GENETIC TRANSFORMATION AND HYBRIDIZATION

Expression of toxin co-regulated pilus subunit A (TCPA)of Vibrio cholerae and its immunogenic epitopes fused to choleratoxin B subunit in transgenic tomato (Solanum lycopersicum)

Manoj Kumar Sharma Æ Nirmal Kumar Singh Æ Dewal Jani Æ Rama Sisodia ÆM. Thungapathra Æ J. K. Gautam Æ L. S. Meena Æ Yogendra Singh ÆAmit Ghosh Æ Akhilesh Kumar Tyagi Æ Arun Kumar Sharma

Received: 5 July 2007 / Accepted: 30 September 2007 / Published online: 26 October 2007

� Springer-Verlag 2007

Abstract For protection against cholera, it is important to

develop efficient vaccine capable of inducing anti-toxin as

well as anti-colonizing immunity against Vibrio cholerae

infections. Earlier, expression of cholera toxin B subunit

(CTB) in tomato was reported by us. In the present

investigation, toxin co-regulated pilus subunit A (TCPA),

earlier reported to be an antigen capable of providing anti-

colonization immunity, has been expressed in tomato.

Further, to generate more potent combinatorial antigens,

nucleotides encoding P4 or P6 epitope of TCPA were fused

to cholera toxin B subunit gene (ctxB) and expressed in

tomato. Presence of transgenes in the tomato genome was

confirmed by PCR and expression of genes was confirmed

at transcript and protein level. TCPA, chimeric CTB-P4

and CTB-P6 proteins were also expressed in E. coli. TCPA

protein expressed in E. coli was purified to generate anti-

TCPA antibodies in rabbit. Immunoblot and GM1-ELISA

verified the synthesis and assembly of pentameric chimeric

proteins in fruit tissue of transgenic tomato plants. The

chimeric protein CTB-P4 and CTB-P6 accumulated up to

0.17 and 0.096% of total soluble protein (TSP), respec-

tively, in tomato fruits. Whereas expression of TCPA,

CTB-P4 and CTB-P6 in E. coli can be utilized for devel-

opment of conventional vaccine, expression of these

antigens which can provide both anti-toxin as well as anti-

colonization immunity, has been demonstrated in plants, in

a form which is potentially capable of inducing immune

response against cholera infection.

Keywords Cholera � Fusion protein � TCPA � Tomato �Transgenic

Abbreviations

ctxB Cholera toxin B subunit gene

CTB Cholera toxin B subunit

IPTG Isopropyl-b-D-thiogalactoside

P4 Peptide 4

P6 Peptide 6

tcpA Toxin-coregulated pilus subunit A gene

TCP Toxin-coregulated pilus

TSP Total soluble protein

Communicated by P. Lakshmanan.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-007-0464-y) contains supplementarymaterial, which is available to authorized users.

M. K. Sharma � N. K. Singh � D. Jani � R. Sisodia �A. K. Tyagi � A. K. Sharma (&)

Department of Plant Molecular biology, University of Delhi

South Campus, New Delhi 110021, India

e-mail: arun@genomeindia.org

M. Thungapathra � J. K. Gautam � A. Ghosh

Institute of Microbial Technology, Chandigarh 160036, India

L. S. Meena � Y. Singh

Institute of Genomics and Integrative Biology, Mall Road,

Delhi 110007, India

Present Address:M. Thungapathra

Post Graduate Institute of Medical Education and Research,

Chandigarh, India

Present Address:J. K. Gautam

School of Medicine, University of Virginia, Charlottesville, USA

Present Address:A. Ghosh

Indian Institute of Advanced Research, Gandhinagar 382011,

Gujarat, India

123

Plant Cell Rep (2008) 27:307–318

DOI 10.1007/s00299-007-0464-y

Introduction

The plant-expressed antigens have significant potential to

serve as vaccine as they do not pose the risk of causing the

disease as possible with the traditional vaccines where

killed or attenuated disease causing organisms are injected

in the human system. The lower cost of production of

vaccine antigen in plants is also likely to help in making

the vaccine available to the poorest of the poor. A large

number of potential antigen proteins have been produced in

plants and several plant-expressed antigens have also been

tested in human as immunogens with significant effects

(Tacket et al. 1998, 2000, 2004; Kapusta et al. 1999; Yu-

sibov et al. 2002; Sharma et al. 2004; Thanavala et al.

2005). Cholera, a diarrheal disease is caused by Vibrio

cholerae that colonizes in the human intestine and secretes

cholera toxin. Cholera toxin B subunit (CTB) interacts with

hydrophilic carbohydrate moiety of the monosialosylgan-

glioside GM1. This binding is very specific because of the

high affinity interaction (Cai and Yang 2003). CTB is non-

toxic, and it is a component of a widely licensed oral

cholera vaccine (Holmgren and Svennerholm 1990). Pre-

vious studies have reported the expression of pentameric B

subunit in various plant species (Arakawa et al. 1997;

Wang et al. 2001; Jani et al. 2002, 2004; Kang et al. 2004,

2006; Mishra et al. 2006). Further, administration of plant

tissue expressing CTB has been shown to induce serum and

mucosal CTB-specific antibodies in mice (Arakawa et al.

