish themselves, they typically grow very quickly. In general, the slow maturing Nicotiana varieties also produced the highest levels of biomass. As a result, a strong positive correlation exists between flowering time and the

six Nicotiana varieties in an open-field study, along with their ability to produce heterologous proteins and alkaloids, to establish the best plant platform for the commercial production of recombinant proteins.

amount of leaf biomass produced for the Nicotiana species (r = 0.868, P = 0.001). On the other hand, no significant correlation was observed between the height at flowering and the amount of leaf biomass (r = 0.200). With respect to the amount of TSP produced in each Nicotiana variety, a 1.85-fold difference was observed between N. tabacum (cv. TI 95) and N. tabacum (cv. I 64) (Figure 4d). However, the majority of Nicotiana varieties produced very similar levels of TSP. A disadvantage of tobacco expression platforms is the presence of toxic alkaloids, although low-alkaloid varieties exist, such as N. tabacum (cv. 81V9) (Menassa et al., 2001). The alkaloid content varied drastically amongst the 16 selected Nicotiana varieties (Figure 4e). The concentration of alkaloids was 1.58 mg ⁄ g of dried leaf tissue in

Experimental procedures Plant expression vectors The construct used to express human EPO in plants (SPNat EPONat) has been described elsewhere (Conley et al., 2009c). The construct used to express human IL-10 in plants (IL10-ELP) contains a translational fusion with an elastin-like polypeptide (ELP) tag and was described in detail by Conley et al. (2009a). The synthetic APA antibody was directed against P. aeruginosa (strain O6) and consists of a single-chain variable fragment (Hemachandra et al., 2001) fused to the human IgG1 heavy chain constant region (CH1–CH3), which adds effector functions to the antibody (Hu et al., 2003). A (His)6 tag was included for detection of the APA protein. The DNA sequence of the hyperthermostable extracellular a-amylase from P. furiosus was obtained from Dong et al. (1997).



Plant-optimized versions of the APA and PFA genes were designed to mimic the codon usage of highly expressed N. tabacum genes, while avoiding all potentially deleterious processing

(a)

signals and destabilizing motifs according to Joensuu et al. (2009). The APA synthetic gene was constructed using a combined ligase chain reaction ⁄ polymerase chain reaction (LCR ⁄ PCR) approach (Au et al., 1998) with a set of overlapping oligonucleotides designed by the web-based program Gene2Oligo (Rouillard et al., 2004), whereas the PFA gene was synthesized by the company Geneart (Toronto, Canada). Briefly, the EPO, IL-10, APA and PFA coding sequences were placed under the control of the dualenhancer cauliflower mosaic virus 35S promoter (Kay et al., 1987) and nopaline synthase terminator (Bevan et al., 1983) and targeted to the ER.

Agrobacterium-mediated transient expression and transgenic plant generation (b)

(c)

(d)

(e)

For transient expression, the Agrobacterium strains were used to infiltrate the leaves of 6- to 10-week-old greenhouse-grown Nicotiana plants as previously described (Conley et al., 2009c). The plants were infiltrated when 2–4 true and fully grown leaves were present on the plants to ensure that all Nicotiana varieties were infiltrated at a similar developmental stage. In summary, the induced Agrobacterium suspensions were adjusted to a final optical density at 600 nm (OD600) of 1.0 and then directly injected into the intercellular spaces of intact leaves using a 1-mL syringe. After infiltration, the plants were maintained under greenhouse conditions, and the infiltrated leaf panels were sampled 4 days post-transfection during the daytime. For the transient analysis of 52 Nicotiana species and cultivars (Trial 1), which were grown and maintained in an off-site greenhouse, six leaves were agroinfiltrated (three plants, two leaves per plant) with the EPO agrobacterial strain. The off-site greenhouse had limited temperature control and was typically maintained at approximately 25–30 C during the day and 15–20 C at night and had a 16-h photoperiod. For the transient analysis of 16 selected species and cultivars of Nicotiana (Trial 2), which were grown and maintained in an on-site greenhouse, twelve individual leaves were agroinfiltrated (six plants, two leaves per plant) with both the EPO and IL-10 agrobacterial strains. The temperature of the on-site greenhouse was maintained at 20 C with a 16-h photoperiod. Tissue samples from the individually infiltrated leaf panels served as biological replicates and were analysed

