Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

Plant Biotechnology Journal

(2009)

7

, pp. 183–199 doi: 10.1111/j.1467-7652.2008.00389.x

© 2008 Her Majesty the Queen in Right of CanadaJournal compilation © 2008 Blackwell Publishing Ltd

183

Blackwell Publishing LtdOxford, UKPBIPlant Biotechnology Journal1467-76441467-7652© 2008 Blackwell Publishing LtdXXXOriginal Article

Plant recombinant EPO attenuates kidney cell death

Andrew J. Conley

et al.

Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

Andrew J. Conley

1

, Kanishka Mohib

2

, Anthony M. Jevnikar

2,3,

* and Jim E. Brandle

4,5

1

Department of Biology, University of Western Ontario, London, ON, Canada, N6A 5B7

2

Departments of Medicine and Microbiology and Immunology, University of Western Ontario, London, ON, Canada, N6A 5A5

3

Transplantation Immunology Group, Lawson Health Research Institute, London, ON, Canada, N6A 5A5

4

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, Canada, N5V 4T3

5

Vineland Research and Innovation Centre, Vineland Station, ON, Canada, L0R 2E0

Summary

Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective

activities in addition to its well-established role in red blood cell production. Unfortunately,

conventional mammalian cell cultures are unlikely to meet the anticipated market demands

for recombinant EPO because of limited capacity and high production costs. Plant

expression systems may address these limitations to enable practical, cost-effective delivery

of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in

tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results

indicated that targeting to the endoplasmic reticulum (ER) provided the highest

accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in

tobacco leaves. The codon optimization of the human EPO gene for plant expression had

no clear advantage; furthermore, the human EPO signal peptide performed better than a

tobacco signal peptide. In addition, we found that glycosylation was essential for the

stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide

fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy

showed that apoplast and ER-targeted EPO were correctly localized, and

N

-glycan analysis

demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on

ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity

and was able to protect kidney epithelial cells from cytokine-induced death

in vitro

.

These findings demonstrate that tobacco plants may be an attractive alternative for the

production of large amounts of biologically active EPO.

Received 23 July 2008;

revised 10 October 2008;

accepted 13 October 2008

.

*

Correspondence

(fax (519)663-8808;

e-mail: [email protected])

Keywords:

apoptosis, EPO, human

erythropoietin, kidney injury, molecular

farming, transgenic tobacco.

Introduction

Human erythropoietin (EPO) is produced in the kidneys and

is the principal hormone responsible for maintaining the

circulating erythrocyte mass (Lin

et

al

., 1985). Recombinant

human EPO (rhEPO) is an important biopharmaceutical

that is used extensively in anaemia caused by renal failure,

chemotherapy and acquired immunodeficiency syndrome

(AIDS). In addition, rhEPO had a market value of $12 billion

in 2006, clearly a product of considerable value to the

pharmaceutical industry (Tucker and Yakatan, 2008).

EPO is a 166-amino-acid glycoprotein existing as a hetero-

geneous mixture of glycoforms, with 40% of the 34–39-kDa

molecular weight consisting of three

N

-linked [asparagine-24

(Asn24), Asn38 and Asn83] and one

O

-linked [serine-126

(Ser126)] carbohydrate chain (Egrie

et

al

., 1986). Studies

have shown that EPO’s glycocomponent is important for the

protein’s stability, solubility, biosynthesis, secretion and

in vivo

bioactivity, but is not required for the hormone’s

in vitro

interaction with its receptor (Wasley

et

al

., 1991; Yamaguchi

et

al

., 1991). In particular, the terminal sialic acids located on

the

N

-linked oligosaccharides are not required for the

in vitro

184

Andrew J. Conley

et al.

© 2008 Her Majesty the Queen in Right of CanadaJournal compilation © 2008 Blackwell Publishing Ltd,


,

7

, 183–199

bioactivity, but their presence is essential for the

in vivo

bioactivity of EPO, because they prolong the protein’s

circulating half-life in the bloodstream (Spivak and Hogans,

1989; Takeuchi

et

al

., 1989).

EPO exerts its haematopoietic activity through interaction

with the homodimeric EPO receptor [(EPOR)

2

] located on

erythroid progenitor cells in the bone marrow (Matthews

et

al

., 1996). However, recently, the tissue expression of EPOR

has been determined to be widespread, including neurones,

endothelial cells, kidney cells and cardiomyocytes, suggesting

an alternative and perhaps more important biological activity

for EPO (Juul

et

al

., 1998; Masuda

et

al

., 1999). Consistent with

this, EPO has recently been shown to possess remarkable

tissue-protective activities in preclinical injury models of the

spinal cord, kidney, retina, brain and heart. EPO’s broad

efficacy in a wide range of injury models is related to multiple

cytoprotectant pathways, which are activated during disease

or tissue injury. These include the inhibition of programmed

cell death (apoptosis), attenuation of inflammatory responses,

stimulation of angiogenesis and direct recruitment of stem

cells (reviewed by Ghezzi and Brines, 2004). Since the chronic

use of rhEPO as a tissue-protective therapeutic is likely to

cause undesirable side-effects, such as increased blood

pressure and thrombosis because of its haematopoietic

activity (Stohlawetz

et

al

., 2000), recent studies have identified

EPO derivatives that are tissue protective, yet lack haematopoietic

activity. Carbamylated EPO (CEPO), desialylated EPO (asialoEPO)

and an EPO-R103E mutant have all shown tissue-protective

activity without exerting a haematopoietic effect (Erbayraktar

et

al

., 2003; Leist

et

al

., 2004); however, these modifications

would lead to substantial production costs that would

probably prevent their practical use as a tissue injury

prevention therapeutic.

Recombinant EPO has been made in a variety of systems,

including bacteria (Lee-Huang, 1984), yeast (Hamilton

et

al

.,

2006), insect cells (Kim

et

al

., 2005) and the milk of transgenic

goats (Toledo

et

al

., 2006). However, all rhEPO used therapeu-

tically is currently generated in mammalian cell culture (Goto

et

al

., 1988), which is technically complex and expensive.

Limitations imposed by the mammalian cell culture system

reduce the quantities of EPO that can be produced and limit

its therapeutic potential. The utilization of plants as production

systems may lower that barrier because of the potential for

low production costs and the rapid scalability associated

with plant-made biopharmaceuticals. Plants also offer other

advantages over conventional expression systems for the

production of recombinant proteins, such as the absence of

human pathogens, the ability to properly fold and assemble

complex multi-subunit proteins, and the potential for direct

oral administration of unprocessed or partially processed

plant material (Ma

et

al

., 2003).

EPO has been produced in tobacco BY2 cell lines (Matsumoto

et

al

., 1995), tobacco and

Arabidopsis

plants (Cheon

et

al

., 2004)

and the moss

Physcomitrella patens

(Weise

et

al

., 2007).

Matsumoto

et

al

. (1995) were the first to demonstrate that

plant-derived EPO exhibited

in vitro

haematopoietic activity,

but found that it lacked

in vivo

activity, most probably

because of the absence of the sialic acids required for this

function. However, our interest is focused on the tissue-

protective activity of EPO and the plant’s natural ability to

produce asialylated EPO, which has already been shown to

possess tissue-protective activity without excessive haemato-

poiesis (Erbayraktar

et

al

., 2003).

Many approaches have been used to increase the concen-

tration of biopharmaceuticals in plant tissues. Targeting

the recombinant protein to the appropriate plant tissue and

subcellular compartment within the cell appears to be critical

in reaching suitable expression levels (reviewed by Streatfield,

2007). The subcellular location is also important because of

its impact on the glycosylation profile of the recombinant

protein, which may affect its potential immunogenicity when

administered to humans. Human and plant cells produce the

same high-mannose-type

N

-glycans within the endoplasmic

reticulum (ER), whereas complex-type

N

-glycans, which are

attached to proteins as they pass through the late Golgi

apparatus of the secretory pathway, are structurally different

in plants and animals (reviewed by Gomord

et

al

., 2005).

These differences lead to complex

α

(1,3)-fucose and

β

(1,2)-xylose glycan motifs on plant glycoproteins that

are immunogenic when administered to rodents (Cabanes-

Macheteau

et

al

., 1999; Bardor

et

al

., 2003).

The use of fusion proteins, such as ubiquitin (Hondred

et

al

., 1999),

β

-glucuronidase (Gil

et

al

., 2001; Dus Santos

et

al

.,

2002) and human immunoglobulin (Ig)

α

-chains (Obregon

et

al

., 2006), is another common approach to enhance

recombinant protein accumulation in plants. More recently,

elastin-like polypeptide (ELP) fusions have been found to

increase significantly the accumulation levels of human

interleukin-10 and murine interleukin-4 (Patel

et

al

., 2007),

spider silk proteins (Scheller

et

al

., 2004; Patel

et

al

., 2007)

and the full-size anti-human immunodeficiency virus type 1

(anti-HIV-1) antibody 2F5 (Floss

et

al

., 2008) in tobacco leaves,

and single-chain variable fragment (scFv) antibodies (Scheller

et

al

., 2006) in tobacco seeds. ELPs are synthetic biopolymers

made from a repeating pentapeptide ‘VPGXG’ sequence,

which occur in all mammalian elastin proteins (Raju and

Anwar, 1987). ELPs are also valuable for bioseparation, as

they have been shown to act as thermally responsive tags for

Plant recombinant EPO attenuates kidney cell death

185

© 2008 Her Majesty the Queen in Right of CanadaJournal compilation © 2008 Blackwell Publishing Ltd,


,

7

, 183–199

the temperature-based, non-chromatographic separation of

recombinant proteins (Meyer and Chilkoti, 1999; Lin

et

al

., 2006).

The availability of large quantities of biologically active EPO

will be essential for the cost-effective, widespread use of EPO

as a therapeutic in tissue injury prevention. In this article, we

evaluate a number of strategies to enhance the yield of EPO

in plant tissue. These strategies include the plant optimization

of the human EPO gene, the use of human vs. plant signal

peptides, the removal of glycosylation sites and the use of ELP

translational fusions. In addition, the effects of subcellular

location on EPO yield and glycosylation profile are examined.

Finally, we demonstrate the increased receptor-binding affinity

of plant recombinant EPO (prEPO) relative to rhEPO and, for the

first time, we show the tissue-protective biological activity of

prEPO using an

in vitro

model of kidney epithelial cell death.