1998a). V. cholerae needs to colonize in the human intes-

tine to cause the disease. The presence of toxin co-

regulated pilus has been shown to be essential for coloni-

zation (Taylor et al. 1987; Herrington et al. 1988). It has

been found that tcpA mRNA is upregulated during human

infection (Larocque et al. 2005) and anti-TCPA immune

responses have been found to occur in over 90% of indi-

viduals infected with V. cholerae O1 El Tor in Bangladesh

(Hang et al. 2003; Asaduzzaman et al. 2004). Pilin is made

up of TCPA subunits that form bundled filaments at the

bacterial surface. The gene tcpA encodes major pilin sub-

unit of the toxin-coregulated pilus (TCP) of V. cholerae

and sera raised against TCPA have been shown to protect

infant mice passively against challenge with strains having

TCP (Sun et al. 1990; Voss et al. 1996). DNA sequences of

tcpA of El Tor O1 and O139 strains have been found to be

identical (Rhine and Taylor 1994) and therefore serum

against TCP would provide immunity against O1 as well as

O139 strains. Various epitopes have been described within

the TCP protein, of which epitope P4 and P6 have been

shown to provide the level of protective immunity against

challenge, which is comparable to the one obtained by

using complete protein (Sun et al. 1997). This, together

with the data obtained from volunteers from Bangladesh,

infected with V. cholerae, suggests that TCPA subunit

protein and its immunogenic epitopes are the potential

candidates for developing anti-colonizing immunity

against V. cholerae infections. The inclusion of TCPA

protein with CTB in the vaccine would be expected to

protect against the toxin secreted by the pathogen as well as

colonization of the intestine by the pathogen. Therefore,

the expression of TCPA protein and chimeric protein

consisting of CTB and epitopes of TCPA in transgenic

plants is an attractive and promising approach towards the

development of plant vaccine against cholera. It has been

shown in the past that for successful pentamerization of

CTB chimeric molecules, a flexible linker peptide is

required that allows the molecular movements between

CTB and conjugated peptide (Clements 1990). Glycine

codon, GGC and proline codon, CCG have been preferred

in the sequence encoding this tetrapeptide (Gly-Pro-Gly-

Pro) because these codons are less frequently used in

plants. This is likely to slow down translation of mRNA

and assist in proper folding of the CTB peptide moiety

without significant stearic hindrance from the fusion part-

ner (Lipscombe et al. 1991; Kim et al. 2004b).

In the present study, we report expression of TCPA in

tomato, which can be combined with CTB expressed in

tomato to form an effective edible vaccine. Further, chi-

meric antigens having CTB and P4 or P6 epitopes of TCPA

have also been expressed in tomato. Oligomerization of

expressed chimeric proteins in E. coli and tomato, and their

interaction with GM1-ganglioside have also been addressed.

Materials and methods

For all DNA and RNA related experiments, standard pro-

tocols as described by Sambrook et al. (1989) were used,

unless mentioned otherwise. Sequence of primers used for

PCR amplification is provided in Table 1. All amplified

products were sequenced to validate their sequences.

Vector for expression of tcpA gene in plants

The gene encoding TCPA protein was amplified using

gene-specific primers ETF, adding BamHI site and Kozak

sequence to 50 end and ETR1, adding SEKDEL to 30 end.

The amplified DNA was used as template to re-amplify the

gene using same forward primer and ETR2 reverse primer

that added SacI site to 30 end. The amplified product was

cloned in pUC19 vector to generate pUC19TCPA vector.

For the expression of tcpA gene in plants, it was taken out

of pUC19TCPA vector with BamHI and SacI and cloned in

pCAMBIACTB vector, replacing CTB coding cassette

(Jani et al. 2002) to generate pCAMBIATCPA vector.

BamHI–EcoRI fragment from pCMABIATCPA vector,

308 Plant Cell Rep (2008) 27:307–318

123

i.e., tcpA along with its terminator, was moved to

pCAMBIACTBE2L vector, replacing CTB coding cassette,

to generate pCAMBIATCPAE2L vector (Fig. 1a). This

vector was then introduced into Agrobacterium tumefac-

iens strain LBA4404 through chemical transformation and

used for plant transformation.

Vectors for expression of fusion product of CTB and P4

or P6 epitopes of TCP in plants

The DNA fragments encoding for P4 (145–168 amino

acids) and P6 (174–199 amino acids) epitopes of TCPA

protein of V. cholerae were amplified using gene-specific

primers. Primer EP4F or EP6F added Kpn I restriction site

to 50 end of respective DNA fragments and EP4R1 or

EP6R1 added sequence encoding SEKDEL to 30 end of the

respective DNA fragments encoding two epitopes. These

amplified p4 or p6 DNA fragments were used as template

for re-amplification, using same forward primers along

with EP4R2 or EP6R2 reverse primers used for P4 or P6

epitope, respectively, that added SacI to 30 ends of the

sequences encoding these epitopes. The gene encoding

CTB protein was amplified using forward primer, CPFF,

which added BamHI restriction site and Kozak sequence,

and reverse primer CPFR1, which incorporated a flexible

hinge region coding for tetrapeptide Gly-Pro-Gly-Pro at 30

terminus. The amplified ctxB gene fragment was used as

template for re-amplification, using forward primer, CPFF

and reverse primer, CPFR2 that added Kpn1 site to 30 end

after hinge region. It was cloned in pUC19 to generate

pUC19CTB-H vector. The amplified fragments coding for

epitope P4 and P6 were cloned in pUC19CTB-H vector

using KpnI and SacI to generate pUC19CTB-P4 and

pUC19CTB-P6 vectors, respectively. The ctxB-p4 and

ctxB-p6 fusion gene fragments were cloned in pCAMBI-

ACTB vector (Jani et al. 2002) using BamHI and SacI sites

to generate pCAMBIACTB-P4 and pCAMBIACTB-P6

vectors, respectively. The CaMV35S promoter controlling

the expression of fusion gene in pCMABIACTB-P4 vector

was replaced with 35S promoter with double enhancer

sequence from pBSKE2L (Jani 2003) vector using BamHI

and HindIII sites, generating pCAMBIACTB-P4E2L vector

(Fig. 1b). Since P6 coding sequence has HindIII site, the

BamHI–EcoRI fragment from pCMABIACTB-P6 vector,

i.e., ctxB-p6 with its terminator, was moved to pCAM-

BIACTBE2L vector to generate pCAMBIACTB-P6E2L

vector, replacing ctxB gene (Fig. 1c). These vectors were

introduced into A. tumefaciens strain AGL 1 and were used

for plant transformation.

