ORIGINAL PAPER

Expression and purification of an anti-Foot-and-mouthdisease virus single chain variable antibody fragmentin tobacco plants

J. J. Joensuu Æ K. D. Brown Æ A. J. Conley ÆA. Clavijo Æ R. Menassa Æ J. E. Brandle

Received: 10 November 2008 / Accepted: 20 March 2009 / Published online: 3 April 2009

� Her Majesty the Queen in Right of Canada 2009

Abstract Low-cost recombinant antibodies could

provide a new strategy to control Foot-and-mouth

disease virus (FMDV) outbreaks by passive immuni-

zation of susceptible animals. In this study, a single

chain variable antibody fragment (scFv) recognizing

FMDV coat protein VP1 was expressed in transgenic

tobacco plants. To enhance the accumulation of scFv

protein, the codon-usage of a murine hybridoma-

derived scFv gene was adjusted to mimic highly

expressed tobacco genes and fused to an elastin-like

polypeptide (ELP) tag. This scFv–ELP fusion accu-

mulated up to 0.8% of total soluble leaf protein in

transgenic tobacco. To recover scFv–ELP protein

from the leaf extract, a simple and scalable purifica-

tion strategy was established. Purified scFv–ELP

fusion was cleaved to separate the scFv portion.

Finally, it was shown that the purified scFv proteins

retained their capacity to bind the FMDV in the

absence or presence of ELP fusion.

Keywords Foot-and-mouth disease, FMD �Elastin-like polypeptide, ELP �Single chain variable antibody fragment, scFv �Codon optimization � Transgenic tobacco

Introduction

Foot-and-mouth disease (FMD) affects all domesti-

cated cloven-hoofed animals (especially bovine,

porcine, and ovine) as well as wild ruminants. The

causative Foot-and-mouth disease virus (FMDV), an

aphthovirus of the Picornaviridae family is highly

contagious, and FMD is the most significant con-

straint to international trade in live animals and

animal products today (Grubman and Baxt 2004).

Despite the fact that current whole-virus-based vac-

cines remain an important means of mitigation in

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-009-9257-0) containssupplementary material, which is available to authorized users.

J. J. Joensuu � K. D. Brown � A. J. Conley �R. Menassa (&) � J. E. Brandle

Agriculture and Agri-Food Canada, 1391 Sandford Street,

London, ON N5V 4T3, Canada

e-mail: Rima.Menassa@agr.gc.ca

A. J. Conley

Department of Biology, University of Western Ontario,

London, ON N6A 5B7, Canada

A. Clavijo

Canadian Food Inspection Agency, Winnipeg,

MB R3E 3M4, Canada

J. E. Brandle

Vineland Research and Innovation Centre,

Vineland Station, ON L0R 2E0, Canada

Present Address:J. J. Joensuu

VTT Technical Research Centre of Finland,

P.O. Box 1000, 02044 VTT Espoo, Finland

123

Transgenic Res (2009) 18:685–696

DOI 10.1007/s11248-009-9257-0

areas where the disease is endemic (Doel 2003), there

are several limitations that restrict their utility either

as a prophylactic treatment or to control outbreaks in

FMDV-free areas. Those limitations are: (1) high-

containment facilities are required for the vaccine

production, (2) current measures to discriminate

between infected and vaccinated animals are

inadequate, (3) vaccinated animals can develop a

sub-clinical carrier state during outbreaks, and (4)

vaccination cannot always protect susceptible ani-

mals quickly enough to prevent infection (Grubman

and Baxt 2004). As a consequence most FMDV-free

countries do not vaccinate against FMD and the

recent outbreaks have been controlled by mass

culling of all infected and suspected animals (Cottam

et al. 2008; Davies 2002; Yang et al. 1999). FMDV

outbreaks have a severe impact on the economy of

effected countries, for example it was estimated that

the FMD epidemic in the United Kingdom in 2001

led to the destruction of over 4 million animals with a

total cost of several billion pounds sterling (Davies

2002).

In order to limit FMD outbreaks, passive immu-

nization with low-cost recombinant antibodies is an

attractive alternative to protect susceptible animals in

protection zones surrounding the infection sites.

Transgenic plants carry a great potential for the

large-scale production of various recombinant pro-

teins, including monoclonal antibodies or derived

antibody fragments (Ma et al. 2005).

Plants have advantages over conventional expres-

sion systems including low production costs, rapid

scalability, the absence of animal pathogens and

the ability to correctly fold and assemble complex

multimeric proteins (Twyman et al. 2003). In

particular, tobacco is a well-established system for

recombinant protein production, it combines the

ease of transformation with a high biomass yield

and the platform is based on leaves, which removes

the need for flowering; thus minimizing the risk of

gene leakage into the environment through pollen or

seed dispersal (Rymerson et al. 2002). Notably,

tobacco addresses many regulatory barriers because

it is a non-food, non-feed crop, therefore eliminating

the risk of plant-made recombinant proteins entering

the food chain (Menassa et al. 2001). Although

tobacco is inherently biosafe, the low yield of some

recombinant proteins in tobacco leaves often limits

economical production (Doran 2006). Furthermore,

the presence of tobacco alkaloids may preclude it

from oral delivery, so the target protein must be

purified prior to administration, which can contrib-

ute to greater than 80% of the product cost (Kusnadi

et al. 1997). Therefore, a strategy is needed for

increasing the accumulation of recombinant proteins

in plants, while also assisting in their subsequent

purification.