Results

Transient expression of EPO in various tobacco

subcellular compartments

Three plant expression vectors were constructed that targeted

prEPO to the apoplast, ER and chloroplast (Figure 1a). The

human endogenous EPO signal peptide (SP

EPO

) was fused to

the mature native EPO sequence (EPO

Nat

) to direct the protein

into the secretory pathway (secEPO) and, finally, into the

apoplast. Retention to the ER (SP

EPO

·EPO

Nat

) was achieved

by adding an ER retrieval signal (KDEL) to the C-terminus of

the apoplast-targeted secEPO construct. For chloroplast

targeting, the transit peptide from the tobacco small subunit

RuBisCo gene was fused to EPO (chEPO). To aid in the detection

and processing of the recombinant protein, a tobacco etch

virus (TEV) protease site, StrepII purification tag and c-Myc

detection tag were attached to the C-terminus of the EPO

coding sequence. All coding sequences were introduced into

the plant binary expression vector pCaMterX and placed

under the control of the enhanced cauliflower mosaic virus

(CaMV) 35S promoter, a tCUP translational enhancer and the

nopaline synthase (nos) terminator.

These constructs were agroinfiltrated into tobacco leaves,

and the concentration of prEPO was quantified using a

sandwich enzyme-linked immunosorbent assay (ELISA). Of

the three subcellular compartments tested, prEPO from the

ER-retained construct (SP

EPO

·EPO

Nat

) accumulated to the

highest level [88 ng/mg total soluble protein (TSP)], which

was seven times higher than the apoplast-targeted construct

(secEPO) and 185 times higher than the chloroplast-targeted

construct (Figure 1b). Clearly, the subcellular location of

Figure 1 Transient expression of plant recombinant erythropoietin (prEPO) targeted to the apoplast, endoplasmic reticulum (ER) and chloroplasts of Nicotiana tabacum leaves. (a) Structure of the genetic constructs used for the expression of prEPO in tobacco leaves. SP-EPO, endogenous human EPO signal peptide; TP-RuB, transit peptide from the tobacco small subunit RuBisCo gene; EPO (Native), native EPO mature sequence; StrepII, purification tag; TEV, tobacco etch virus protease site; c-Myc, detection tag; KDEL, ER retention signal. (b) The concentration of prEPO measured by enzyme-linked immunosorbent assay (ELISA) from leaf sectors harvested 4 days post-infiltration. Each column represents the mean value (n = 6), and the standard deviation is represented by error bars. TSP, total soluble protein. (c) Western blot analysis of transiently expressed prEPO targeted to the apoplast (lane 1), ER (lane 2) and chloroplasts (lane 3) of tobacco leaves. Tobacco extracts from wild-type (lane 4) and empty vector-agroinfiltrated wild-type (lane 5) tissue demonstrate the presence of a nonspecific band (c. 38 kDa) indicated by an arrow. Thirty micrograms of TSP were loaded into each lane (1–5), and 5 ng of recombinant human EPO (rhEPO) was used as a positive control (lane 6).

186 Andrew J. Conley et al.

© 2008 Her Majesty the Queen in Right of CanadaJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 183–199

prEPO greatly affects its accumulation, with the ER being the

best of the compartments tested.

Western blot analysis also showed the presence of different

prEPO forms in the various subcellular compartments. When

using a polyclonal EPO antibody, the apoplast-, ER- and

chloroplast-targeted prEPO were detected as 28-, 31- and

19-kDa bands, respectively (Figure 1c). Occasionally, a 38-kDa

Agrobacterium-specific band was detected (Figure 1c, lanes

1, 3, 5) in the plant extracts after agroinfiltration, but this band

was not observed in wild-type tobacco tissue (lane 4). The

larger size of SPEPO·EPONat relative to secEPO can be attributed

to the presence of additional C-terminal amino acids (c. 3.7 kDa),

whereas chEPO was considerably smaller, probably because

of the absence of glycans. In general, prEPO was significantly

smaller than rhEPO produced in Chinese hamster ovary

(CHO) cells, which migrates as a broad band (c. 33–39 kDa)

because of its microheterogeneity of glycosylation (Egrie

and Browne, 2001). Differential glycosylation between plants

and humans, particularly the lack of sialic acids on plant

glycoproteins, is probably responsible for the major size

discrepancy between prEPO and rhEPO.

Transient expression of EPO in the ER of tobacco leaves

To achieve high levels of EPO protein translation, a synthetic

version of the human EPO gene (EPOOpt) was constructed to

reflect the codon usage and optimal codon context of highly

expressed tobacco genes (Campbell and Gowri, 1990; Chiapello

et al., 1998). Furthermore, sequences encoding potential cryptic

splice sites, polyadenylation signals, destabilizing AT-rich

regions, strong secondary structure and TATA-box-like

elements were removed (Koziel et al., 1996; Gutierrez et al.,

1999). Consecutive strings of A + T and C + G were also

avoided, together with CG and TA dinucleotides and regions

with strong transcript secondary structure (Figure S1a, see

online ‘Supporting Information’). In its entirety, the optimi-

zation of the EPO gene resulted in changes to 24% of the

nucleotides in 59% of the codons and a decrease in the

G + C content from 59% to 48% (Figure S1b).

A collection of ER-targeted EPO expression vectors was

constructed in order to evaluate the effect of a plant and

mammalian signal peptide [SPTob (plant) and SPEPO (human)],

four EPO variants (EPONat, EPOOpt, EPOR103, EPOAgly) and the

presence of an ELP fusion partner on prEPO accumulation

and biological activity (Figure 2a). The EPO mutant EPOR103

was created by changing a single amino acid (R103E) of

the EPOOpt gene with the purpose of separating EPO’s

cytoprotective bioactivity from its haematopoietic bioactivity.

In addition, aglycosylated EPO (EPOAgly) was created by

inactivating all glycosylation sites of EPOOpt by substitution

mutagenesis of the target asparagines with lysine, and the

target serine with valine. EPOAgly provides an alternative

approach to separate EPO’s biological activities, whilst also

reducing the risk of plant glycan immunogenicity when

administered to humans.

Agrobacterium-mediated transient assays and quantitative

ELISA were performed on the series of ER-targeted EPO

constructs. For each combination of signal peptide and ELP

fusion, the EPONat variant tended to have the highest con-

centration of prEPO, followed by EPOOpt, EPOR103 and EPOAgly

(Figure 2b). The optimization of the human EPO sequence had

no effect on the accumulation of prEPO in the absence of an

ELP fusion partner. In the presence of an ELP fusion partner, the

optimized gene had a detrimental effect on the prEPO con-

centration relative to the native gene. On average, the removal

of glycosylation sites resulted in prEPO concentrations that

were 1/250th of their glycosylated counterparts. In the case of

EPONat, the presence of ELP increased the concentration of prEPO

twofold when combined with the tobacco signal peptide

(SPTob). Without ELP, the native human signal peptide (SPEPO)

produced a higher level of EPONat when compared with the

tobacco signal peptide. However, the two positive effects

were not additive in the case of SPEPO·EPONat·ELP. For the

EPOOpt, EPOR103 and EPOAgly variants, comparable levels of

prEPO accumulation were observed, irrespective of the signal

peptide and ELP fusion arrangement.

To confirm the integrity of transiently expressed prEPO,

Western blot analysis was performed on the protein extracts.

The aglycosylated expression constructs were not included

because of their extremely low levels of accumulation. Many

of the EPO protein bands were over-exposed to allow for the

detection of less concentrated EPO proteins, thus preventing

a quantitative comparison between these expression

constructs via Western blots. As shown in Figure 2c, the EPO

anti-sera reacted with a single protein, yielding bands of the

expected size. Longer exposures of the same blots revealed

slightly smaller immunoreactive bands (data not shown),

which could represent either degradation products or the

partial glycosylation of prEPO. However, all recombinant

bands shifted to a smaller single band after deglycosylation

(data not shown), suggesting that prEPO exists as a mixture

of different glycoforms in the plant.

Generation of transgenic tobacco plants expressing

EPO

To complement the transient expression analyses of prEPO,

stable transgenic tobacco plants were generated using

Plant recombinant EPO attenuates kidney cell death 187


Agrobacterium-mediated transformation. Four-hundred and

five independent transgenic tobacco lines were regenerated

from 13 EPO expression constructs. As expected, the prEPO

concentration varied significantly among transgenic plants

(Figure 3a), which was attributed to the chromosomal

position effects associated with random gene insertion

(Hobbs et al., 1990; Krysan et al., 2002). The level of prEPO

approached 500 ng/mg TSP (i.e. 0.05% of TSP) in the highest

expressing lines. The range of EPO accumulation within a

given population expressing the same genetic construct

was greatest for SPEPO·EPONat at 210-fold, and lowest for

SPTob·EPOOpt·ELP at ninefold, with an average difference of

Figure 2 Transient expression analysis of endoplasmic reticulum (ER)-targeted plant recombinant erythropoietin (prEPO) in tobacco leaves. (a) Schematic representation of a series of 16 EPO expression constructs targeted to the ER of tobacco leaves. SP-Tob, PR1b tobacco secretory signal peptide; SP-EPO, endogenous human EPO signal peptide; EPO (Native), native EPO mature sequence; EPO (Tobacco-optimized), tobacco-optimized EPO sequence; EPO (R103), function-altered EPO mutant (R103E); EPO (Aglycosylated), aglycosylated EPO sequence; TEV, tobacco etch virus protease site; HIS, (His)6 purification tag; StrepII, purification tag; c-Myc, detection tag; KDEL, ER retention signal; ELP, elastin-like polypeptide tag (28 × VPGVG). (b) Accumulation of prEPO in leaf extracts following transient expression by agroinfiltration. TSP, total soluble protein. (c) Western blot analysis of transiently expressed prEPO in the ER of tobacco. Total protein extracts (20 μg/lane) from the designated leaf sector (lanes 1–12) and wild-type control (lane 13) were separated by 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted to nitrocellulose and probed with anti-EPO serum. Lane 14, recombinant human EPO (rhEPO) (3 ng).



80-fold across all individual populations. For comparison, the

mean of the five transformants from each population with

the highest concentration of prEPO was used to demonstrate

the effects of signal peptides, EPO variants, subcellular

localization and presence of an ELP fusion on prEPO protein

production (Figure 3b).

The trends observed during the transient analyses corre-

lated well with those observed in the stable transgenic plants,

with some minor discrepancies. For each combination of

signal peptide and ELP fusion, the EPOOpt variant tended to

produce the most prEPO protein, followed by EPONat and

EPOR103. Thus, tobacco optimization of the human EPO

sequence had a more positive effect in stable plants than for

the transient analyses, but still did not have any practical

impact on prEPO protein accumulation in tobacco plants. The

alteration of the EPO primary sequence (EPOR103) decreased

significantly the EPO concentration by more than twofold.

To evaluate the effect of glycosylation on EPO accumulation,

29 transgenic SPTob·EPOAgly plants were generated. The top five

plants for this construct produced prEPO at a concentration

of 0.12 ng/mg TSP, which is an 850-fold decrease relative to

their glycosylated counterparts (data not shown). The retention

of EPO in the ER of transgenic plants (SPEPO·EPONat) produced

seven times more prEPO than in the apoplast-targeted construct

(secEPO), which was consistent with the transient analysis.