Vectors for expression of fusion of CTB with P4

or P6 epitope of TCPA in E. coli

For bacterial expression, ctxB gene excluding sequence

coding for signal peptide was amplified, using the forward

primer, CPFF, which added BamHI restriction site to 50 end

and reverse primer CPFR1, which incorporated a flexible

hinge region coding for tetrapeptide Gly-Pro-Gly-Pro at 30

terminus. The amplified ctxB gene fragment was used as

template for re-amplification of the gene using same for-

ward primer and reverse primer CPFR2 that added SacI site

after hinge region. This ctxB-H fragment was cloned in

pQE30 vector to generate pQE30CTB-H vector. DNA

fragments encoding P4 and P6 epitopes were amplified

using primer pairs PP4F, PP4R and PP6F, PP6R, respec-

tively, which added SacI site to 50 ends and SmaI site to 30

ends. These amplified products were then cloned into

pQE30CTB-H vector to generate pQE30CTB-P4 and

pQE30CTB-P6 vectors. Both plasmids were introduced

into E. coli strain SG13009[pREP4]. Transformed bacteria

were grown at 37�C to OD600 of 0.5. Cells were induced

using 1 mM IPTG and grown further for 4 h. Lysate of

induced bacterial culture was analyzed by polyacrylamide

gel electrophoresis.

Table 1 List of the primers used

Identity Sequence

CPFF 50-CTTAGGATCCACACCTCAAAATATTACTG-30

CPFR1 50-GCC CGG GCCATTAGCCATACTAATTGC-30

CPFR2 50-TATTATGAGCTCCGGGCCCGGGCCATTA-30

CEFF 50-ATGTAGGATCCACCATGATTAAATTAAAAT-30

EP4F 50-ATCCGGTACCGCTGATCCTGGTGATTTCG-30

EP4R1 50-AAGTTCATCTTTTTCAGATGCAATGGACTTAA

TTACGC-30

EP4R2 50-CTTAGAGCTCTCAAAGTTCATCTTTTTCAG-30

PP4F 50-ATCCGAGCTCGCTGATCTTGGTGATTTCG-30

PP4R 50-GGATCCCCGGGTGCAATGGACTTAATTACGA-30

EP6F 50-ATATGGTACCTTAAACCTAACTAATATCAC-30

EP6R1 50-AAGTTCATCTTTTTCAGAACTGTTACCAAAAG

CTACTG-30

EP6R2 50-ATATGAGCTC TCA AAGTTCATCTTTTTCAG-30

PP6F 50-ATCCGAGCTCTTAAACCTAACTAATATCAC-30

PP6R 50-GGATCCCGGGACTGTTACCAAAAGCTACTG-30

PTF 50-AGGGATCCACATTACTCGAAGTAATCA-30

PTR 50-ATGAGCTCACTGTTACCAAAAGCTACTG-30

ETF 50-ATAGGATCCACCATGGCATTATTAAAACAGC

TTTTTAAG-30

ETR1 50-TAGTTCATCTTTCTCCGAACTGTTACCAAAAGC-30

ETR2 50-TTAGAGCTCTCATAGTTCATCTTTCTCCGAACTG-30

Plant Cell Rep (2008) 27:307–318 309

123

Plant transformation

Tomato (Solanum lycopersicum var. Pusa Ruby) seeds

were surface-sterilized with 4% sodium hypochlorite

solution for 12 min and germinated on MS nutrient med-

ium (Murashige and Skoog 1962), supplemented with the

organic components of B5 medium (Gamborg et al. 1968).

Transformation was done as described earlier (Jani et al.

2002) with some modifications. A. tumefaciens strain

AGL1 harboring pCAMBIATCPAE2L, pCAMBIACTB-

P4E2L or pCAMBIACTB-P6E2L, was used for plant

transformation and pre-cultured cotyledon explants were

co-cultivated with Agrobacterium for 72 h.

b-Glucuronidase (GUS) assay and PCR analysis

of transgenic plants

b-Glucuronidase expression was checked by histochemical

staining of leaf tissue of putative transgenic plants as

described by Chaudhury et al. (1995). The chlorophyll in

green tissues was bleached out by incubation in ace-

tone:ethanol (1:3) before observation of GUS activity. For

PCR analysis, genomic DNA was isolated from leaves of

transgenic tomato plants as described earlier (Dellaporta

et al. 1983). The presence of ctxB-p4, ctxB-P6 fusion gene

and tcpA gene was determined by PCR analysis, using

gene-specific primers mentioned in section on construction

of vectors. For PCR, 100 ng of genomic DNA was used as

template and thermo-cycling conditions were as follow:

94�C for 40 s, 58�C for 40 s and 72�C for 45 s for a total of

25 cycles. PCR products were analyzed on 1.2% agrose

gel.

Northern analysis of transgenic plants

Total RNA from the leaves of untransformed and trans-

formed tomato plants was isolated using TRI

REAGENTTM from SIGMA, following the instructions

from manufacturer. For each sample, 15 lg of RNA was

electrophoresed on 1.2% formaldehyde agrose gel and

transferred to nylon membrane by capillary transfer.