Figure 4 Agronomic analyses of the various Nicotiana varieties. For the purpose of this paper, flowering was defined as the point in time where the first emergence of a flower petal was visible. The flowering time of the Nicotiana varieties is represented by the number of days of growth that took place after the seeds were placed into soil (a), whereas the flowering height was measured from the base of the plant to the flower bud at the flowering time point (b). The plants were stripped of their leaves at flowering to determine their total leaf biomass (c). To determine the amount of total soluble protein produced in the Nicotiana varieties, a single leaf disc was taken from eight leaves of each plant, sampled from older, mature and young leaves and pooled together for the analysis (d). For each Nicotiana variety, all leaves from the population of plants were pooled together and analysed (n = 3) to determine their alkaloid concentration (e). Each column represents the mean value of 10 wild-type plants (a–d) and the standard deviation is represented by error bars.



separately, with the average of the six or twelve leaf panels used to represent the concentration of a given recombinant protein in a Nicotiana species or cultivar. For the generation of stable transgenic plants, the EPO, IL-10, APA and PFA expression constructs were introduced into 16 selected species and cultivars of Nicotiana using Agrobacteriummediated transformation of leaf discs according to the method of Horsch et al. (1985). Primary transformants were grown on-site in a greenhouse for 4–6 weeks following transfer to soil.

Afterwards, the gel was stained with an iodine solution (10 mM iodine, 100 mM potassium iodide) until a clear zone appeared, indicating starch degradation. After washing with water, the gels were subsequently scanned and the concentration of recombinant PFA was analysed by using image densitometry with TOTALLAB TL100 software (Nonlinear Dynamics, Durham, NC, USA). The band intensities were compared to lanes containing known amounts of purified PFA derived from bacterial expression.

Plant sample preparation

Acknowledgements

For the transient analysis of Trial 1, each sample (n = 6) consisted of a single 7-mm leaf disc taken from each of the six infiltrated leaves and pooled together. For the transient analysis of Trial 2, each sample (n = 12) consisted of four 7-mm leaf discs taken from an individually infiltrated leaf. For each transgenic plant, a single 7-mm leaf disc was taken from each of the first four expanded leaves and used to represent the concentration of recombinant protein in the whole plant. For each sample, TSP was extracted and prepared as described in detail by Conley et al. (2009c).

The authors thank Dr Brian McGarvey and Bob Pocs for the tobacco alkaloid analysis and Alex Molnar for his assistance

Quantification of EPO, IL-10, APA and PFA protein levels The concentration of EPO in plant leaf extracts was determined by sandwich enzyme-linked immunosorbant assay (ELISA) according to Conley et al. (2009c). The quantification of plant recombinant IL-10 in leaf extracts was achieved by human IL-10 ELISA using antibodies and standard protein according to the manufacturer’s instructions (BD Biosciences, Mississauga, ON, Canada). The quantification of APA in leaf extracts was achieved by sandwich ELISA. Nunc-Immuno MaxiSorp surface plates (Nalge Nunc, Rochester, MN, USA) were coated with a 1 : 1000 dilution of custom-made anti-APA rabbit serum (Invitrogen, Carlsbad, CA, USA) diluted in carbonate buffer (0.05 M, pH 9.6) and incubated overnight at 4 C. The wells were blocked with 3% BSA in PBS for 2 h at room temperature. Plant extracts were diluted in PBS (1 ⁄ 10) and incubated on the plate overnight at 4 C. The plate was then incubated with a 1 : 1000 dilution of mouse anti-HIS antibody (27471001; GE Healthcare, Uppsala, Sweden) diluted in PBS for 1 h at room temperature. Next, the plates were incubated with a 1 : 1000 dilution of HRP-conjugated goat anti-mouse IgG (170-6516; Bio-Rad, Hercules, CA, USA) diluted in PBS for 1 h at room temperature. The plates were washed five times between all incubation steps with PBS containing 0.05% Tween-20. The plates were developed by the addition of ABTS substrate (A-1888; Sigma, St Louis, MO, USA), and the absorbance was measured at 405 nm with a Bio-Rad 550 microplate reader. Recombinant APA was expressed and purified from Escherichia coli and used to generate the standard curve. The concentration of PFA was determined by a starch degradation assay according to Wang et al. (2007). After the proteins were resolved by 10% SDS-PAGE, the gel was incubated in acetate buffer (50 mM sodium acetate, pH 5.5) for 15 min at 40 C. The gel was then transferred into acetate buffer containing 1% soluble starch and incubated for 15 min at 95 C.

with the preparation of the figures. This research was supported by the Agriculture and Agri-Food Canada A-Base Funding Programme. We thank the Natural Sciences and Engineering Research Council (NSERC) Postgraduate Scholarship for providing financial support to A.J.C.

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