The presence of an ELP fusion partner increased significantly

the amount of prEPO (3.5-fold) for all EPO variants utilizing the

tobacco signal peptide, which contrasts with the transient

analysis, where an increase was observed only for EPONat.

The ELP tag also doubled the level of EPOR103 utilizing the

human signal peptide. The human signal peptide (SPEPO)

had a positive effect (3.5-fold) on the accumulation of

EPONat and EPOOpt; however, the presence of ELP did not

increase significantly the prEPO concentration for these

constructs any further, which was consistent with the

transient analysis.

Western blot analysis was also performed on the prEPO

proteins from stable transgenic lines in order to verify their

sizes and to allow for comparison with their counterparts from

transient expression. In all instances, single immunoreactive

bands of the expected size were detected from the transgenic

plants (Figure 3c), although partially glycosylated forms of

prEPO could be detected in longer exposures of the blots (data

not shown). The prEPO produced in stable transgenic tobacco

plants is similar in size to that obtained from agroinfiltrated

tobacco tissue.

Figure 3 Expression of erythropoietin (EPO) in transgenic tobacco plants. (a) Enzyme-linked immunosorbent assay (ELISA) of plant recombinant EPO (prEPO) protein concentration in leaf tissue of stable transgenic plants carrying various EPO expression vectors. Two samples (each sample contains a single leaf disc from each of the first four expanded leaves from each plant) were taken from each individual transformant for ELISA. (b) The average of the top five prEPO-expressing plants is represented for each expression construct. (c) Western blot analysis of EPO transgenic tobacco plants (lanes 1–13) and the non-transgenic control (lane 14). Total soluble protein (TSP) (30 μg/lane) was extracted from the leaf tissue of selected individual plants, fractionated by 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted to nitrocellulose and probed with a polyclonal EPO antibody. Lane 15, rhEPO (5 ng).



Glycosylation analysis of plant-derived EPO

To characterize the glycosylation of prEPO, transgenic plant

extracts were treated with various glycosidases, followed by

Western blot analysis. The apoplast-targeted prEPO (secEPO)

was fully resistant to digestion by endoglycosidase H (EndoH),

suggesting that secEPO acquires a complex glycan structure

when transported through the Golgi apparatus (Figure 4a,

lanes 1 and 2). Conversely, treatment with EndoH resulted in

a mobility shift for SPEPO·EPONat and its ELP fusion partner

(SPEPO·EPONat·ELP), consistent with the glycosylation pattern

of ER-retained glycoproteins. As expected, rhEPO accumu-

lated in an EndoH-resistant form, suggesting the presence

of complex glycans. After peptide N-glycosidase F (PNGaseF)

treatment, no mobility shift was observed for secEPO

(Figure 4b, lanes 1 and 2), indicating the presence of a core

α(1,3)-linked fucosyl residue, which is expected for secreted

plant glycoproteins. After PNGaseF digestion, SPEPO·EPONat

migrated as a smaller band of about 23 kDa (includes 4 kDa

of additional C-terminal amino acids). This is nearly identical

to that of N-deglycosylated rhEPO protein standard (c.

20.5 kDa), which still contains an O-linked oligosaccharide

chain (c. 2 kDa). Partial deglycosylation demonstrated that all

three potential glycosylation sites on prEPO were occupied

(data not shown). From these results, the smaller size of

prEPO can be attributed to smaller plant-specific N-glycans

relative to mammalian-produced EPO. Furthermore, enzymatic

treatment with α-neuraminidase or O-glycosidases demon-

strated that prEPO lacks sialic acid residues and O-linked

oligosaccharides (data not shown). Taken together, these

results demonstrate that apoplast-targeted EPO (secEPO)

contains complex-type N-glycans, with a core α(1,3)-linked

fucose residue. In contrast, the KDEL-tagged prEPO proteins

were sensitive to EndoH and PNGaseF, suggesting a high-

mannose oligosaccharide structure indicative of ER local-

ization with efficient retention/retrieval from the cis-Golgi.

Subcellular localization of recombinant EPO

in tobacco leaves

To verify the subcellular localization of prEPO, green fluo-

rescent protein (GFP) was used as a C-terminal fusion, and

the resulting constructs (Figure 5a) were agroinfiltrated into

tobacco leaves and examined by confocal laser scanning

microscopy. Weak secEPO·GFP fluorescence was detected

in the apoplast of epidermal plant cells (Figure 5b), which was

confirmed by FM4-64 labelling of adjacent plasma mem-

branes (Figure 5b, inset). The addition of a C-terminal KDEL

sequence to secEPO·GFP resulted in bright SPEPO·EPONat·GFP

fluorescence, which resembled a typical reticulate pattern

(Boevink et al., 1996), consistent with ER localization (Figure 5c).

Furthermore, fluorescence was absent from the apoplastic

space (Figure 5c, inset). SPTob·EPONat·GFP (Figure 5d) showed a

very similar localization pattern to SPEPO·EPONat·GFP, demon-

strating that the plant and human signal peptides target EPO

to the secretory pathway in a similar fashion. To allow for

easier visualization, the laser intensity gain was increased

for the apoplast-targeted proteins relative to the ER-

targeted proteins. As controls, the same three localization

constructs were synthesized without the EPO genetic

component, and their localization was indistinguishable from

the experimental constructs (data not shown).

EPOR-binding analysis of plant-produced EPO

To examine prEPO’s ability to bind human EPOR, an indirect

ELISA was developed. Briefly, the microtitre plates were coated

with anti-EPOR antibody and then incubated with the same

set of plant extracts and standards as used when determining

the concentration of prEPO (Figures 1b and 2b); however, a

saturating amount of EPOR was added to the buffer used

for sample dilutions. Anti-EPO antibodies were then utilized

for the detection of the bound EPOR:EPO complexes. The

EPOR-binding assay demonstrated that the accumulation of

Figure 4 Deglycosylation of plant recombinant erythropoietin (prEPO). (a) Total protein extracts (30 μg/lane) from agroinfiltrated plant tissue (lanes 1–8) were incubated for 24 h in the presence (+) or absence (–) of endoglycosidase H (EndoH) and then analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-EPO serum. Recombinant human EPO (5 ng) was used as a positive control (lanes 9 and 10). (b) As described in (a), except that peptide N-glycosidase F (PNGaseF) was used.



prEPO in planta (Figure 6a) followed the same construct-

dependent expression trends as observed in Figures 1b and

2b. However, the absolute values of prEPO accumulation

detected by the EPOR-binding assay were significantly

different from the quantitative ELISA data, suggesting that

prEPO has a different binding affinity than rhEPO to human

EPOR. The standard curve comprising rhEPO was used as the

common reference point in both the EPOR-binding assay and

the quantitative EPO ELISA, allowing for a comparison

between the two assays. Every EPO variant (EPONat, EPOOpt,

EPOR103, EPOAgly and secEPO) demonstrated a very consistent

enhancement or reduction in EPOR binding, regardless of

the signal peptide utilized or the presence of an ELP fusion

partner (Figure 6b). Therefore, the presence of an ELP fusion

partner appears to have no effect on the receptor binding of

prEPO. The four EPONat and EPOOpt expression constructs

showed twofold higher binding affinity than rhEPO for EPOR.

The successful engineering of the EPOR103 variant to possess

a lower affinity for the haematopoietic receptor was achieved,

as a single amino acid change decreased its affinity for EPOR

by threefold, resulting in an overall sixfold decrease from its

unmodified plant-produced counterpart. In the aglycosylated

EPOAgly variants, the protein bound EPOR with a fourfold

higher affinity than did the mammalian-produced rhEPO.

Finally, the apoplast-targeted secEPO had a 1.5-fold higher

affinity for EPOR. Together, these results clearly show the

efficient binding of tobacco-produced EPO to human EPOR,

which is an important step towards demonstrating the

biological activity necessary for therapeutic applications.

Biological activity of plant-derived EPO in an in vitro

model of tissue injury

Although prEPO has been shown to bind human EPOR with

a high affinity, it is critical to assess the recombinant protein’s

in vitro biological activity. To enrich for the prEPO protein,

which started at a concentration of 0.02% of TSP, and to

remove potentially inhibitory substances from the plant

extracts, prEPO was purified to 20% of TSP by StrepII affinity

chromatography prior to biological activity analysis. The

purified SPEPO·EPONat protein’s ability to prevent the death of

renal tubular epithelial cells (TECs) was compared with that

of commercial rhEPO standards. As shown in Figure 7, a basal

level of cell death (c. 20%) occurred in the untreated medium

control as a result of serum removal during the 24-h culture

period. The addition of the pro-inflammatory cytokine

interferon-γ (IFN-γ) greatly increased the level of cell death to

approximately 45% in TECs supplemented with purified

wild-type tobacco eluate. However, equivalent amounts of

prEPO or rhEPO were able to reduce significantly the level of

Figure 5 Subcellular localization of plant recombinant erythropoietin (prEPO) in tobacco epidermal cells by confocal microscopy. (a) Diagram of constructs used for the visualization of prEPO in transgenic tobacco leaves with their predicted subcellular location indicated. SP-EPO, endogenous human EPO signal peptide; SP-Tob, PR1b tobacco secretory signal peptide; EPO (Native), native EPO mature sequence; GFP, enhanced green fluorescent protein; KDEL, endoplasmic reticulum (ER) retention signal. (b) Localization constructs were agroinfiltrated into tobacco leaves and visualized by confocal microscopy. The secEPO·GFP was detected in the apoplast, which was further validated with a merged image (inset) of GFP fluorescence in the apoplast (green) with FM4-64 dye, which labels the plasma membrane (red). (c, d) Human and plant signal peptides are equally capable of targeting prEPO to the ER in the presence of a C-terminal KDEL sequence. The typical reticulate network of the ER was observed; however, no GFP fluorescence could be detected in the apoplast (inset). Bar, 10 μm. Bar for insets, 2.5 μm.



TEC death to near baseline values (24% and 21%, respectively).

These results demonstrate that plant-derived EPO is biologi-

cally active and could function as a general tissue-protective

cytokine.

Discussion

In addition to its haematopoietic effects on the bone marrow,

EPO acts as a general tissue-protective cytokine that has the

potential to treat numerous diseases and injuries, including

stroke, myocardial infarction, spinal cord injury and acute

kidney failure (Ghezzi and Brines, 2004). In this study, for the

first time, prEPO has been shown to protect kidney cells from

inflammatory-induced death.

To enhance the level of EPO accumulation in planta, the

human EPO gene was optimized for tobacco expression and

targeted to several subcellular compartments. Furthermore,

the efficiency of two signal peptides was compared and

the presence of an ELP fusion partner was evaluated.