The pCAMBIACTB-P4E2L, pCAMBIACTB-P6E2L or

pCAMBIATCPAE2L vectors were digested with BamHI–

SacI enzymes to generate ctxB-p4, ctxB-p6 fusion gene or

tcpA gene fragments, respectively. These genes were used

as probe after labeling with [32P] radioisotope, using

Megaprime DNA Labelling System of Amersham Biosci-

ences or Gene Image AlkPhos direct labeling and detection

system of Amersham Biosciences, which produces light

signal in an enzyme catalyzed reaction.

c

pCAMBIATCPE2L

L Border

35S ter

npt II 35S

Eco RI

Nos terSac I

ER tcpA

BamH I

KZ

Hind IIICatalase intron

Nos terR Border

gus35SE2L 35S

a

pCAMBIACTB-P4E2L

L Border

35S ter

npt II 35S

Eco RI

Nos terSac I

ER

BamH I

KZ

Hind IIICatalase intron

Nos terR Border

gusp4 ctxB

Kpn I

35S35SE2L

b

pCAMBIACTB-P6E2L

L Border

35S ter

npt II 35S

Eco RI

Nos terSac I

ER

BamH I

KZ

Hind IIICatalase intron

Nos terR Border

gusp6 ctxB

Kpn I

35S35SE2L

Fig. 1 Diagrammatic representations of T-DNA regions of plant

expression vectors. a pCAMBIATCPAE2L, b pCAMBIACTB-

P4E2L, c pCAMBIACTB-P6E2L. npt II Neomycin phosphotransfer-

ase gene, ctxB cholera toxin B subunit gene from Vibrio cholerae,tcpA toxin co-regualted pilus protein A gene from Vibrio choleare, p4

and p6 tcpA gene fragments encoding P4 and P6 epitopes of TCP, gusb-glucoronidase gene from E. coli, 35S CaMV35S promoter, 35SE2LCaMV35S promoter with double enhancer and leader peptide from

alfalfa mosaic virus, KZ Kozak sequence, ER endoplasmic retention

signal, nos ter nopaline synthase gene terminator

310 Plant Cell Rep (2008) 27:307–318

123

Protein extraction and detection of recombinant

proteins

Total soluble proteins were extracted from the leaves or

fruits of wild/transgenic tomato plants and were used for

immuno-detection of recombinant protein as described by

Jani et al. (2002). Protein extraction buffer was supple-

mented with cocktail of protease inhibitor from SIGMA as

recommended by the manufacturer. Total soluble protein

(40–80 lg) from transformed and untransformed plants

was resolved on either non-reducing 12% sodium dodecyl

sulphate (SDS) polyacrylamide gel without boiling the

samples or in reducing conditions after boiling the samples,

along with 30–60 ng of purified CTB protein. Rabbit anti-

CTB antibody at 1:8,000 dilutions, anti-TCPA antibody at

1:4,000 dilution and goat anti-rabbit IgG, conjugated to

horseradish peroxidase (Sigma A-9169) at 1:10,000 dilu-

tions were used for immunodetection. The gene encoding

TCPA protein was expressed in E. coli and TCPA protein

was purified by electro-elution. TCPA was further purified

using Qiagen’s column and purified protein was used to

generate anti-TCPA antibodies.

Analysis of binding of CTB-P4 and CTB-P6 to

GM1-ganglioside and quantification of fusion protein

To determine the binding potential and expression level of

plant-derived CTB-P4 and CTB-P6 fusion proteins, GM1-

ELISA was performed as described by Jani et al. (2002).

Primary anti-cholera toxin antibody at 1:5,000 dilution and

anti-rabbit IgG (secondary antibody) conjugated to horse-

radish peroxidase at 1:6,000 dilutions were used. The

expression levels were calculated by averaging the

expression levels of each sample in two or more experi-

ments done with triplicate samples. As a control BSA was

used in place of GM1, and binding of the plant derived

recombinant protein for GM1 receptor and BSA was

checked.

Estimation of TCPA level in transgenic plants

The TCPA protein expression level was estimated by

measuring the density of the band corresponding to TCPA

in the western blot image, using ImageMasterTM VDS

Software, Version 2.0, Pharmacia Biotech. The western

image was changed to grey scale. Density of the bands was

measured following the manufacturer’s instructions.

Expression level was calculated by comparing the trans-

genic protein band density with the density of band of

known amount of purified TCPA protein.

Segregation analysis of transgenic plants

Seeds harvested from the transgenic plants were surface-

sterilized and inoculated on ½MS medium. Germinated

seedlings were transferred to MS medium, supplemented

with 400 mg/l kanamycin in the culture bottles and grown

under a 16 h light/8 h dark photoperiod. The seedlings

were evaluated for resistance and sensitivity to the antibi-

otic after 3 weeks of their growth on selection medium.

The resistant seedlings were transferred to soil for hard-

ening. The observations were checked for their statistical

significance and Chi-square test was performed to find out

the best fit Mendalian segregation pattern.

Results

Production of transgenic plants expressing CTB-P4,

CTB-P6 fusion proteins or TCPA protein

Binary vectors for the expression of CTB-P4 and CTB-P6

fusion proteins contained fusion of 393 bp gene fragment

of ctxB, encoding cholera toxin B subunit having the Ko-

zak sequence at 50 end and an extra 12 bp sequence

encoding hinge peptide at 30 end with 72 or 78 bp frag-

ment, encoding P4 or P6 epitope of TCPA from V.

cholerae. This chimeric gene was driven by CaMV35S

promoter and double enhancer sequence. A sequence

encoding the endoplasmic reticulum retention signal

SEKDEL was added to the 30 end of fusion gene. The

sequence encoding tetrapeptide hinge was added to the 30

end of ctxB gene to generate intramolecular flexibility

between the CTB and P4 or P6 epitopes that ultimately

would reduce structural constraints on CTB oligomeriza-

tion. For the transformation of plants with tcpA, ctxB-p4 or

ctxB-p6 genes, plant transformation constructs pCAMBI-

ATCPAE2L, pCAMBIACTB-P4E2L and pCAMBIACTB-

P6E2L, respectively, were used (Fig. 1). These constructs

also had nptII gene under control of CaMV35S promoter

for selecting the transgenic plants on kanamycin, and gus

gene coding for the reporter enzyme.