Synthetic elastin-like polypeptides (ELPs) are

biopolymers composed of the repeating pentapeptide

sequence VPGXG, where the guest residue X can be

any amino acid except proline (Urry 1988). In an

aqueous solution, ELPs are soluble below their

transition temperature, and become reversibly insol-

uble if heated above their transition temperature

(Tt; Urry 1997). This thermally responsive property

of ELP is also effective when ELP is fused to various

partners, enabling a simple nonchromatographic

method for protein purification called ‘inverse tran-

sition cycling’ (ITC; Meyer and Chilkoti 1999).

Interestingly, in addition to functioning as a method

of purification, ELP fusions have been also shown to

enhance the accumulation of recombinant proteins in

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

and seeds (Scheller et al. 2006).

In this work, we evaluated plant codon optimiza-

tion and ELP fusion strategies to enhance the

accumulation of an anti-FMDV single chain variable

antibody fragment (scFv) in transgenic tobacco

plants. In addition, a simple and scalable purification

strategy is described to recover plant-made scFvs.

Materials and methods

Purification of FMDV particles and production

of anti-FMDV mAb

Foot-and-mouth disease virus strain A24/Cruzeiro

was obtained from the World Reference Laboratory

for FMD at the Institute for Animal Health, Pirbright,

UK. FMDV was grown in baby hamster kidney cells

(BHK-21) in Glasgow’s minimum essential medium

supplemented with 1% fetal bovine serum, 2 mM

L-glutamine, and 50 lg/ml gentamycin. FMDV was

harvested 24 h post-infection and clarified by centri-

fugation at 18009g for 30 min. About 10 mM

2-bromoethylamine hydrobromide (BEI) was used

to inactivate the virus for 24 h at 37�C. After 24 h,

686 Transgenic Res (2009) 18:685–696

123

any remaining BEI was inactivated using sodium

thiosulphate to a final concentration of 2%.

The inactivated virus culture was clarified with

chloroform extraction (10% v/v) and concentrated by

mixing the supernatant with 50% polyethylene glycol

(PEG, MW 8000) to a final concentration of 7.5% and

stirred for 12 h at 4�C. The precipitate obtained by

centrifugation was resuspended in Tris–NaCl buffer

(150 mM NaCl, 50 mM Tris, pH 7.8). The concen-

trated virus was layered onto a continuous 15–45%

sucrose density gradient and ultracentrifuged at

96,0009g for 2.5 h at 4�C. The band containing

purified virus particles was collected and stored at

-70�C. All the handling of the live and inactivated

FMDV was performed under biosafety level 3

containment at the National Centre for Foreign

Animal Disease (Winnipeg, Canada).

Purified, inactivated A24/Cruzeiro FMDV was

used as the antigen for the immunization of mice.

The immunization protocol and mAb production are

described elsewhere (Yang et al. 2008). Briefly,

female BALB/C mice were immunized with 20 lg of

inactivated FMDV in an equal volume of TiterMax

Gold (TiterMax USA Inc., Norcross, USA) subcuta-

neously. Two identical boosts were given at 4 week

intervals. Finally, the mice were boosted with the

same dose of antigen in phosphate-buffered saline

(PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM

Na2HPO4, 1.8 mM KH2PO4, pH 7.5) by intravenous

injection 4 days before fusion. Immunized spleen

cells were fused with myeloma cells (P3X63

Ag8.653). After 2 weeks, hybridoma supernatants

were screened using an antigen capture ELISA using

concentrated FMDV (Ferris et al. 1988).

Creation of a high expression codon usage table

for tobacco

Complete coding DNA sequences (CDS) encoding

nuclear genes from Nicotiana tabacum were

extracted from the GenBank DNA sequence database

(release 134, accessed Feb 15th, 2003) using

the WWW-Query interface (http://pbil.univ-lyon1.fr/

search/query.html). The initial dataset of 1,114

sequences was further selected for base pair length

greater than or equal to 300. The sequence annota-

tions of the remaining 1,061 CDS were scanned to

facilitate the removal of all viral and transposable

element DNA coding sequences. Only legitimate

complete protein coding sequences in the remaining

1,031 CDS were allowed in the dataset based on the

criteria that each CDS must contain a start and stop

codon with no detectable frame shifts in the inter-

vening sequence. The final dataset contained 952

CDS representing 1,096,921 nucleotides and 365,640

codons. Correspondence analysis (COA) was per-

formed according to the instructions of CodonW

software (Peden 1999), available at http://codonw.

sourceforge.net) to identify genes displaying a codon

bias. A specific subset of 113 genes displaying a

codon bias and corresponding to genes expected to be

highly expressed in tobacco according to (Sawant

et al. 2001; Sawant et al. 1999) was used to create a

codon usage table (CUT). The high expression CUT

was then entered into the DNABuilder (Lasergene 6

software, DNASTAR Inc. Madison, USA) to con-

figure a backtranslation software tool. Codons used at

a frequency of less than 10% in tobacco or which

contain potential inhibitory motifs such as CG dinu-

cleotides or CXG triplets were set to 0% of

synonymous codon usage for the particular amino

acid and the frequency of the remaining codons

adjusted accordingly.