Agrobacterium-mediated transient expression in Nicotiana

tabacum was used as a convenient method to rapidly test

many different expression constructs (Wroblewski et al.,

2005) prior to the resource-intensive process of generating

transgenic plants. Although, many novel production systems

rely on the transient expression of recombinant proteins in

plants (Huang and Mason, 2004; Marillonnet et al., 2005),

our goal was the generation of stable transgenic plants for

field production (Brandle, 2004). Our results show that the

experiments conducted using transient analyses were

predictive of those conducted using stable transgenic plants.

The use of a rigorous replicated experimental design can be

credited for improving the utility of transient analyses as a

predictor of performance in stable plants.

EPO was targeted to the apoplast, ER and chloroplast in

order to assess the optimal subcellular location for high-level

accumulation of prEPO. Although secretion to the apoplast

has been demonstrated as a means of EPO production in

plants (Matsumoto et al., 1995; Cheon et al., 2004; Weise

et al., 2007), Matsumoto et al. (1995) found that the apoplast

is not a suitable storage environment because EPO remains

attached to the plant cell wall which, when combined with

the high level of proteolytic activity in the apoplast (Fiedler

et al., 1997), could explain the limited accumulation of EPO

in this compartment. The addition of a carboxy-terminal

KDEL motif to a secreted protein in plants results in its

accumulation in the ER lumen (Schouten et al., 1996). Our

results correspond with previous reports demonstrating

that secreted recombinant proteins are more stable and

Figure 6 Plant-derived erythropoietin (EPO) binds to the human EPO receptor (EPOR). (a) For each expression construct, the equivalent samples as used in Figures 1b and 2b were analysed by an EPOR-binding assay to determine the quantitative receptor binding of plant recombinant EPO (prEPO) relative to recombinant human EPO (rhEPO). TSP, total soluble protein. (b) The difference in relative binding affinity of prEPO and rhEPO to the human EPOR was determined by comparing the ratio of EPO accumulation detected in the EPOR-binding assay (a) with the quantitative enzyme-linked immunosorbent assay (ELISA) results (Figures 1b and 2b). The standard curve comprising rhEPO was used in both assays and served as the common reference point, allowing for comparison between the two assays. A value of 1.0 represents an equal ability of prEPO and rhEPO to bind EPOR.

Figure 7 Plant-derived erythropoietin (EPO) exhibits tissue-protective biological activity. Renal tubular epithelial cells (TECs) were treated with the indicated samples, and the level of cell death was analysed with a FACSCalibur flow cytometer. The data are presented as the mean ± standard deviation of triplicate samples and are representative of three separate experiments. IFN, interferon-γ ; prEPO, plant recombinant EPO; rhEPO, recombinant human EPO.



accumulate to higher levels when targeted to the ER lumen

(Huang et al., 2001; Menassa et al., 2001). The ER provides

the appropriate environment for complex post-translational

modifications, such as glycosylation and disulphide bond

formation, to occur (Hwang et al., 1992), which are both

important for EPO’s stability. We decided to target EPO to

the chloroplasts because they have been shown to accumulate

high levels of recombinant proteins (Van Molle et al., 2007).

As an additional benefit, the chloroplasts should generate an

aglycosylated form of prEPO, which eliminates potentially

immunogenic plant glycans. However, the chloroplast-

targeted EPO accumulated to very low concentrations,

probably a result of a lack of glycosylation (Wasley et al.,

1991).

After concluding that the ER was the best location for

prEPO accumulation, we optimized the human EPO gene

sequence for tobacco expression. Many bacterial and human

gene sequences have been successfully optimized for increased

plant expression. However, our tobacco-optimized EPO gene

did not enhance the level of prEPO accumulation when

compared with the native EPO gene. Our results are supported

by examples demonstrating that the optimization of hetero-

logous eukaryotic genes for expression in plants (Rouwendal

et al., 1997; Lonsdale et al., 1998) is less impressive than the

optimization of bacterial genes for plant expression (Perlak

et al., 1991; Kang et al., 2004).

Although it is common practice to replace the human

signal peptide with a plant signal peptide when producing

secreted human proteins in plants, it is probably not essential,

as the recognition of N-terminal signal peptides is highly

conserved amongst eukaryotes (Blobel et al., 1979; Rapoport

et al., 1996). The exchange of human for plant signal peptides

has been shown to enhance significantly the accumulation of

certain proteins (Sijmons et al., 1990; Schaaf et al., 2005);

however, our data suggest that the endogenous human

EPO signal peptide performed better than the tobacco signal

peptide for EPO expression. Other examples of human signal

sequences functioning more efficiently than plant signal

sequences in plants also exist (Ma et al., 2005).

The fusion of an ELP partner has been shown to increase

the yield of several recombinant target proteins in transgenic

tobacco leaves (Patel et al., 2007; Floss et al., 2008) and

seeds (Scheller et al., 2006). In this study, the fusion of ELP to

EPO had a negligible effect on many of the prEPO expression

constructs, although the presence of an ELP fusion partner

significantly enhanced the accumulation of specific EPO

constructs. However, the enhancement of EPO accumulation

when utilizing an ELP fusion was much lower than for other

recombinant proteins, which showed up to a 100-fold increase

in the concentration of the target protein (Patel et al., 2007).

The beneficial effect of ELP on recombinant protein accumu-

lation is probably protein specific but, in our case, the limited

effect of ELP fusion on EPO accumulation may be a result of

the potential toxicity of EPO-ELP to the plant (Cheon et al.,

2004). Within 4 days of agroinfiltration, complete tissue

necrosis could be observed within regions of the infiltrated

leaf panels. Moreover, the production of EPO-ELP in transgenic

tobacco plants retarded their vegetative growth and resulted

in malformed leaves with necrotic lesions. As a result, plant cells

expressing high levels of EPO-ELP during transient analysis

or transgenic plant generation simply die, thus limiting the

concentration of prEPO in planta. The C-terminal ELP tag had

no effect on the receptor-binding affinity of EPO, demon-

strating that ELP should have no effect on the biological

activity of recombinant proteins, as was recently demon-

strated for antibody–ELP fusions (Floss et al., 2008).

The size of prEPO was significantly smaller than that of

mammalian CHO cell-derived rhEPO because of differences

in glycosylation patterns. The N-glycans of EPO are important

for the stability, solubility, transport and biological activities

of the protein (Yamaguchi et al., 1991). Differences in glycosy-

lation between plants and humans could be a potential

barrier to the therapeutic use of prEPO for humans because

of the presence of immunogenic α(1,3)-fucose and β(1,2)-

xylose motifs on plant glycoproteins (Tekoah et al., 2004).

We chose to remove the glycosylation sites of EPO in

consideration of the potential immunogenicity issues, and to

evaluate the role of N-linked glycans on the accumulation

and receptor-binding affinity of the aglycosylated form. Our

aglycosylated prEPO was made by introducing lysine residues

at each of the N-linked glycosylation sites and by changing

the serine at the O-glycosylation site to valine. These sub-

stitutions have been shown previously to increase the stability

and solubility of aglycosylated EPO, without affecting the

protein structure (Cheetham et al., 1998; Narhi et al., 2001).

However, the absence of plant glycans dramatically reduced

the level of prEPO accumulation in plant leaves, suggesting

that glycosylation is important for correct folding, biosynthesis,

stability and/or secretion of EPO in planta. Regardless, the

binding affinity to EPOR actually increased. Our work differs

from other reports, which demonstrate that the removal of

N-glycans has a limited effect on the stability, folding, assembly

and activity of plant-derived antibodies (Nuttall et al., 2005;

Rodriguez et al., 2005). Therefore, glycosylation may be

more important for the accumulation of cytokines than for

the accumulation of antibodies in plants. As an alternative

approach to prevent the addition of immunogenic complex

plant glycans to prEPO, we fused an ER retention signal to



EPO in order to restrict glycosylation to exclusively high-

mannose-type N-glycans (Triguero et al., 2005; Petruccelli

et al., 2006). The ER-retained prEPO accumulated in an EndoH-

and PNGaseF-sensitive form, indicating the absence of

complex N-glycans, including the immunogenic α(1,3)-fucose

and β(1,2)-xylose motifs known to be attached to glycans

when passing through the medial or trans-Golgi compartments

(Pagny et al., 2000; Sriraman et al., 2004). Moreover, the

presence of the ER retention signal was solely responsible

for efficient ER retrieval, as untagged secreted prEPO was

both EndoH and PNGaseF resistant, thus demonstrating

EPO’s ability to be fully secreted. In addition, no significant

difference in glycosylation pattern was observed between

prEPO produced by transient expression or stable transfor-

mation. The absence of O-glycan on prEPO should be of little

concern, as O-glycosylation has no essential role in either the

in vitro or in vivo biological activity of the cytokine (Higuchi

et al., 1992). However, terminal sialic acids on the oligosac-

charide chains of EPO are essential for in vivo haematopoietic

bioactivity, as they prevent the hepatic elimination of the

protein and extend the circulating half-life of EPO (Fukuda

et al., 1989). Therefore, all commercially available rhEPOs for

the treatment of anaemia are produced in mammalian cell

cultures (Jacobs et al., 1985; Lin et al., 1985; Fibi et al., 1995),

which are able to synthesize sialyated glycoproteins. However,

reports have shown that asialoEPO, made by the enzymatic

desialylation of rhEPO, provides tissue protection without

inducing red blood cell production (Erbayraktar et al., 2003).

A potential explanation for this effect is that prolonged

periods of high circulating levels of EPO are needed to induce

haematopoiesis, but brief exposure to EPO is sufficient to

initiate tissue-protective programmes (Erbayraktar et al., 2003).

Therefore, an inability to produce sialic acid may be a beneficial

feature of EPO production in plants, as a simple and straight-

forward means of separating EPO’s tissue-protective activity

from its haematopoietic activity.

We developed an EPOR-binding ELISA which allowed us

to compare the relative binding affinities of prEPO and rhEPO

to human EPOR. The aglycosylated prEPO has a fourfold

higher receptor-binding affinity than fully glycosylated rhEPO,

which is similar to the increased receptor-binding activity

shown for aglycosylated Escherichia coli-produced EPO (Delorme

et al., 1992). In addition, our prEPO lacking sialic acid residues

exhibited a twofold increase in binding affinity, which has

also been demonstrated previously for desialylated EPO

(Elliott et al., 2004a). As the EPO molecule is positively charged

and EPOR is negatively charged at physiological pH, evidence

suggests that the glycans may shield the attractive electro-

static interactions present between EPO- and EPOR-binding

surfaces (Darling et al., 2002). This is particularly true in the

case of sialic acid, which is highly negatively charged, thus

decreasing the affinity of mammalian-produced EPO for

the negatively charged EPOR via charge repulsion (Elliott

et al., 2004b). Therefore, prEPO without sialic acid residues

should bind more efficiently than rhEPO to human EPOR.