Ten days old cotyledons from S. lycopersicum var. Pusa

Ruby were transformed with ctxB-p4 or ctxB-p6 vectors

through Agrobacterium-mediated transformation using

AGL1 strain and tcpA vector was transferred to tomato

plants using LBA4404 strain. For the expression of each

fusion protein in plants, 20 independent transgenic kana-

mycin resistant tomato lines were produced. Eight

independent transgenic kanamycin resistant tomato lines

expressing TCPA protein were generated. Based upon GUS

expression, 15 out of 20 transgenic lines for each fusion

protein and all 8 lines carrying tcpA gene were selected for

further evaluation.

Plant Cell Rep (2008) 27:307–318 311

123

Detection of the transgenes and their transcripts

Genomic DNA was isolated from leaf tissue of transgenic

plants. PCR amplification of ctxB-p4 and ctxB-p6 fusion

genes was done using forward primer of ctxB and reverse

primer of p4 or p6, respectively. For amplification of tcpA

gene, primer pair of ETF and PTR was used. PCR products

of expected sizes, i.e., 504 bp for ctxB-p4 gene (Fig. 2a),

510 bp for ctxB-p6 gene (Fig. 2b) and 674 bp for tcpA

gene (Fig. 2c), were obtained. Genomic DNA from

untransformed plants did not show any amplification.

Transgenic tomato plants did not show any major mor-

phological difference compared with wild type plants.

Transgene-specific transcripts were also detected in

northern analysis performed on total RNA isolated from

the leaves of transgenic plants. Blots were probed with

enzyme-labeled or radiolabeled ctxB-p4, ctxB-p6 or tcpA

gene fragments. Certain degree of variation in levels of

transgene specific mRNAs was observed among indepen-

dent transgenic events for all the genes, i.e., ctxB-p4, ctxB-

p6 and tcpA (Fig. 3).

Immunoblot analysis of plant-expressed antigenic

proteins

To check the expression of recombinant proteins in trans-

genic plants, total soluble protein was extracted from the

fruits and probed with polyclonal antibody raised against

TCPA or CTB. Polyclonal antiserum against CTB was

purchased from Sigma. For raising TCPA-specific antisera,

TCPA protein was expressed in E. coli strain

SG13009[pREP]. Lysate from culture induced with 1 mM

IPTG for 4 h showed clear band for induced TCPA protein

on SDS-polyacrylamide gel (supplementary figure S1a).

The TCPA protein was purified by electro-elution, fol-

lowed by affinity purification, using Ni-NTA matrices from

Qiagen and the purity was confirmed on gel (supplemen-

tary figure S1b). The purified TCPA protein was used to

raise anti-sera. Chimeric proteins CTB-P4 and CTB-P6

were also expressed in E. coli. Although the expression

level of chimeric proteins CTB-P4 or CTB-P6 was low,

induced bands at *64 kDa representing multimeric pro-

teins CTB-P4 or CTB-P6 were observed on the coomassie

stained SDS-polyacrylamide gel (supplementary figure

S1c). Purified TCPA protein served as control for immu-

nological analysis of plant-expressed TCPA protein.

The expression level of CTB-P4 and CTB-P6 fusion

proteins varied among the plants. Though these proteins

were barely detectable in few of the plants, there was sig-

nificant accumulation in other plants (Fig. 4). The expected

molecular weight of the pentameric chimeric protein CTB-

P4 and CTB-P6 is 75.2 and 77.5 kDa, respectively. In the

transgenic fruits, CTB-P4 and CTB-P6 fusion proteins were

detected to be of *76 kDa (Fig. 4a, b). Corresponding

bands were not detected in protein extracts from untrans-

formed plants. Molecular weights of CTB-P4 and CTB-P6

suggests that fusion proteins were organized in pentameric

form. The western analysis of boiled samples was also

conducted. Total protein from the transgenic plants

expressing B subunit of cholera toxin was also used as

control. As expected the molecular weights of the mono-

meric CTB-P4 and CTB-P6 fusion peptides were found to

be higher than that of plant-expressed CTB monomer in the

western blot due to addition of 24 or 26 amino acids of P4 or

Fig. 2 PCR amplification of transgenes from tomato plants. a ctxB-

p4, b ctxB-p6, c tcpA. For PCR 100 ng genomic DNA was used with

gene-specific primers. B blank, C +ve control, L length standards, WTwild type, transgenic plant lines are numbered

Fig. 3 Detection of transgene-specific transcripts by northern hybrid-

ization. a ctxB-p4, b ctxB-p6, c tcpA. Northern transfer was carried

out using 20 lg of total RNA, which was hybridized with [32P]

labeled or enzyme labeled probe for respective transgene. Lower

panel shows the methylene blue or ethidium bromide stained

ribosomal RNA (rRNA) for indicating quantity and quality of RNA

employed. WT wild type, transgenic plant lines are numbered

312 Plant Cell Rep (2008) 27:307–318

123

P6 epitopes, respectively, and 4 amino acids of the hinge

region (Fig. 4d). Variable accumulation of TCPA protein

was also detected in fruit tissue of transgenic lines analyzed

(Fig. 4c). The expected molecular weight of TCPA protein

is 20.9 kDa whereas plant-produced recombinant TCPA

was detected to be of 25 kDa.

GM1-receptor binding assay and quantification of

recombinant proteins in transgenic plants

GM1-ganglioside, a receptor of pentameric cholera toxin B

subunit, was used to check the biological activity and

quantity of the oligomeric fusion proteins. Like bacteria-

purified CTB, plant-produced fusion proteins demonstrated

strong affinity for GM1-ganglioside and did not bind to

BSA (Fig. 5a). The expression level was calculated by

comparing the OD value obtained using protein extracts

from transgenic plants with OD values obtained using

known amount of standard purified protein and the protein

level was expressed as per cent of total soluble protein in

the sample. The expression level of pentameric CTB-P4

fusion protein varied from 0.007 to 0.17% TSP in the fruit

tissue (Fig. 5b), whereas expression level of CTB-P6

fusion protein varied from 0.019 to 0.096% TSP in the fruit

tissue (Fig. 5c) of various transgenic lines.