Expression of native and tobacco-optimized scFv

constructs in tobacco plants

The anti-FMDV scFv sequences encoding the variable

light and heavy regions were obtained from F24G2

murine mAb (Berry et al. 2004) to construct the native

scFv (scFvnat). The coding sequence for the scFv gene

was optimized for expression in N. tabacum by reverse

translating the protein sequence, including the Pr1b

secretory signal sequence (Cutt et al. 1988) using the

N. tabacum high expression dataset CUT (Table S1,

see online supplementary material). The backtranslat-

ed sequence was then analyzed for the presence of

several potential inhibitory elements that were subse-

quently removed manually by conservative codon

replacement (Table S2, see online supplementary

material). The synthetic plant-optimized gene (scFvopt)

was constructed using a combined ligase chain reac-

tion (LCR)/PCR approach (Au et al. 1998) utilizing a

set of overlapping oligonucleotides designed by the

web-based program Gene2Oligo (Rouillard et al.

2004). The native and tobacco-optimized scFv

gene sequences have been deposited into GenBank

(FJ392581 and FJ392582). To create the ELP fusion

Transgenic Res (2009) 18:685–696 687

123

constructs (scFvnat-ELP and scFvopt-ELP), 28 repeats

of the pentapeptide VPGVG were added to the C-

terminus of the native and tobacco-optimized scFv

genes. A tobacco etch virus (TEV) cut site (ENLYFQ/

G) was included between the fusion partners. As well,

a StrepII purification tag (WSHPQFEK) and an

endoplasmic reticulum retention signal (KDEL) were

added to the C-terminal end of the recombinant

polypeptides.

For plant expression, scFv genes were moved

into the plant binary expression vector pCaMterX

(Laurian Robert, personal communication). The cod-

ing sequences were under the control of the dual-

enhancer cauliflower mosaic virus (CaMV) 35S

promoter (Kay et al. 1987), and the nopaline synthase

(nos) terminator. The expression constructs were

electroporated into Agrobacterium tumefaciens strain

EHA105 (Hood et al. 1993). For transient expression,

the Agrobacterium strains were infiltrated into leaves

of 10- to 14-week old N. tabacum plants as described

previously (Kapila et al. 1997; Yang et al. 2000). To

account for plant to plant variability, leaf to leaf

variability, and position on a leaf, comparably sized

leaves from nine different plants were agroinfiltrated

for each expression construct. As well, the agroinfil-

trated panels were systematically distributed across

the leaves’ surface. After infiltration, the plants were

maintained in a controlled growth chamber at 22�C,

with a 16 h photoperiod for 4 days and the individual

infiltrated panels were sampled separately for ELISA

analysis as well as pooled samples were collected for

immunoblotting. The stable transgenic plants were

generated as described previously (Miki et al. 1999)

by using low-alkaloid tobacco (cv. 81V9; Menassa

et al. 2001). Primary transformants (T0) were grown

in a greenhouse and the first four true leaves were

sampled once they reached 25 cm in length and used

to represent the concentration of recombinant protein

in the whole plant. Seeds were collected from the

transgenic tobacco lines with the highest scFv

concentration and used to produce the subsequent

T1 generation by self-fertilization.

Plant protein extraction

For each sample, total soluble protein (TSP) was

extracted from four 7 mm leaf discs (approximate

fresh weight of 25 mg) of transgenic and wild-type

plants by homogenization with a Mixer Mill MM 300

(Retsch, Haan, Germany). The resulting frozen

powdered leaves were then resuspended at 4�C in

300 ll of extraction buffer (PBS, 0.1% Tween-20,

1 mM EDTA, 100 mM ascorbic acid, 1 mM PMSF

and 1 lg/ml leupeptin). The extract was clarified

twice by centrifugation at 20,0009g for 10 min at

4�C. The TSP concentration was measured according

to the method of Bradford using the Bio-Rad reagent

(Bio-Rad, Hercules, USA) with bovine serum albu-

min as a standard (Bradford 1976).

Immunoblot analysis

For immunoblot analysis, plant extracts were resolved

on a 10% SDS polyacrylamide gel and then transferred

to a nitrocellulose membrane by semi-dry electroblot-

ting. The membranes were blocked with 5% non-fat

milk powder (w/v) in Tris-buffered saline-Tween

(TBST, 50 mM Tris, 150 mM NaCl, 0.5% Tween

20, pH 7.5) overnight at 4�C. The membranes were

incubated with a 1:1,000 dilution of anti-scFv rabbit

serum for 1 h at room temperature with gentle shaking.