Leist et al. (2004) have demonstrated that a cytoprotectant

EPO analogue lacking haematopoietic activity can be designed

by mutating amino acid 103 (i.e. R103E). This amino acid

does not interfere with the protein’s conformation, but is

critical for binding to the haematopoietic receptor (Grodberg

et al., 1993). As expected, the ability of our prEPO(R103E)

mutant to bind EPOR was reduced by sixfold, although its

biological activity has yet to be tested. This prEPO(R103E)

mutant could also be used to further separate EPO’s tissue-

protective activity from its haematopoietic activity when

produced in plants.

Matsumoto et al. (1995) and Weise et al. (2007) have demon-

strated that plant-derived EPO induces the proliferation of

EPO-sensitive cell lines; however, in the report by Matsumoto

et al. (1995), in vivo haematopoietic activity was absent,

and was attributed to the rapid removal of prEPO from the

circulation because of a lack of sialic acid residues. To our

knowledge, this is the first report demonstrating that

prEPO possesses tissue-protective bioactivities by reducing

the susceptibility of renal TECs to cytokine-induced cell

death. TECs comprise more than 75% of renal parenchymal

cells, and their susceptibility to injury directs the long-term

function of allografts, as tubular injury can be a primary cause

of nephron loss. Augmenting the endogenous capacity of

TECs to resist injury would be a useful strategy in renal

transplantation, as well as in other forms of organ injury in

which epithelial cells play a prominent role (Du et al., 2004).

As there are no current therapeutic agents that specifically

protect epithelial cells from inflammation-induced injury or

death, there has been growing interest in the use of novel

strategies for tissue injury attenuation, including EPO. Therefore,

recent studies have demonstrated that EPO protects the

kidney against injury caused by ischaemia–reperfusion by

utilizing its anti-apoptotic action, which may translate into

improved long-term function of the kidney (reviewed by

Johnson et al., 2006a). Therefore, prEPO could be administered

to renal allograft recipients or to the perfusion solutions of

kidney organs to attenuate renal allograft injury caused by

ischaemia, toxins and immunological rejection (Johnson et al.,

2006b; Spandou et al., 2006). EPOR is also expressed by

gut epithelial cells (Juul et al., 1999). Furthermore, EPO has

been shown to decrease apoptosis of gut epithelial cells

(Cuzzocrea et al., 2004; Guneli et al., 2007), suggesting that



prEPO may have a benefit in inflammatory bowel disease by

direct oral delivery, which has been shown for other plant-

produced cytokines (Menassa et al., 2007). Although current

levels of EPO expression in plants are relatively low, we believe

that tobacco can produce an economically feasible amount

of prEPO, as only low doses of EPO are needed to exert a

therapeutic response.

In summary, this study demonstrates that higher accumu-

lation levels of EPO can be achieved in the ER than in the

apoplast or chloroplasts. Optimization of the human EPO

gene for plant expression has no effect, and the human EPO

signal peptide is processed more efficiently than the tobacco

signal peptide. Glycosylation is very important for the stability

of EPO in planta, whereas the presence of an ELP fusion has

a limited effect on EPO concentration. The transient and

stable expression analyses are well correlated, and the

maximum concentration of EPO approaches 0.05% of TSP in

tobacco leaves. The apoplast- and ER-targeted EPO proteins

are correctly targeted to their respective subcellular com-

partments, and complex immunogenic glycans are present

only on apoplast-targeted EPO and absent from ER-targeted

EPO. Most importantly, our prEPO binds to human EPOR

with an increased affinity, and also has anti-apoptotic activity,

which may reflect a capacity to prevent tissue injury in human

disease. These results suggest that tobacco plants can provide

an efficient and less expensive means of producing a tissue-

protective EPO analogue that does not possess the potentially

harmful side-effects associated with excessive haematopoietic

activity. Future work will focus on increasing the concentration

of EPO in planta and on the evaluation of prEPO’s biological

activity in various in vivo models of tissue disease and injury.

Experimental procedures

Construction of plant expression vectors

A plant-optimized gene (SPTob·EPOOpt) with a PR1b secretory signalpeptide from tobacco (Cutt et al., 1988) was designed to mimic thecodon usage of highly expressed N. tabacum genes, whilst avoidingall potentially deleterious processing signals and destabilizing motifs.Briefly, the entire synthetic gene was constructed using a combinedligase chain reaction/polymerase chain reaction (LCR/PCR) approach(Au et al., 1998) with a set of overlapping oligonucleotides designedby the web-based program Gene2Oligo (Rouillard et al., 2004). Thisapproach was also employed to synthesize the native human EPOcDNA sequence with its own signal peptide (SPEPO·EPONat), thechloroplast transit peptide (TPRuB) and the ELP and GFP fusionpartners. The minor nucleotide changes necessary to generateSPTob·EPOR103 and SPTob·EPOAgly were performed by substituting oneor more oligonucleotides in the LCR mix, followed by reassembly.To assist in subsequent cloning steps, BamHI and EcoRI restrictionsites were included at the 5′ and 3′ ends of the completed constructs,

respectively. In addition, a KasI site was added to the 3′ end of thevarious EPO genes to create a two-amino-acid linker (glycine-alanine)and to allow for in-frame ligation with various C-terminal fusions,protease cleavage sites, ER retention signals and purification tags.The secEPO and chEPO constructs were synthesized by removingthe appropriate 3′ sequence from SPEPO·EPONat by PCR amplification.The remaining expression constructs were created by interchangingpreviously synthesized construct components using overlap extensionPCR combined with restriction enzyme digestion of C-terminalelements, followed by in-frame ligation. Once completed, the con-structs were digested with BamHI and EcoRI, cloned into pBluescriptfor sequencing and then moved into the plant binary expressionvector pCaMterX (Laurian Robert, Agriculture and Agri-Food Canada,Ottawa, pers. commun.). The coding sequences were under the controlof the dual-enhancer CaMV 35S promoter (Kay et al., 1987), a tCUPtranslational enhancer (Wu et al., 2001) and the nos terminator. Theexpression constructs were electroporated into Agrobacteriumtumefaciens strain EHA105 (Hood et al., 1993) and then used for planttransformation. All expression vector sequences have been depositedin GENBANK (Figure S2, see online ‘Supporting Information’).

Agrobacterium-mediated transient expression assays

and tobacco transformation

For transient expression, the Agrobacterium strains were used toinfiltrate the leaves of 10–14-week-old N. tabacum plants. Agrobac-terium was prepared for leaf infiltration as described previously(Kapila et al., 1997; Yang et al., 2000). Briefly, the Agrobacteriumsuspensions were adjusted to a final optical density at 600 nm(OD600) of 2.4 and then injected into the abaxial air spaces of intactleaves just under the epidermal surface using a 1-mL syringe. Toaccount for plant to plant variability, leaf to leaf variability andposition on a leaf, comparably sized leaves from six different plantsof similar age were agroinfiltrated for each expression construct. Inaddition, the agroinfiltrated panels were systematically distributedacross the leaf surface. After infiltration, the plants were maintainedin a controlled growth chamber at 22 °C with a 16-h photoperiodfor 4 days, and the individual infiltrated panels were collected andanalysed separately.

For the generation of stable transgenic plants, expression constructswere introduced into low-alkaloid tobacco (cv. 81V9) (Menassa et al.,2001) using Agrobacterium-mediated transformation of leaf discs,as described by Miki et al. (1999). Primary transformants were grownin a glasshouse for 4–6 weeks following transfer to soil. The first fourtrue leaves were sampled once they had reached 25 cm in length,and were used to represent the concentration of recombinantprotein in the whole plant. Seeds were collected from the transgenictobacco lines with the highest EPO concentration, and were used toform subsequent generations by self-fertilization and the selection ofhomozygous lines.

Plant protein extraction

For each sample, TSP was extracted from four 7-mm leaf discs(approximate fresh weight, 25 mg) of transgenic and wild-type plantsby homogenization with a Mixer Mill MM 300 (Retsch, Haan, Germany).The resulting frozen powdered leaves were then resuspended at4 °C in 300 μL of extraction buffer [phosphate-buffered saline (PBS),



pH 7.4, 0.1% Tween-20, 2% polyvinylpolypyrrolidone (PVPP), 1 mM

ethylenediaminetetraacetic acid (EDTA), 100 mM ascorbic acid, 1 mM

phenylmethylsulphonylfluoride (PMSF) and 1 μg/mL leupeptin].The extract was clarified twice by centrifugation at 20 000 g for10 min at 4 °C. The TSP concentration was measured according tothe method of Bradford (1976) using Bio-Rad reagent (Bio-Rad,Hercules, CA, USA), with bovine serum albumin as a standard.

Quantification of EPO protein levels by ELISA

The quantification of prEPO in tobacco leaf extracts was achievedby sandwich ELISA. Nunc-Immuno MaxiSorp surface plates (NalgeNunc, Rochester, NY, USA) were coated with 2 μg/mL of mouseanti-EPO monoclonal antibody (01350; Stem Cell Technologies,Vancouver, BC, Canada) diluted in disodium phosphate buffer(0.1 M, pH 9.0), and incubated overnight at 4 °C. The wells wereblocked with 2.7% ELISA Blocking Reagent (Roche, Mannheim,Germany) in PBS for 1 h at room temperature. Plant extracts wereserially diluted in blocking buffer (PBS containing 2.7% ELISABlocking Reagent and 0.05% Tween-20) and incubated on the plateovernight at 4 °C. The plate was then incubated with 4 μg/mL ofrabbit anti-EPO antibody (E2531; Sigma, St. Louis, MO, USA) dilutedin blocking buffer for 1.5 h at room temperature. Next, the plateswere incubated with a 1 : 1000 dilution of horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG (170-6515; Bio-Rad) dilutedin blocking buffer for 1 h at room temperature. The plates werewashed five times between incubation steps with PBS containing 0.05%Tween-20. The plates were developed by the addition of 2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulphonic acid (ABTS) substrate (A-1888;Sigma), and the absorbance was measured at 405 nm with a Bio-Rad550 microplate reader. To generate a standard curve, recombinanthuman EPO (CRE600B; Cell Sciences, Canton, MA, USA) was dilutedwith blocking buffer to concentrations between 0.125 and 8 ng/mL,and processed as described above. Two individual samples weretaken from every transgenic plant or agroinfiltrated leaf panel andtreated independently during ELISA.

Western blot analysis

For Western blot analysis, plant extracts were resolved on a 12%sodium dodecylsulphate (SDS) polyacrylamide gel and then transferredto a nitrocellulose membrane by semi-dry electroblotting. Themembranes were blocked with 1% Western Blocking Reagent(Roche) in Tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl,pH 7.5) overnight at 4 °C. The membranes were incubated with a1 : 500 dilution of rabbit anti-EPO antibody (E3455-10; USBiological,Swampscott, MA, USA) for 1 h at room temperature with gentleshaking. The primary antibody was detected with a 1 : 5000 dilutionof HRP-conjugated goat anti-rabbit IgG (Bio-Rad) and visualizedusing an ECL kit (GE Healthcare, Mississauga, ON, Canada), accordingto the manufacturer’s instructions. The membranes were washed fourtimes between each step with TBS containing 0.05% Tween-20, and allantibodies were diluted in TBS with 0.5% Western Blocking Reagent.