The expression level of TCPA protein in the transgenic

plants was measured by densitometry. The intensity of the

plant-expressed TCPA protein band in the western blot was

compared to intensity of band of known amount of purified

TCPA from bacteria. The TCPA protein accumulated up to

0.12% TSP in the fruit tissue of transgenic tomato plants

(Fig. 5c).

Inheritance and segregation analysis

Transgenic lines P6-43 (CTB-P6), P4-55 and P4-76 (CTB-

P4) and TCP-6 (TCPA), which showed higher expression

of the antigens, were used for analysis of inheritance to

progeny. The expression of nptII gene, which confers

resistance to kanamycin, was used to study the inheritance

and segregation pattern of the transgene. The transgene was

found to be inherited following a Mendelian pattern of

segregation (Table 2).

The presence of transgenes was checked in T1 progeny

plants through PCR amplification using gene-specific

primers. PCR products of expected sizes i.e., 504 bp for

ctxB-p4 gene (Fig. 6a) and 510 bp for ctxB-p6 gene

(Fig. 6b) were amplified. The expression of CTB-P4

(Fig. 6c) and CTB-P6 (Fig. 6d) was also evaluated at

protein level in progeny plants. Although minor variations

are apparent, the levels were generally similar between

parents and their progenies (Fig. 5b, c).

Stability of recombinant protein

The stability of the plant-synthesized recombinant protein

was checked by GM1 ELISA on the protein extracts from

the lyophilized tissue stored at 4�C and room temperature

for three months. During the process of lyophilization 20%

loss of protein activity was recorded. The level of recom-

binant CTB-P4 or CTB-P6 protein in lyophilized powder,

after three months of storage at 4�C was 80–90% of the

initial amount at the time of storage and *75% recombi-

nant protein was detected in the sample stored at room

temperature for same period (Fig. 7).

Discussion

The production of potential vaccine antigens, with a

capacity to provide protective immunity against pathogen,

in transgenic plants is safe and inexpensive. The technol-

ogy has promising future for the development of vaccines

against infectious diseases, especially those, which are

Fig. 4 Detection of recombinant protein in fruit tissue of tomato

plants. a For detection of CTB-P4, 40 lg of TSP from fruit tissue

along with 60 ng of purified CTB were used during immunoassay. bFor detection of CTB-P6, 40 lg of TSP from fruit tissue along with

30 ng of purified CTB were used during immunoassay. c For

detection of TCPA, 50 lg of TSP from fruit tissue along with 60 ng

of purified TCPA were used during immunoassay. d For detection of

monomeric fusion peptides, 80 lg of TSP from transgenic plants

expressing fusion protein as well as CTB alone, along with 20 ng of

purified CTB were used after boiling for 5 min with b-mercaptoeth-

anol. Chimeric proteins and TCPA were detected using CTB- and

TCPA-specific antibodies, respectively. P purified protein, PC plant

expressing CTB protein, transgenic plant lines are numbered, UTuntransformed, WT wild type

Plant Cell Rep (2008) 27:307–318 313

123

caused by the pathogens that make their entry in the body

through mucosal surfaces. Cholera is caused by V. cholerae

that colonizes the intestine. CTB and TCPA have been

described as protective antigens against the infection. Sera

raised against CTB and TCPA were shown to protect infant

mice passively against the challenge (Elson and Ealding

1984; Clemens et al. 1986; Sun et al. 1990; Voss et al.

1996). Recently, it has been shown that transcutaneous

immunization with TCPA induces protective immunity

against V. cholerae O1 challenge in mice (Rollenhagen

et al. 2006). It had been reported earlier that larger size of

the fusion partner and position of the fusion, i.e., at NH2- or

COOH-terminal, affects the pentamerizing ability or

induces conformational changes that lead to reduced GM1

binding efficiency of CTB or no binding at all (Lipscombe

et al. 1991). Also, it has been found that linking the peptide

to NH2-terminal end of CTB may reduce its GM1 binding

efficiency as N-terminus lies near to GM1 binding pocket of

CTB (Zhang et al. 1995). Tomato plant was chosen to

express TCPA and its two epitopes P4 or P6 fused to CTB

as it generates abundant biomass and edible tissue, and can

grow in tropical and subtropical regions of the world where

the need of the cheaper vaccines is more. The palatability

of raw tomato fruits makes it an attractive host for the

production of vaccine antigens, as the tissue containing

antigens needs to be delivered uncooked. Transgenic

tomato fruits can be powdered after freeze-drying and can

be stored in dry conditions. It would protect the recombi-

nant protein against degradation during storage. The

approach to purify recombinant vaccine antigens from

plants would be expensive whereas oral delivery of the

plant made vaccine in processed form would be economi-

cally feasible. Tomato has been used to express antigen

proteins earlier also for similar reasons (McGarvey et al.

1995; Sandhu et al. 2000; Jani et al. 2002; Walmsley and

Arntzen 2003; Pogrebnyak et al. 2005; Saldana et al. 2006;

Shchelkunov et al. 2006; Jiang et al. 2007). In addition to

being source of purified V. cholerae antigens, live E. coli

cells expressing TCPA, CTB-P4 and CTB-P6 may also find

utility as components of cholera vaccine.