The primary antibody was detected with a 1:10,000

dilution of alkaline phosphatase-conjugated goat anti-

rabbit IgG (Promega, Madison, USA) and visualized

using NBT/BCIP (Promega) as substrate. The mem-

branes were washed four times between each step with

TBST and all antibodies were diluted in TBST with

3% non-fat milk powder. The concentration of scFv in

plant extracts was quantified from immunoblots by

densitometry with TotalLab TL100 software (Nonlin-

ear USA Inc., Durham, USA) using purified plant

scFv-ELP as a standard.

Quantification of scFv-ELP protein

levels by ELISA

The quantification of scFv-ELP in tobacco leaf

extracts was achieved by sandwich enzyme-linked

immunosorbent assay (ELISA). Nunc-Immuno Max-

iSorp surface plates (Nalge Nunc, Rochester, USA)

were coated with anti-ELP rabbit serum diluted

1/4,000 in sodium carbonate buffer (0.06 M, pH

9.6) and incubated overnight at 4�C. The wells were

blocked with blocking buffer (1% BSA, 0.1% Tween

20 in PBS) for 30 min at 37�C. Plant extracts were

diluted 1/10 in blocking buffer and incubated on the

plate overnight at 4�C. The plate was then incubated

688 Transgenic Res (2009) 18:685–696

123

with anti-ELP guinea pig serum diluted 1/2,000 in

blocking buffer for 1 h at 37�C. Next, the plates were

incubated with a 1/5,000 dilution of alkaline phos-

phatase-conjugated goat anti-guinea pig IgG (Sigma,

A-5062) diluted in blocking buffer for 1 h at 37�C.

The plates were washed five times between incuba-

tion steps with PBS containing 0.05% Tween-20. The

plates were developed by the addition of 4-nitro-

phenyl phosphate substrate (Sigma, N-9389) and the

absorbance was measured at 405 nm with a Bio-Rad

550 microplate reader. To generate a standard curve,

purified plant scFv-ELP was mixed with non-trans-

genic plant extract and diluted with blocking buffer to

concentrations between 1–500 ng/mL and processed

as described above. All samples were analyzed as

duplicates.

Purification of scFv from tobacco leaves

The highest expressing transgenic scFvopt-ELP plant

line was vegetatively propagated and the leaves of

fully grown plants were harvested and stored at

-20�C until extraction. Frozen tobacco leaves

(660 g) were homogenized with 2 l of extraction

buffer using a CB-6 commercial blender (Waring

Inc., Torrington, USA). The extract was filtered

through Delnet mesh (DelStar technologies Inc.,

Middletown, USA) and centrifuged at 20,0009g for

10 min at 4�C. To precipitate unwanted plant

proteins, the pH of the supernatant was adjusted to

2.8 with HCl and stirred for 10 min before increasing

the pH back to 7.1 with NaOH. The extract was

clarified by centrifugation at 20,0009g for 10 min at

4�C and the supernatant was allowed to warm to

22�C. To trigger the phase transition of ELP, NaCl

was added to 4.5 M, and centrifuged at 20,0009g for

10 min at 22�C. The pellet was resuspended with

100 ml of ice-cold PBS and the suspension was

clarified twice by centrifugation at 20,0009g for

10 min at 4�C. The supernatant was filtered through a

0.45 lm syringe filter and applied to a 10 ml

Streptactin Macroprep column (IBA GmbH, Gottin-

gen, Germany) followed by washing and elution

according to the manufacturer’s instructions. The

fractions containing scFv-ELP were pooled (30 ml)

and dialyzed against PBS at 4�C and concentrated

with Jumbosep (Pall Life Sciences, Mississauga,

Canada) centrifugable units with a 10 kDa cutoff.

Glycerol was added to 15% and aliquots were stored

at -80�C. The concentration of purified scFv-ELP was

determined from GelCode (Pierce, Thermo Fisher

Scientific Inc., Rockford, USA) stained 10% SDS–

page gels by densitometry with TotalLab TL100

software (Nonlinear USA Inc.) using BSA as a

standard.

Cleavage of the scFv–ELP fusion protein

The TEV recognition site between the scFv and ELP

tag was cleaved with the AcTEV protease containing a

(His)6-purification tag (Invitrogen, Burlington, Can-

ada) according to the manufacturer’s instructions at

4�C overnight. The digestion mixture was dialyzed

against PBS at 4�C. To remove the uncleaved

scFv-ELP and AcTEV, the dialyzate was supple-

mented with the Ni-NTA (Qiagen Inc., Mississauga,

Canada) and Streptactin (IBA) sepharoses (1/8 of final

volume of each) and incubated at 4�C for 1 h with

gentle shaking. The cleaved scFv was then recovered

from the supernatant after centrifugation twice at

20,0009g for 10 min at 4�C.

Binding of scFv and scFv-ELP to FMDV particles

The binding of purified scFv proteins to FMDV was

determined by ELISA prior to and after the removal

of ELP fusion. Nunc-Immuno ELISA plates (Nalge

Nunc, 269620) were coated with inactivated A24/

Cruzeiro particles in 0.06 M sodium carbonate buffer

(pH 9.6) for 1 h at 37�C. Equal dilution of plain

culture supernatant was used as negative control.