Enzymatic deglycosylation of EPO

Total plant protein extracts and recombinant EPO were deglycosylatedwith PNGaseF (New England Biolabs, Ipswich, MA, USA) or EndoH

(Sigma) for 24 h at 37 °C, according to the manufacturer’s instructions.PNGaseF cleaves all high-mannose, hybrid and complex-typeoligosaccharides from N-linked glycoproteins, except for thoseglycans containing a core α(1,3)-linked fucose residue. EndoH isable to remove high-mannose N-linked glycans, but not complex-type glycans, from glycoproteins. In addition, the Enzymatic ProteinDeglycosylation Kit (Sigma) was employed to remove any potentialO-linked glycans and sialic acids from the protein samples, as describedby the manufacturer. Control samples were treated the same, exceptthat no enzyme was added. Finally, the samples were analysedby sodium dodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblotted as described in the previous section.

Confocal microscopy

For transient expression of the GFP constructs, the appropriateAgrobacterium strains were infiltrated into tobacco leaves as describedabove. For staining of the plasma membrane, a 5-μg/mL solution ofFM4-64 (Invitrogen, Burlington, ON, Canada) was injected intothe abaxial surface of the leaf, and the resulting tissue was excisedand mounted in FM4-64 solution. A Leica TCS SP2 confocal laserscanning microscope equipped with a 63× water immersionobjective (Leica Microsystems, Wetzlar, Germany) was used toexamine the subcellular localization of GFP fluorescence andFM4-64 staining. For the simultaneous imaging of GFP and FM4-64,the two fluorophores were excited with a 488-nm argon laser lineand fluorescence was detected at 500–525 nm and 635–700 nm,respectively.

EPOR-binding assay

The binding capacity of prEPO to human EPOR (E0643; Sigma) wasassessed by modifying the above quantitative ELISA protocol. Briefly,microtitre plates were coated with 4 μg/mL of mouse anti-EPORantibody (MAB3071; R&D Systems, Minneapolis, MN, USA) in disodiumphosphate buffer and incubated overnight at 4 °C. Subsequently,the wells were blocked with 2.7% ELISA Blocking Reagent in PBS for1 h at room temperature. Soluble plant extracts and EPO standardswere serially diluted in blocking buffer containing 200 ng/mL ofEPOR and incubated on the plate overnight at 4 °C. The remainderof the procedure was performed as for the quantitative ELISAdescribed above.

Purification and analysis of biological activity

of plant EPO

For the purification of prEPO, 1 kg of frozen tobacco leaves washomogenized in 3 L of cold extraction buffer. The homogenate wasfiltered through two layers of Miracloth (EMB Biosciences, Mississauga,ON, Canada), and the extract was clarified twice by centrifugation at15 000 g for 15 min at 4 °C. To enrich for prEPO, the supernatantwas filtered through a PLHK 100-kDa and a PLGC 10-kDa Prep/ScaleTFF cartridge (Millipore, Burlington, MA, USA). The resulting 10-kDaretentate was further concentrated using a Jumbosep spin columnwith a 10-kDa cut-off (Pall Corporation, Mississauga, ON, Canada),and then passed through a 0.22-μm membrane filter. Solubleproteins were then applied to a Strep-Tactin MacroPrep column(IBA, St. Louis, MO, USA) and prEPO was eluted according to the



manufacturer’s instructions. The biological activity of prEPO wasassessed by its ability to prevent the cellular death of renal TECs,according to Du et al. (2004) with minor modifications. TECs (2.5 × 105)were added to 24-well plates in triplicate and cultured overnight incomplete K1 medium containing serum. After confluence wasreached, the medium was replaced with fresh K1 medium without serumor growth factors to arrest cell division. TECs were then pretreatedfor 5 h with 70 ng/mL of prEPO or commercial recombinant EPO(Amgen, Thousand Oaks, CA, USA) diluted in K1 medium. To promotecell death, IFN-γ (BD Biosciences, Mississauga, ON, Canada) wasadded to the medium, together with additional EPO (70 ng/mL), andthe TECs were cultured for an additional 24 h. TEC monolayers werereleased from the plates by a brief incubation with trypsin-EDTAsolution (Sigma), and then incubated with Annexin-V conjugatedwith fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D(7-AAD) (BD Biosciences). The level of cell death was determinedwith a FACSCalibur flow cytometer and analysed by CellQuestsoftware (BD Biosciences).

Acknowledgements

The authors wish to thank Laura Slade for technical assistance

and Alex Molnar for assistance with the preparation of the

figures. Thanks are due to Dr Jussi Joensuu, Dr Rima Menassa,

Dr Patrick Telmer and Alex Richman for critical comments on

the manuscript and helpful discussions. This research was

supported by the Agriculture and Agri-Food Canada Matching

Investment Initiative Programme. We thank the Natural

Sciences and Engineering Research Council (NSERC) Postgraduate

Scholarship for providing financial support to A.J.C.

References

Au, L.C., Yang, F.Y., Yang, W.J., Lo, S.H. and Kao, C.F. (1998) Genesynthesis by a LCR-based approach: high-level production ofleptin-L54 using synthetic gene in Escherichia coli. Biochem.Biophys. Res. Commun. 248, 200–203.

Bardor, M., Faveeuw, C., Fitchette, A.C., Gilbert, D., Galas, L., Trottein,F., Faye, L. and Lerouge, P. (2003) Immunoreactivity in mammalsof two typical plant glyco-epitopes, core alpha (1,3)-fucose andcore xylose. Glycobiology, 13, 427–434.

Blobel, G., Walter, P., Chang, C.N., Goldman, B.M., Erickson, A.H.and Lingappa, V.R. (1979) Translocation of proteins acrossmembranes: the signal hypothesis and beyond. Symp. Soc. Exp.Biol. 33, 9–36.

Boevink, P., Santa-Cruz, S., Harris, N. and Oparka, K.J. (1996)Virus-mediated delivery of the green fluorescent protein to theendoplasmic reticulum of plant cells. Plant J. 10, 935–941.

Bradford, M.M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein–dye binding. Anal. Biochem. 72, 248–254.

Brandle, J. (2004) The field evaluation of transgenic crops engineeredto produce recombinant proteins. In: Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins (Fischer, R. and Schillberg,S., eds), pp. 69–76. Weinheim: Wiley-VCH.

Cabanes-Macheteau, M., Fitchette-Laine, A.C., Loutelier-Bourhis,C., Lange, C., Vine, N.D., Ma, J.K., Lerouge, P. and Faye, L. (1999)

N-Glycosylation of a mouse IgG expressed in transgenic tobaccoplants. Glycobiology, 9, 365–372.

Campbell, W.H. and Gowri, G. (1990) Codon usage in higher plants,green algae, and cyanobacteria. Plant Physiol. 92, 1–11.

Cheetham, J.C., Smith, D.M., Aoki, K.H., Stevenson, J.L., Hoeffel,T.J., Syed, R.S., Egrie, J. and Harvey, T.S. (1998) NMR structure ofhuman erythropoietin and a comparison with its receptor boundconformation. Nat. Struct. Biol. 5, 861–866.

Cheon, B.Y., Kim, H.J., Oh, K.H., Bahn, S.C., Ahn, J.H., Choi, J.W.,Ok, S.H., Bae, J.M. and Shin, J.S. (2004) Overexpression of humanerythropoietin (EPO) affects plant morphologies: retardedvegetative growth in tobacco and male sterility in tobacco andArabidopsis. Transgenic Res. 13, 541–549.

Chiapello, H., Lisacek, F., Caboche, M. and Henaut, A. (1998)Codon usage and gene function are related in sequences ofArabidopsis thaliana. Gene, 209, GC1–GC38.

Cutt, J.R., Dixon, D.C., Carr, J.P. and Klessig, D.F. (1988) Isolationand nucleotide sequence of cDNA clones for the pathogenesis-related proteins PR1a, PR1b and PR1c of Nicotiana tabacum cv.Xanthi nc induced by TMV infection. Nucleic Acids Res. 16, 9861.

Cuzzocrea, S., Mazzon, E., Di Paola, R., Patel, N.S., Genovese, T.,Muia, C., De Sarro, A. and Thiemermann, C. (2004) Erythropoietinreduces the development of experimental inflammatory boweldisease. J. Pharmacol. Exp. Ther. 311, 1272–1280.

Darling, R.J., Kuchibhotla, U., Glaesner, W., Micanovic, R., Witcher,D.R. and Beals, J.M. (2002) Glycosylation of erythropoietin affectsreceptor binding kinetics: role of electrostatic interactions.Biochemistry, 41, 14 524–14 531.

Delorme, E., Lorenzini, T., Giffin, J., Martin, F., Jacobsen, F., Boone,T. and Elliott, S. (1992) Role of glycosylation on the secretionand biological activity of erythropoietin. Biochemistry, 31,9871–9876.

Du, C., Jiang, J., Guan, Q., Yin, Z., Masterson, M., Parbtani, A.,Zhong, R. and Jevnikar, A.M. (2004) Renal tubular epithelial cellself-injury through Fas/Fas ligand interaction promotes renalallograft injury. Am. J. Transplant. 4, 1583–1594.

Dus Santos, M.J., Wigdorovitz, A., Trono, K., Rios, R.D., Franzone,P.M., Gil, F., Moreno, J., Carrillo, C., Escribano, J.M. and Borca,M.V. (2002) A novel methodology to develop a foot and mouthdisease virus (FMDV) peptide-based vaccine in transgenic plants.Vaccine, 20, 1141–1147.

Egrie, J.C. and Browne, J.K. (2001) Development and characterizationof novel erythropoiesis stimulating protein (NESP). Br. J. Cancer,84(Suppl. 1), 3–10.

Egrie, J.C., Strickland, T.W. and Lane, J. (1986) Characterization andbiological effects of recombinant human erythropoietin. Immuno-biology, 172, 213–224.

Elliott, S., Chang, D., Delorme, E., Eris, T. and Lorenzini, T. (2004a)Structural requirements for additional N-linked carbohydrate onrecombinant human erythropoietin. J. Biol. Chem. 279, 16 854–16 862.

Elliott, S., Egrie, J., Browne, J., Lorenzini, T., Busse, L., Rogers, N. andPonting, I. (2004b) Control of rHuEPO biological activity: the roleof carbohydrate. Exp. Hematol. 32, 1146–1155.