Various epitopes have been described within the TCPA

protein, of which epitope P4 and P6 have been shown to

provide protective immunity against the challenge (Sun

et al. 1997). Peptides P4 and P6 were selected as fusion

partner and linked to C-terminal end of CTB through a

hinge linker. Endoplasmic reticulum retention signal

sequence was added to C-terminus of CTB-hinge-P4/P6

fusion. The endoplasmic reticulum compartment has been

reported to facilitate oligomerization of labile toxin B

subunit (LT-B) of E. coli, which is very similar to CTB and

targeting to ER was also found to enhance the expression

of LT-B (Haq et al. 1995). In the present study, chimeric

proteins CTB-P4 and CTB-P6 were expressed in tomato

plant through Agrobacterium-mediated transformation.

The aim was to produce a safe and inexpensive subunit

vaccine in tomato plants, which can be delivered through

oral route and could provide immunity against the toxic

secretions of V. cholerae as well as against its colonization

in the intestine. Also, the complete coding sequence of

a

b

0.20

0.25

0.00

0.05

0.10

0.15

CTB 0.2 ng

CTB 0.8 ng

P4 76 T1 3

P4 76 T1 4

P4 76 T1 1

4

P4 76 T1 1

8

Sample Identity

Control

Ab

sorb

ance

GM1 BSA

T0 Fruits T1 Fruits

0.02

0.06

0.10

0.14

0.18

0.22

% T

SP

Control

P4 55 P4 62

P4 158

P4 163P4 76

P4 55 T1 1

P4 55 T1 5

P4 76 T1 3

P4 76 T1 4

P4 76 T1 14

P4 76 T1 18

CTB-P4

c

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Contro

l

P6 14

P6 27

P4 35

P6 43

P6 52

P6 43

T1 1

P6 43

T1 2

P6 43

T1 3

P6 43

T1 4

TCP 3

TCP 6

T1 fruitsT0 fruits T0 fruits

% T

SP

CTB-P6 TCP A

Fig. 5 Quantification of recombinant proteins from fruits of trans-

genic plants. a Demonstration of specific affinity of GM1-ganglioside

for CTB-P4 chimeric protein expressed in tomato plants. A plate was

coated with GM1 or BSA and incubated with purified CTB, and total

soluble protein from leaf tissue of the wild type and transgenic plants.

b CTB-P4 fusion protein from the fruit tissue of T0 and T1 generation

plants. c CTB-P6 fusion protein from the fruit tissue of T0 and T1

generation plants and TCPA protein from fruit tissue of T0 generation

plants. One microgram total soluble protein from fruit was used for

GM1-ELISA, TSP total soluble protein

314 Plant Cell Rep (2008) 27:307–318

123

tcpA was transferred to tomato plants to express complete

TCPA subunit. TCPA was expressed in tomato with a view

that fruits expressing TCPA can be mixed with fruits

expressing CTB (Jani et al. 2002) to develop an efficient

vaccine against cholera. The presence of recombinant

CTB-P4 and CTB-P6 fusion proteins was checked by

western blot analysis using anti-CTB antibodies. For the

detection of TCPA, anti-TCPA antibodies were generated.

TCPA protein was expressed in E. coli. The protein was

purified to homogeneity and used for raising of antibodies.

The plant-expressed fusion proteins CTB-P4 and CTB-P6

were detected to be of 76-kDa molecular weight, in the

western blot analysis, corresponding to weight of penta-

meric proteins. It shows that the fusion of TCPA epitopes

to CTB did not interfere with tertiary structure of CTB

needed for pentamerization. CTB-P4 and CTB-P6 mono-

mer peptides were detected in the western blot after boiling

the samples for 5 min in reducing conditions before elec-

trophoresis. The molecular weight of plant-expressed

fusion proteins was found to be 76 kDa whereas the bac-

teria-expressed fusion proteins were detected to be of

64 kDa. An increase in the molecular weight of plant-

expressed fusion proteins may be due to addition of

SEDKEL peptide and pos-translational modifications

occurring in the plant cells. Earlier, higher molecular

weight of plant-expressed CTB has been found to be due to

glycosylation in the plant cells (Mishra et al. 2006). If the

samples were not boiled and non-reducing conditions were

used, purified CTB protein or recombinant fusion proteins

were detected at pentameric positions in the denaturing gel.

It might be attributed to the significant hydrophobic inter-

actions of the oligomers.

GM1-ganglioside has been characterized to be the

receptor of CTB protein in vivo (de Haan et al. 1998) and

for binding of CTB to its receptor to take place, pentameric

structure of CTB is essential (Tsuji et al. 1995). Since

pentameric structure of cholera toxin is essential for its

immuno-reactive characteristics (Jobling and Holmes

2002), it is very important to ensure that fusion of any

peptide to CTB does not impair the ability of CTB to form

pentamer. Upon oral immunization, plant-produced CTB

protein has been shown to induce CTB-specific immune

responses in mice and behaved like the native CTB with

regard to its effects on T-cell proliferation and cytokine

levels (Arakawa et al. 1997; Jani et al. 2002; Jiang et al.

2007). In the present work, the plant-produced oligomeric

fusion protein was tested for its ability to bind to GM1-

ganglioside receptor. The total soluble protein preparations

from transformed and untransformed plants were analyzed

for receptor binding using CTB-specific antibodies. The

Table 2 Segregation analysis of kanamycin resistance in the T1 progeny of selected transformed tomato plants expressing CTB-P4, CTB-P6

fusion protein or TCPA protein

Line no. No. of seeds

germinated

No. of kanamycin

resistant seeds

No. of kanamycin

sensitive seeds

v2 value Probability Segregation

ratio

P4–55 121 97 24 1.72 0.10 \ P \ 0.25 3:1

P4–76 133 103 30 0.423 0.5 \ P \ 0.75 3:1

P6–43 75 61 14 2.35 0.10 \ P \ 0.25 3:1

TCP-6 110 86 24 0.593 0.25 \ P \ 0.50 3:1

Fig. 6 Molecular characterization of T1 progeny plants expressing

CTB-P4 or CTB-P6 chimeric proteins. The fusion gene ctxB-p4 (a) or

ctxB-p6 (b) was amplified from genomic DNA of transgenic plants.