After blocking the plates overnight at 4�C with

blocking buffer (PBS, 0.05% Tween 20, 1% BSA),

equimolar amounts of purified scFv-ELP and scFv

proteins in blocking buffer were added and incubated

1 h at 37�C. The plates were then incubated with anti-

scFv rabbit serum diluted 1/1,000 in blocking buffer

for 1 h at 37�C and developed as described above. All

samples were analyzed as triplicates.

Statistical analysis

Statistical analysis (SPSS 12.0 for Windows) was

performed using a one-way ANOVA, after confirm-

ing the normal distribution of the data with

Lilliefors’s test. Comparisons among the pairs of

means were done with a Tukey test. Statistical

significance was defined as P \ 0.05.

Transgenic Res (2009) 18:685–696 689

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Results and discussion

Transient and stable expression of scFv

in the endoplasmic reticulum of tobacco leaves

The aim of this study was to provide a proof-of-

concept for expression of a functional anti-FMDV

antibody in plants. We have chosen the format of a

single chain antibody fragment (scFv) targeted

against FMDV VP1 coat protein. Although, plants

are capable to assemble full-size antibodies, scFvs

can accumulate to higher levels while providing

similar antigen binding properties required for

FMDV neutralization (Ma et al. 2005). In the future,

the format of scFv will allow us to easily attach the

constant regions of different model and target species

to gain the effector functions and avidity of full-size

antibodies to be expressed as a single polypeptide.

The effect of codon-optimization, as well as the

presence of an ELP fusion partner on the accumula-

tion of the scFv was evaluated in tobacco plants by

Agrobacterium-mediated transient gene expression

and stable transformation. Four plant expression

vectors (scFvnat, scFvopt, scFvnat-ELP, and scFvopt-

ELP) were constructed to target the expression of the

anti-FMDV single chain antibody to the endoplasmic

reticulum (ER). To direct the recombinant proteins

into the secretory pathway, a tobacco Pr1b signal

peptide was fused to the scFv sequences. Retention

to the ER was achieved by adding an ER retrieval

signal (KDEL) to the C-terminus of the recombinant

polypeptides.

Genes of foreign origin may have sub-optimal

codon composition for the plant translational machin-

ery (Adang et al. 1993; Horvath et al. 2000; Perlak

et al. 1991). Here, a tobacco codon correspondence

analysis was completed to create a codon usage table

(Table S1, see online supplementary material) to

mimic the codon usage of highly expressed tobacco

genes. By using this codon table, a tobacco-optimized

gene encoding an anti-FMDV-scFv antibody was

synthesized while avoiding potentially deleterious

processing signals and destabilizing motifs (Table S2,

see online supplementary material). In its entirety, the

optimization of the scFv gene resulted in changes to

24% of the nucleotides in 59% of the codons, and a

decrease in the G ? C content from 50 to 43%.

Prior to the laborious generation of transgenic

plants, Agrobacterium-mediated transient expression

in N. tabacum was used to rapidly test the different

expression constructs. The ELP fusion strategy has

been previously shown to increase the yield of several

recombinant target proteins in transgenic tobacco

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

(Scheller et al. 2006). All four constructs (scFvnat,

scFvopt, scFvnat-ELP, and scFvopt-ELP) were agroin-

filtrated into tobacco leaves and the concentration of

scFv was quantified from an anti-scFv-immunoblot

by densitometry (Fig. 1a) or by enzyme-linked immu-

nosorbent assay (ELISA; Fig. 1b).

The anti-scFv-immunoblot showed the smaller

size of scFv (migrating at about 38 kDa), relative to

the scFv–ELP fusion constructs (50 kDa; Fig. 1a).

Interestingly, both scFv–ELP fusion constructs

(scFvnat-ELP and scFvopt-ELP) accumulated to much

higher levels relative to their counterparts without

ELP (scFvnat and scFvopt). The highest accumula-

tion [0.1% of total soluble protein (TSP)] was

detected with scFvopt-ELP, which is 1.3 times higher

than scFvnat-ELP (0.08% TSP), 14 times higher

than scFvopt (0.007% TSP), and 20 times higher than

scFvnat (0.005% TSP), respectively. The effect of

codon-optimization on scFv-ELP accumulation was

further analyzed with a double sandwich anti-ELP

ELISA (Fig. 1b). This quantification confirmed the

results from immunoblot, and showed a significant

(P \ 0.001), but a relatively low increase (approxi-

mately 1.6-fold) in scFv accumulation of 0.08% of

TSP for scFvopt-ELP than 0.05% of TSP for scFvnat-

ELP.