Erbayraktar, S., Grasso, G., Sfacteria, A., Xie, Q.W., Coleman, T.,Kreilgaard, M., Torup, L., Sager, T., Erbayraktar, Z., Gokmen, N.,Yilmaz, O., Ghezzi, P., Villa, P., Fratelli, M., Casagrande, S., Leist, M.,Helboe, L., Gerwein, J., Christensen, S., Geist, M.A., Pedersen, L.O.,Cerami-Hand, C., Wuerth, J.P., Cerami, A. and Brines, M. (2003)Asialoerythropoietin is a nonerythropoietic cytokine with broad



neuroprotective activity in vivo. Proc. Natl. Acad. Sci. USA, 100,6741–6746.

Fibi, M.R., Hermentin, P., Pauly, J.U., Lauffer, L. and Zettlmeissl, G.(1995) N- and O-glycosylation muteins of recombinant humanerythropoietin secreted from BHK-21 cells. Blood, 85, 1229–1236.

Fiedler, U., Phillips, J., Artsaenko, O. and Conrad, U. (1997) Optimizationof scFv antibody production in transgenic plants. Immunotechnology,3, 205–216.

Floss, D.M., Sack, M., Stadlmann, J., Rademacher, T., Scheller, J.,Stoger, E., Fischer, R. and Conrad, U. (2008) Biochemical andfunctional characterization of anti-HIV antibody-ELP fusionproteins from transgenic plants. Plant Biotechnol. J. 6, 379–391.

Fukuda, M.N., Sasaki, H., Lopez, L. and Fukuda, M. (1989) Survivalof recombinant erythropoietin in the circulation: the role ofcarbohydrates. Blood, 73, 84–89.

Ghezzi, P. and Brines, M. (2004) Erythropoietin as an antiapoptotic,tissue-protective cytokine. Cell Death Differ. 11(Suppl. 1), S37–S44.

Gil, F., Brun, A., Wigdorovitz, A., Catala, R., Martinez-Torrecuadrada,J.L., Casal, I., Salinas, J., Borca, M.V. and Escribano, J.M. (2001)High-yield expression of a viral peptide vaccine in transgenicplants. FEBS Lett. 488, 13–17.

Gomord, V., Chamberlain, P., Jefferis, R. and Faye, L. (2005) Biophar-maceutical production in plants: problems, solutions andopportunities. Trends Biotechnol. 23, 559–565.

Goto, M., Akai, K., Murakami, A., Hashimoto, C., Tsuda, E., Ueda, M.,Kawanishi, G., Takahashi, N., Ishimoto, A., Chiba, H. and Sasaki, R.(1988) Production of recombinant human erythropoietin inmammalian cells: host-cell dependency of the biological activityof the cloned glycoprotein. Bio/Technology, 6, 67–71.

Grodberg, J., Davis, K.L. and Sykowski, A.J. (1993) Alanine scanningmutagenesis of human erythropoietin identifies four aminoacids which are critical for biological activity. Eur. J. Biochem. 218,597–601.

Guneli, E., Cavdar, Z., Islekel, H., Sarioglu, S., Erbayraktar, S., Kiray,M., Sokmen, S., Yilmaz, O. and Gokmen, N. (2007) Erythropoietinprotects the intestine against ischemia/reperfusion injury in rats.Mol. Med. 13, 509–517.

Gutierrez, R.A., MacIntosh, G.C. and Green, P.J. (1999) Currentperspectives on mRNA stability in plants: multiple levels andmechanisms of control. Trends Plant Sci. 4, 429–438.

Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y.,Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H., Choi, B.K., Hopkins,D., Wischnewski, H., Roser, J., Mitchell, T., Strawbridge, R.R.,Hoopes, J., Wildt, S. and Gerngross, T.U. (2006) Humanization ofyeast to produce complex terminally sialylated glycoproteins.Science, 313, 1441–1443.

Higuchi, M., Oh-eda, M., Kuboniwa, H., Tomonoh, K., Shimonaka,Y. and Ochi, N. (1992) Role of sugar chains in the expression ofthe biological activity of human erythropoietin. J. Biol. Chem. 267,7703–7709.

Hobbs, S.L.A., Kpodar, P. and DeLong, C.M.O. (1990) The effect ofT-DNA copy number, position and methylation on reporter geneexpression in tobacco transformants. Plant Mol. Biol. 15, 851–864.

Hondred, D., Walker, J.M., Mathews, D.E. and Vierstra, R.D. (1999)Use of ubiquitin fusions to augment protein expression intransgenic plants. Plant Physiol. 119, 713–724.

Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993) NewAgrobacterium helper plasmids for gene transfer to plants.Transgenic Res. 2, 208–218.

Huang, Z. and Mason, H.S. (2004) Conformational analysis ofhepatitis B surface antigen fusions in an Agrobacterium-mediatedtransient expression system. Plant Biotechnol. J. 2, 241–249.

Huang, Z., Dry, I., Webster, D., Strugnell, R. and Wesselingh, S. (2001)Plant-derived measles virus hemagglutinin protein inducesneutralizing antibodies in mice. Vaccine, 19, 2163–2171.

Hwang, C., Sinsky, A.J. and Lodish, H.F. (1992) Oxidised redox stateof glutathione in the endoplasmic reticulum. Science, 257, 3747–3753.

Jacobs, K., Shoemaker, C. and Rudersdorf, R. (1985) Isolation andcharacterization of genomic and cDNA clones of human erythro-poietin. Nature, 313, 806–810.

Johnson, D.W., Forman, C. and Vesey, D.A. (2006a) Novel reno-protective actions of erythropoietin: new uses for an old hormone.Nephrology, 11, 306–312.

Johnson, D.W., Pat, B., Vesey, D.A., Guan, Z., Endre, Z. and Gobe,G.C. (2006b) Delayed administration of darbepoetin or erythro-poietin protects against ischemic acute renal injury and failure.Kidney Int. 69, 1806–1813.

Juul, S.E., Yachnis, A.T. and Christensen, R.D. (1998) Tissue distributionof erythropoietin and erythropoietin receptor in the developinghuman fetus. Early Hum. Dev. 52, 235–249.

Juul, S.E., Joyce, A.E., Zhao, Y. and Ledbetter, D.J. (1999) Why iserythropoietin present in human milk? Studies of erythropoietinreceptors on enterocytes of human and rat neonates. Pediatr. Res.46, 263–268.

Kang, T.J., Han, S.C., Jang, M.O., Kang, K.H., Jang, Y.S. and Yang,M.S. (2004) Enhanced expression of B-subunit of Escherichia coliheat-labile enterotoxin in tobacco by optimization of codingsequence. Appl. Biochem. Biotechnol. 117, 175–187.

Kapila, J., De-Rycke, R. and Angenon, G. (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. PlantSci. 122, 101–108.

Kay, R., Chan, A., Daly, M. and McPherson, J. (1987) Duplication ofCaMV 35S promoter sequences creates a strong enhancer forplant genes. Science, 236, 1299–1302.

Kim, Y.K., Shin, H.S., Tomiya, N., Lee, Y.C., Betenbaugh, M.J. and Cha,H.J. (2005) Production and N-glycan analysis of secreted humanerythropoietin glycoprotein in stably transfected Drosophila S2cells. Biotechnol. Bioeng. 92, 452–461.

Koziel, M.G., Carozzi, N.B. and Desai, N. (1996) Optimizing expressionof transgenes with an emphasis on post-transcriptional events.Plant Mol. Biol. 32, 393–405.

Krysan, P.J., Young, J.C., Jester, P.J., Monson, S., Copenhaver, G.,Preuss, D. and Sussman, M.R. (2002) Characterization of T-DNAinsertion sites in Arabidopsis thaliana and the implications forsaturation mutagenesis. OMICS, 6, 163–174.

Lee-Huang, S. (1984) Cloning and expression of human erythropoietincDNA in Escherichia coli. Proc. Natl. Acad. Sci. USA, 81, 2708–2712.

Leist, M., Ghezzi, P., Grasso, G., Bianchi, R., Villa, P., Fratelli, M., Savino,C., Bianchi, M., Nielsen, J., Gerwien, J., Kallunki, P., Larsen, A.K.,Helboe, L., Christensen, S., Pedersen, L.O., Nielsen, M., Torup, L.,Sager, T., Sfacteria, A., Erbayraktar, S., Erbayraktar, Z., Gokmen,N., Yilmaz, O., Cerami-Hand, C., Xie, Q.W., Coleman, T., Cerami,A. and Brines, M. (2004) Derivatives of erythropoietin that aretissue protective but not erythropoietic. Science, 305, 239–242.

Lin, F.K., Suggs, S., Lin, C.H., Browne, J.K., Smalling, R., Egrie, J.C.,Chen, K.K., Fox, G.M., Martin, F., Stabinsky, Z., Badrawi, S.M.,



Lai, P.H. and Goldwasser, E. (1985) Cloning and expressionof the human erythropoietin gene. Proc. Natl. Acad. Sci. USA, 82,7580–7584.

Lin, M., Rose-John, S., Grotzinger, J., Conrad, U. and Scheller, J. (2006)Functional expression of a biologically active fragment ofsoluble gp130 as an ELP-fusion protein in transgenic plants:purification via inverse transition cycling. Biochem. J. 398, 577–583.

Lonsdale, D.M., Moisan, L.J. and Harvey, A.J. (1998) The effect ofaltered codon usage on luciferase activity in tobacco, maize andwheat. Plant Cell Rep. 17, 396–399.

Ma, J.K., Drake, P.M. and Christou, P. (2003) The production ofrecombinant pharmaceutical proteins in plants. Nat. Rev. Genet.4, 794–805.

Ma, S., Huang, Y., Davis, A., Yin, Z., Mi, Q., Menassa, R., Brandle,J.E. and Jevnikar, A.M. (2005) Production of biologically activehuman interleukin-4 in transgenic tobacco and potato. PlantBiotechnol. J. 3, 309–318.

Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V. and Gleba,Y. (2005) Systemic Agrobacterium tumefaciens-mediatedtransfection of viral replicons for efficient transient expression inplants. Nat. Biotechnol. 23, 718–723.

Masuda, S., Nagao, M. and Sasaki, R. (1999) Erythropoietic, neuro-trophic, and angiogenic functions of erythropoietin and regulationof erythropoietin production. Int. J. Hematol. 70, 1–6.

Matsumoto, S., Ikura, K., Ueda, M. and Sasaki, R. (1995) Character-ization of a human glycoprotein (erythropoietin) produced incultured tobacco cells. Plant Mol. Biol. 27, 1163–1172.

Matthews, D.J., Topping, R.S., Cass, R.T. and Giebel, L.B. (1996) Asequential dimerization mechanism for erythropoietin receptoractivation. Proc. Natl. Acad. Sci. USA, 93, 9471–9476.

Menassa, R., Nguyen, V., Jevnikar, A. and Brandle, J. (2001) Aself-contained system for the field production of plantrecombinant interleukin-10. Mol. Breed. 8, 177–185.