For PCR, 100 ng of genomic DNA from transformed as well as

untransformed plants was used. For detection of CTB-P4 fusion

protein (c), 40 lg of TSP from fruit tissue along with 60 ng of native

CTB were used. For detection of CTB-P6 fusion protein (d), 40 lg of

TSP from fruit tissue along with 30 ng of native CTB were used.

Rabbit anti-CTB antibodies were used for immunoassay. B Blank, C+ve control, L length standards, P purified protein, PC plant

expressing CTB protein, WT wild type, transgenic lines are numbered

Plant Cell Rep (2008) 27:307–318 315

123

multimeric protein from the transformed plants showed

strong affinity towards GM1-ganglioside as compared to

BSA, whereas the protein from untransformed plants did

not show any affinity to either BSA or GM1-ganglioside.

This suggests that recombinant CTB-P4 and CTB-P6

fusion proteins derived from transgenic tomato plants were

pentameric and retained their ability to interact with GM1-

ganglioside.

Transgenic progeny inherited transgenes in a manner

expected from Mendelian segregation of a single locus

gene. All the transgenic plants selected on kanamycin were

found to be PCR positive. The progeny plants expressing

CTB-P4 or CTB-P6 were characterized at protein level.

The CTB-P4 expression in the T1 progeny members of P4-

55 was comparable to its parent whereas the progeny of P4-

76 showed some variations in expression pattern. Similarly,

among T1 progeny members of P6-43, some reduction in

expression level was observed for certain plants whereas

other plants had expression level that was comparable to

their parent. Overall there were no major differences in the

expression levels between plants of T0 generation and

plants of their T1 progeny.

Up to 1.5% TSP expression level of CTB has been

achieved through nuclear transformation of ctxB gene

(Arakawa et al. 1997; Wang et al. 2001; Jani et al. 2002;

Kang et al. 2004, 2006), but in tomato fruits the achieved

CTB expression level is 0.08% (Jani et al. 2002). For the

development of chimeric CTB proteins in plant tissue,

stability of the peptide conjugated to CTB is another con-

straint in the way of achieving high-level expression of

oligomeric active proteins. The expression levels of chi-

meric proteins with CTB as on of the component, achieved

through nuclear transformation ranges from 0.002 to 0.11%

TSP (Arakawa et al. 1998b; Kim and Langridge 2003; Kim

et al. 2004a, b, c, d; Lee et al. 2004; Choi et al. 2005; Li

et al. 2006). To enhance the chimeric protein expression

level, the gene was expressed under CaMV35S promoter

having duplicated enhancer sequence. Further, leader

sequence of alfalfa mosaic virus was also incorporated in

the construct. Expression levels up to 0.17% TSP for

oligomeric chimeric protein CTB-P4 and 0.096% TSP for

chimeric protein CTB-P6 were recorded in tomato fruits in

T0 generation, as determined by binding of GM1-ganglio-

side to the chimeric proteins. Optimization of coding

sequence of the transgene may further enhance the

expression level of recombinant proteins. Little reduction

in the active protein levels of CTB-P4 and CTB-P6 was

recorded in the lyophilized tissue stored at 4�C or room

temperature for 3 months.

The potential of the TCP as a protective antigen has

been described long back (Sharma et al. 1989). In the

present study, TCPA protein was also successfully

expressed in transgenic tomato plants. The molecular

weight of plant-expressed TCPA protein was 25 kDa

whereas the its expected size is 20.9 kDa. This increase in

size can be attributed to addition of SEKDEL sequence at

C-terminal end, which was added to retain the recombinant

protein in the endoplasmic reticulum or to possible gly-

cosylation of the protein as reported for CTB (Mishra et al.

2006).

The plant-derived CTB has already been shown to have

antigenicity similar to the purified CTB (Wang et al. 2001;

Jani et al. 2004). Fusion of protein epitope to CTB has been

described for the expression of CTB-insulin fusion

(Arakawa et al. 1998b) and CTB-hepatitis C virus epitope

fusion (Nemchinov et al. 2000) and these plant-expressed

chimeric antigens have been shown to elicit immune

response upon oral delivery in the mouse model. The

bacteria-expressed TCPA has been shown to elicit protec-

tive immune response in infant mice. The TCPA protein

has been produced in the plants for the first time. Plant-

derived antigens are very attractive alternatives for the

vaccine production, but use of fusion proteins having

potential immunogenic epitopes of two or more antigens

from a pathogen makes the case of plant vaccines even

more attractive. Cholera toxin B subunit has been used as

an adjuvant to boost the response of other antigens. Fusion

of CTB with the epitopes of TCP and expression of chi-

meric proteins in plants may result into production of

vaccine, which could be more efficient because of both

anti-toxin and anti-colonization immunity.

Acknowledgments The research work is supported by financial

assistance from the Department of Biotechnology, Government of

India, New Delhi. Manoj K. Sharma acknowledges University Grants

0.00

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

CTB-P4 CTB-P6

% T

SP

det

ecte

d

Fresh

Lyo

Lyo + 4°CLyo + RT

Fig. 7 Protein stability in lyophilized fruit tissue. Stability of

recombinant protein in lyophilized fruit tissue, stored at room

temperature and 4�C for 3 months, was quantified using GM1-ELISA.

Wild type fruit was used as negative control. Fresh, initial level in

fresh tissue; Lyo initial level in lyophilized tissue, Lyo+4�Clyophilized tissue stored at 4�C for 3 months, Lyo+RT, lyophilized

tissue stored at room temperature for 3 months. TSP total soluble

protein

316 Plant Cell Rep (2008) 27:307–318

123

Commision, India, for Senior Research Fellowship and Dewal Jani

acknowledges CSIR, India, for Senior Research Fellowship.

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