Based on the results obtained from the transient

expression studies, only the scFv–ELP fusion con-

structs (scFvnat-ELP and scFvopt-ELP) were chosen to

generate stable transgenic tobacco plants. Thirty

independent transgenic lines from the T0 generation

were screened for scFv-ELP accumulation by immu-

noblotting and showed single immunoreactive bands

equal to the size obtained from the transient analysis

(data not shown). The five highest expressing lines

per construct were selfed to generate T1 seed. Ten

plants of each T1 line were screened for scFv-ELP

accumulation by anti-ELP-ELISA. The mean of the

three highest expressing plants were used to represent

the scFv-ELP accumulation within each transgenic

line (Fig. 2). In agreement with the transient expres-

sion analysis, the scFvopt-ELP plants showed a trend

for higher accumulation than the scFvnat-ELP plants.

However, the highest expressing lines in both native

690 Transgenic Res (2009) 18:685–696

123

and tobacco-optimized populations, accumulated

scFv up to 0.8% of TSP (Fig. 2). The high variation

in scFv concentration among the transgenic plants

was expected and is attributed to the chromosomal

position effects associated with random gene

insertion (Hobbs et al. 1990; Krysan et al. 2002).

The observed modest increase in scFvopt accumula-

tion over scFvnat is in accordance with examples

demonstrating that optimization of heterologous

eukaryotic genes for expression in plants (Conley

et al. 2009b; Lonsdale et al. 1998; Rouwendal et al.

1997) is not as important as the optimization of

bacterial genes for plant expression, where up to 100-

fold differences have been reported (Kang et al. 2004;

Perlak et al. 1991).

Being independent from the intrinsic variability in

gene expression normally associated with stable

transgenic plants, agroinfiltration has been shown to

be an efficient technique for the quick evaluation of

genetic constructs for recombinant protein accumu-

lation (Kapila et al. 1997). Combined with a rigorous

replicated experimental design, this method can find

even subtle differences between the expression

constructs, but cannot always predict the absolute

accumulation capacity of transgenic plants. Our

transgenic plants accumulated approximately ten

times more scFv (0.8% vs. 0.08 TSP) than the

agroinfiltrated leaves. This can be, at least in part,

explained by post-transcriptional gene silencing

induced by agroinfiltration (Voinnet et al. 2003).

Schouten et al. (1996) studied the expression of

single chain antibody fragments in different subcel-

lular locations of tobacco plants. They showed that

targeting scFvs to (ER) with a secretory signal

sequence and a carboxy-terminal KDEL motif led

to the highest accumulation of these recombinant

proteins. A similar strategy was used in this study. To

Fig. 1 The transient expression of scFv and scFv–ELP fusion

proteins in tobacco leaf extracts harvested from leaf sectors

(n = 9) 4 days post agroinfiltration. (a) An anti-scFv immu-

noblot analysis showing the difference in size and

accumulation level of native and codon-optimized versions of

scFv (lanes 1–2, 38 kDa) and scFv-ELP (lanes 3–4, 50 kDa)

constructs. Ten micrograms of total soluble protein was loaded

on each lane. The letter C represents tobacco extract from

leaves agroinfiltrated with an empty vector (lane 5). (b) An

anti-ELP ELISA analysis showing the difference in accumu-

lation of native and codon-optimized scFv-ELP constructs.

Each column represents the mean value of nine data points and

the standard error of the mean is represented with error bars.

**, significant difference (P \ 0.001) in accumulation

0

0.2

0.4

0.6

0.8

1

scF

v %

TS

P

scFvnat-ELPscFvopt-ELP

Fig. 2 Expression of scFv-ELP in transgenic tobacco plants.

An anti-ELP-ELISA analysis of scFv protein concentration in

the leaf tissue of stable transgenic plants carrying native or

tobacco codon-optimized versions of the scFv gene. Ten T1

generation plants per transgenic line were sampled (each

sample contains a single leaf disc from each of the first four

expanded leaves from each plant). The five highest expressing

lines for each construct are presented. Columns represent the

mean of the three highest expressing plants within a line. The

standard error of the mean is represented with error bars

Transgenic Res (2009) 18:685–696 691

123

confirm the subcellular localization of recombinant

scFv protein to the ER, green fluorescent protein

(GFP) was used as a C-terminal fusion and the

resulting construct was agroinfiltrated into tobacco

leaves and examined by confocal laser scanning

microscopy. The ER targeted scFv-GFP resembled a

typical reticulate pattern (Boevink et al. 1996)

consistent with ER-localization (data not shown).

Purification of scFv–ELP fusion protein

Many fusion tags have been developed to facilitate

the purification of recombinant proteins using affinity

chromatography techniques (Lichty et al. 2005; Terpe

2003), but these methods are costly and difficult to

scale-up (Waugh 2005). In addition, the complex

plant proteome and the typical low yield [\1% TSP,

(Joensuu et al. 2008)] complicate the purification

scheme of plant-made recombinant proteins.

The thermally responsive property of ELP to

undergo a reversible inverse phase transition from

soluble protein into insoluble hydrophobic aggre-

gates enables a simple nonchromatographic method

for protein purification called ‘inverse transition

cycling’ (ITC; Meyer and Chilkoti 1999). As an

early step in a purification scheme, ITC offers

several benefits as a simple, rapid, scalable and

inexpensive non-chromatographic means of purify-

ing plant recombinant proteins (Menkhaus et al.