Menassa, R., Du, C., Yin, Z.Q., Ma, S., Poussier, P., Brandle, J. andJevnikar, A.M. (2007) Therapeutic effectiveness of orally adminis-tered transgenic low-alkaloid tobacco expressing humaninterleukin-10 in a mouse model of colitis. Plant Biotechnol. J. 5,50–59.

Meyer, D.E. and Chilkoti, A. (1999) Purification of recombinantproteins by fusion with thermally-responsive polypeptides. Nat.Biotechnol. 17, 1112–1115.

Miki, B.L.A., McHugh, S.G., Labbe, H., Ouellet, T., Tolman, J.H. andBrandle, J.E. (1999) Transgenic tobacco: gene expression andapplications. In: Biotechnology in Agriculture and Forestry:Transgenic Medicinal Plants (Bajaj, Y.P.S., ed.), pp. 336–354.Berlin/Heidelberg: Springer-Verlag.

Narhi, L.O., Arakawa, T., Aoki, K., Wen, J., Elliott, S., Boone, T. andCheetham, J. (2001) Asn to Lys mutations at three sites whichare N-glycosylated in the mammalian protein decrease theaggregation of Escherichia coli-derived erythropoietin. ProteinEng. 14, 135–140.

Nuttall, J., Ma, J.K. and Frigerio, L. (2005) A functional antibodylacking N-linked glycans is efficiently folded, assembled andsecreted by tobacco mesophyll protoplasts. Plant Biotechnol. J. 3,497–504.

Obregon, P., Chargelegue, D., Drake, P.M., Prada, A., Nuttall, J.,Frigerio, L. and Ma, J.K. (2006) HIV-1 p24-immunoglobulin fusionmolecule: a new strategy for plant-based protein production.Plant Biotechnol. J. 4, 195–207.

Pagny, S., Cabanes-Macheteau, M., Gillikin, J.W., Leborgne-Castel, N.,Lerouge, P., Boston, R.S., Faye, L. and Gomord, V. (2000) Proteinrecycling from the Golgi apparatus to the endoplasmic reticulumin plants and its minor contribution to calreticulin retention. PlantCell, 12, 739–756.

Patel, J., Zhu, H., Menassa, R., Gyenis, L., Richman, A. and Brandle,J. (2007) Elastin-like polypeptide fusions enhance the accumulationof recombinant proteins in tobacco leaves. Transgenic Res. 16,239–249.

Perlak, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L. and Fischhoff,D.A. (1991) Modification of the coding sequence enhances plantexpression of insect control protein genes. Proc. Natl. Acad. Sci.USA, 88, 3324–3328.

Petruccelli, S., Otegui, M.S., Lareu, F., Tran Dinh, O., Fitchette, A.C.,Circosta, A., Rumbo, M., Bardor, M., Carcamo, R., Gomord, V.and Beachy, R.N. (2006) A KDEL-tagged monoclonal antibody isefficiently retained in the endoplasmic reticulum in leaves, but isboth partially secreted and sorted to protein storage vacuoles inseeds. Plant Biotechnol. J. 4, 511–527.

Raju, K. and Anwar, R.A. (1987) Primary structures of bovine elastina, b, and c deduced from the sequences of cDNA clones. J. Biol.Chem. 262, 5755–5762.

Rapoport, T.A., Jungnickel, B. and Kutay, U. (1996) Protein transportacross the eukaryotic endoplasmic reticulum and bacterial innermembranes. Annu. Rev. Biochem. 65, 271–303.

Rodriguez, M., Ramirez, N.I., Ayala, M., Freyre, F., Perez, L., Triguero,A., Mateo, C., Selman-Housein, G., Gavilondo, J.V. and Pujol, M.(2005) Transient expression in tobacco leaves of an aglycosylatedrecombinant antibody against the epidermal growth factorreceptor. Biotechnol. Bioeng. 89, 188–194.

Rouillard, J.M., Lee, W., Truan, G., Gao, X., Zhou, X. and Gulari, E.(2004) Gene2Oligo: oligonucleotide design for in vitro genesynthesis. Nucleic Acids Res. 32, W176–W180.

Rouwendal, G.J., Mendes, O., Wolbert, E.J. and Douwe de Boer, A.(1997) Enhanced expression in tobacco of the gene encodinggreen fluorescent protein by modification of its codon usage.Plant Mol. Biol. 33, 989–999.

Schaaf, A., Tintelnot, S., Baur, A., Reski, R., Gorr, G. and Decker, E.L.(2005) Use of endogenous signal sequences for transient pro-duction and efficient secretion by moss (Physcomitrella patens)cells. BMC Biotechnol. 5, 30.

Scheller, J., Henggeler, D., Viviani, A. and Conrad, U. (2004) Purificationof spider silk-elastin from transgenic plants and application forhuman chondrocyte proliferation. Transgenic Res. 13, 51–57.

Scheller, J., Leps, M. and Conrad, U. (2006) Forcing single-chainvariable fragment production in tobacco seeds by fusion toelastin-like polypeptides. Plant Biotechnol. J. 4, 243–249.

Schouten, A., Rossien, J., Van Engelen, F.A., De Jong, G.A., Borst-Vrenssen, A.W., Zilverentant, J.F., Bosch, D., Stiekema, W.J.,Gommers, F.J., Schouts, A. and Bakker, J. (1996) The C-terminalKDEL sequence increases the expression level of a single-chainantibody designed to be targeted to both the cytosol and thesecretory pathway in transgenic tobacco. Plant Mol. Biol. 30,781–793.

Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C.,Van Den Elzen, P.J.M. and Hoekema, A. (1990) Production ofcorrectly processed human serum albumin in transgenic plants.Bio/Technology, 8, 217–221.

Spandou, E., Tsouchnikas, I., Karkavelas, G., Dounousi, E., Simeonidou,C., Guiba-Tziampiri, O. and Tsakiris, D. (2006) Erythropoietin



attenuates renal injury in experimental acute renal failureischaemic/reperfusion model. Nephrol. Dial. Transplant. 21, 330–336.

Spivak, J.L. and Hogans, B.B. (1989) The in vivo metabolism ofrecombinant human erythropoietin in the rat. Blood, 73, 90–99.

Sriraman, R., Bardor, M., Sack, M., Vaquero, C., Faye, L., Fischer,R., Finnern, R. and Lerouge, P. (2004) Recombinant anti-hCGantibodies retained in the endoplasmic reticulum of transformedplants lack core-xylose and core-alpha(1,3)-fucose residues. PlantBiotechnol. J. 2, 279–287.

Stohlawetz, P.J., Dzirlo, L., Hergovich, N., Lackner, E., Mensik, C.,Eichler, H.G., Kabrna, E., Geissler, K. and Jilma, B. (2000) Effectsof erythropoietin on platelet reactivity and thrombopoiesis inhumans. Blood, 95, 2983–2989.

Streatfield, S.J. (2007) Approaches to achieve high-level heterologousprotein production in plants. Plant Biotechnol. J. 5, 2–15.

Takeuchi, M., Inoue, N., Strickland, T.W., Kubota, M., Wada, M.,Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A.(1989) Relationship between sugar chain structure and biologicalactivity of recombinant human erythropoietin produced inChinese hamster ovary cells. Proc. Natl. Acad. Sci. USA, 86, 7819–7822.

Tekoah, Y., Ko, K., Koprowski, H., Harvey, D.J., Wormald, M.R.,Dwek, R.A. and Rudd, P.M. (2004) Controlled glycosylation oftherapeutic antibodies in plants. Arch. Biochem. Biophys. 426,266–278.

Toledo, J.R., Sanchez, O., Segui, R.M., Garcia, G., Montanez, M.,Zamora, P.A., Rodriguez, M.P. and Cremata, J.A. (2006) Highexpression level of recombinant human erythropoietin in themilk of non-transgenic goats. J. Biotechnol. 123, 225–235.

Triguero, A., Cabrera, G., Cremata, J.A., Yuen, C.T., Wheeler, J. andRamirez, N.I. (2005) Plant-derived mouse IgG monoclonalantibody fused to KDEL endoplasmic reticulum-retention signalis N-glycosylated homogeneously throughout the plant withmostly high-mannose-type N-glycans. Plant Biotechnol. J. 3,449–457.

Tucker, J. and Yakatan, S. (2008) Biogenerics 2007: how far have wecome? J. Comm. Biotechnol. 14, 56–64.

Van Molle, I., Joensuu, J.J., Buts, L., Panjikar, S., Kotiaho, M., Bouckaert, J.,Wyns, L., Niklander-Teeri, V. and De Greve, H. (2007) Chloroplastsassemble the major subunit FaeG of Escherichia coli F4 (K88)fimbriae to strand-swapped dimers. J. Mol. Biol. 368, 791–799.

Wasley, L.C., Timony, G., Murtha, P., Stoudemire, J., Dorner, A.J.,Caro, J., Krieger, M. and Kaufman, R.J. (1991) The importance ofN- and O-linked oligosaccharides for the biosynthesis and in vitroand in vivo biologic activities of erythropoietin. Blood, 77, 2624–2632.

Weise, A., Altmann, F., Rodriguez-Franco, M., Sjoberg, E.R., Baumer,W., Launhardt, H., Kietzmann, M. and Gorr, G. (2007) High-levelexpression of secreted complex glycosylated recombinanthuman erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant. Plant Biotechnol. J. 5, 389–401.

Wroblewski, T., Tomczak, A. and Michelmore, R. (2005) Optimizationof Agrobacterium-mediated transient assays of gene expressionin lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259–273.

Wu, K., Malik, K., Tian, L., Hu, M., Martin, T., Foster, E., Brown, D.and Miki, B. (2001) Enhancers and core promoter elements areessential for the activity of a cryptic gene activation sequencefrom tobacco, tCUP. Mol. Gen. Genom. 265, 763–770.

Yamaguchi, K., Akai, K., Kawanishi, G., Ueda, M., Masuda, S. andSasaki, R. (1991) Effects of site-directed removal of N-glycosylationsites in human erythropoietin on its production and biologicalproperties. J. Biol. Chem. 266, 20 434–20 439.

Yang, Y., Li, R. and Qi, M. (2000) In vivo analysis of plant promotersand transcription factors by agroinfiltration of tobacco leaves.Plant J. 22, 543–551.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Tobacco optimization of the human erythropoietin

(EPO) gene. (a) Comparison between the native human EPO

gene and the synthetic tobacco-optimized EPO gene, demon-

strating the removal and avoidance of many deleterious

processing and/or instability signals, whilst mimicking the

codon usage and optimal codon context of highly expressed

tobacco genes. (b) The global genetic changes introduced

during the optimization of the human EPO gene for

enhanced plant expression.

Figure S2 List of the GENBANK accession numbers for the

presented plant expression constructs.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material) should

be directed to the corresponding author for the article.

Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

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