2004; Meyer and Chilkoti 1999). While simulta-

neously purifying and concentrating your protein of

interest, prior to costly downstream processing steps,

such as affinity chromatography, ITC also allows for

a straightforward means for removing toxic water-

soluble alkaloids from tobacco leaf extracts. As

well, aggregated ELP-fusion proteins have been

shown to possess improved stability relative to the

soluble fusion protein, allowing for better long-term

storage and application of the protein (Shamji et al.

2007; Shimazu et al. 2003).

Here, we describe a simple and scalable purifica-

tion scheme for plant-made scFvs (Fig. 3a). First,

suitable physical (temperature) and chemical (pH)

conditions were screened (optimal conditions were

found to be 22�C, pH 2.8) to maintain the scFv–ELP

fusion protein in a soluble state while precipitating

the majority of unwanted plant proteins (Fig. 3b, lane

2). Then, by utilizing the properties of the ELP fusion

partner, the recombinant protein was enriched by ITC

(Fig. 3b, lane 3), and finally scFv-ELP was purified to

a high degree by StrepII affinity chromatography

(Fig. 3b, lane 4). Since scFv–ELP fusion (Fig. 3b,

arrowhead on the right) migrates on SDS–PAGE

almost at equal size as the large subunit of Ribulose-

1,5-bisphosphate carboxylase/oxygenase (Fig. 3b,

arrowhead on the left), the purification of scFv-ELP

was confirmed by analyzing the same samples with

anti-ELP-immunoblotting (Fig. 3c). In total, 1.5 mg

of purified scFv-ELP was recovered from 660 g of

tobacco leaves. This represents approximately 5% of

the soluble scFv-ELP present in the original plant

extract. The ITC step recovered about 30% of the

available scFv-ELP, which resembles a typical

recovery rate of plant recombinant proteins with this

technique (Conley et al. 2009a). The recoveries of the

low-pH precipitation and the StrepII capture were 63

and 25%, respectively.

Previous reports on purification of ELP fusions

from plant extracts have utilized longer ELP100

repeats and higher ITC temperatures (40�C in the

presence of 2 M NaCl) than used in this study (Lin

et al. 2006; Scheller et al. 2004). Recently, we have

shown that while longer ELPs can offer better

recovery in ITC, they can drastically limit the

accumulation of the recombinant proteins in plants

(Conley et al. 2009a). Therefore, we fused the anti-

FMDV scFv to a relatively short ELP28. In order to

simplify the purification procedure, lower ITC

temperatures are advantageous. The transition tem-

perature for ELPs is also dependent on the salt and

protein concentration. Here, the ITC was done at

room temperature in the presence of 4.5 M NaCl,

which was found to be optimal for this scFv–ELP28

fusion. Similar results were obtained with 3.5 M

NaCl at 37�C. Higher salt concentrations increased

the non-specific precipitation of plant proteins (data

not shown). The abundance of many proteases in leaf

extract can often complicate the recovery of plant-

made recombinant proteins (Streatfield 2007). In our

purification strategy, the two nonchromatoraphic

purification steps (low pH and ITC) prior to StrepII

affinity chromatography hindered the activity of plant

proteases or inhibited their co-purification and a

stable fraction of plant-made scFv-ELP was recov-

ered. Conversely, the direct StrepII-capture of scFv-

ELP from plant extract yielded a low recovery and

the purified protein was rapidly degraded upon

storage (data not shown).

692 Transgenic Res (2009) 18:685–696

123

Cleavage of scFv–ELP fusion protein and binding

to FMDV particles

The presence of fusion partners or affinity tags may

affect important characteristics or functions of the

protein to be studied. Removal of the tag from a

protein of interest can be accomplished with a site-

specific protease, and the cleavage process should not

reduce protein activity (Jenny et al. 2003; Terpe

2003). In this study, a seven-amino-acid recognition

site specific for Tobacco etch virus (TEV) protease

was cloned between the scFv and ELP portions of the

scFv–ELP fusion protein.

The high specificity, its activity on a variety of

substrates, and the efficient cleavage at low temper-

ature make the TEV protease an ideal tool for

removing tags from fusion proteins (Parks et al.

1994). Indeed, we observed practically complete

cleavage of scFv–ELP fusion upon overnight diges-

tion at 4�C. Furthermore, the released ELP portion,

remaining uncleaved fusion, and TEV protease were

removed from the cleavage mixture by sepharose

capture using the His-tag on the protease and the

StrepII-tag on the ELP moiety. Fractions from each

step were analyzed on SDS–PAGE gel (Fig. 4a), and

anti-StrepII (Fig. 4b), as well as anti-His-immuno-

blots (Fig. 4c). Uncleaved scFv–ELP fusion migrated

as a 50 kDa band (Fig. 4a, b, lane 2) and the StrepII

antibody revealed a small portion of the undigested

form after the cleavage, as well as the released ELP-

tag (migrating at 11 kDa; Fig. 4b, lane 3). The free

ELP was not visible on the SDS–PAGE gel since it

does not stain with the technique used (Trabbic-

Carlson et al. 2004). The TEV protease (migrating at

28 kDa) in the digestion mixture was detected with

an anti-His antibody as expected (Fig. 4a, c, lane 3).

The appearance of a 35 kDa migrating band follow-

ing the digestion confirmed the release of the scFv

portion from the fusion protein (Fig. 4a, lane 3). The

Fig. 3 Purification of scFv–ELP fusion protein from tobacco

leaf extract. (a) A schematic presentation of the purification

procedure. Ten microlitre fractions of each scFv-ELP purifi-

cation step were separated on 10% SDS–PAGE, and stained for

total protein (b) or detected with anti-ELP-immunoblotting (c).

The total soluble protein in leaf extract is presented in lane 1.

To purify scFv-ELP, the majority of unwanted plant proteins

were first precipitated at low pH (lane 2 showing the

supernatant after precipitation at pH 2.8). Then, scFv-ELP

was enriched with ITC (lane 3 showing the soluble pellet after

the precipitation). Finally, the scFv-ELP was purified by

StrepII-tag affinity chromatography (lane 4 showing the eluate

containing scFv-ELP protein migrating at 50 kDa). The

arrowhead on the left shows the size of the large subunit of

the Ribulose-1,5-bisphosphate carboxylase/oxygenase and the

arrowhead on the right shows the size of the scFv–ELP fusion

protein

b

Transgenic Res (2009) 18:685–696 693

123

incubation with Ni-NTA and Streptactin sepharoses

removed the undigested fusion, released ELP-StrepII,

and TEV protease effectively, and none of these were

detected in the remaining supernatant by immuno-

blotting (Fig. 4b, c, lane 4). The recovered scFv

fraction showed a single band of high purity (Fig. 4a,

lane 4).

The binding of the purified scFv proteins to FMDV

was studied by ELISA analysis prior to and after the

removal of ELP portion. Equimolar amounts of

serially diluted scFv and scFv-ELP proteins were

incubated on FMDV coated plates and probed with

anti-scFv-antiserum (Fig. 5). ELP fusions and ITC

purification generally do not hinder the functionality

of the target protein (Floss et al. 2008; Scheller et al.

2006; Shamji et al. 2007; Trabbic-Carlson et al.

2004). Similarly, our scFv–ELP fusion protein was

able to recognize FMDV particles. However, a higher

binding of scFv to FMDV was observed in the

absence of the ELP portion. To confirm that the ELP

portion of scFv-ELP did not hinder the scFv-antise-

rum recognition, a similar ELISA was performed by

coating scFv and scFv-ELP directly to the plate, and

no significant difference was observed between the

two proteins (data not shown). It has been also

previously shown that ELPs may alter the activity

of the fusion proteins (Meyer and Chilkoti 1999;

Shimazu et al. 2003).

In conclusion, this study demonstrated that an anti-

FMDV scFv antibody fragment can accumulate to

relatively high level (0.8% of leaf TSP) in transgenic

tobacco plants when expressed in fusion with ELP. A

simple and scalable purification strategy was used to

recover scFv-ELP in an active form. This is further

proof that ELP fusion technology provides an effec-

tive alternative for enhancing recombinant protein

yields in plants, while also providing a means for

their subsequent purification. Our future work will

focus on demonstrating the in vivo passive immuni-

zation capacity of these plant-made anti-FMDV scFv

antibodies.

Acknowledgments The authors wish to thank Jamie McNeil,

Lisa Starr, and the staff at the National Centre for Foreign

Animal Disease for technical assistance. Alex Molnar is

Fig. 4 Purification of scFv protein by cleavage of the TEV

protease recognition site between scFv and ELP portions of the

scFv–ELP fusion. The fusion protein was cleaved with TEV

protease and the released ELP portion, uncleaved fusion, and

TEV protease were captured from the cleavage mixture with

Ni-NTA and Streptactin sepharoses. Ten microlitre fractions of

each step were separated on 10% SDS–PAGE gels and stained

for total protein (a), or detected with anti-StrepII (b), and anti-

His (c) immunoblotting. Lane 1; His-tagged TEV protease

(28 kDa). Lane 2; scFv–ELP fusion (50 kDa) before TEV

cleavage. Lane 3; scFv-ELP fusion cleaved with TEV protease.

Lane 4; purified scFv portion (35 kDa) after Ni-NTA and

Streptactin sepharose treatment

0

0.2

0.4

0.6

0.8

1

0 0.08 0.16 0.31 0.63 1.25 2.5 5

scFv µg/ml

OD

405

before ELP cleavage

after ELP cleavage

Fig. 5 The binding of an anti-FMDV scFv with and without

the ELP moiety to FMDV particles. The data is presented as

the mean of triplicate samples and the error bars represent the

standard error of the mean

694 Transgenic Res (2009) 18:685–696

123

acknowledged for his assistance with the preparation of the

figures. This research was supported by Agriculture and Agri-

Food Canada’s Matching Investment Initiative program. The

Academy of Finland is acknowledged for providing a

fellowship for J.J.J., and the Natural Sciences and

Engineering Research Council (NSERC) is thanked for

providing financial support to A.J.C.

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