Molecular cloning, recombinant expression and
IgE-binding epitope of x-5 gliadin, a major allergen in
wheat-dependent exercise-induced anaphylaxis
Hiroaki Matsuo, Kunie Kohno and Eishin Morita
Department of Dermatology, Shimane University School of Medicine, Izumo, Japan
Wheat is one of the most widely cultivated staple
foods for western people. Patient with wheat allergy,
especially wheat-dependent exercise-induced anaphy-
laxis (WDEIA) has increased recently, as there is now
a higher consumption of western style food in Japan
[1,2]. WDEIA is a distinct form of wheat allergy in
which the patient experiences a very severe allergic
reaction in response to intense exercise after ingestion
of wheat [3,4]. Our previous study demonstrated that
exercise and aspirin intake facilitate absorption of the
wheat allergens from the gastrointestinal tract in
patients with WDEIA [5]. It follows that the allergens
transferred into circulating blood cross-link receptor-
bound IgE on mast cells and cause degranulation
followed by release of chemical mediators such as his-
tamine. They induce immediate inflammatory reactions
similar to those of common food allergies such as
urticaria, angioedema, hypotension, and shock.
To diagnose WDEIA, we typically perform an exer-
cise challenge test combined with wheat ingestion for
patients who have episodes of anaphylaxis after wheat
intake. However, the challenge test is unsafe for
patients because an anaphylactic shock is sometimes
provoked in the test. An radioallergosorbent test to
wheat protein or wheat gluten is commercially avail-
able for diagnosis of wheat allergy, but this test is not

was expressed in Escherichia coli by means of the pET system and purified
using RP-HPLC. Western blot analysis and dot blot inhibition assay of
recombinant and native x-5 gliadin purified from wheat flour demonstrated
that recombinant protein had IgE-binding ability. Our results suggest that
the recombinant protein can be a useful tool for identifying patients with
wheat-dependent exercise-induced anaphylaxis in vitro.
Abbreviations
WDEIA, wheat-dependent exercise-induced anaphylaxis.
FEBS Journal 272 (2005) 4431–4438 ª 2005 FEBS 4431
always satisfactory to diagnose WDEIA because of
low sensitivity or the occurrence of false-positive
results [6]. The heterogeneity of antigens used in the
test is considered to be a major cause of these prob-
lems. It has been reported that x-5 gliadin is a major
allergen in patients with WDEIA; the skin prick test
and radioallergosorbent test with x-5 gliadin is consid-
ered to be useful to diagnose WDEIA [6–8].
Common wheat (Triticum aestivum) is a hexaploid
species, in which each cell contains six sets of chromo-
somes and is estimated to have several copies of the
x-5 gliadin gene [9]. In wheat there are at least six dif-
ferent x-5 gliadin proteins; the primary structures of
these is very similar but the contents vary according to
growing districts or cultivated variety [10]. Hence it is
difficult to prepare a homogeneous x-5 gliadin protein
from wheat flour.
In the present study we analyzed IgE-binding epi-
topes in an extra three patients with WDEIA and
cloned the x-5 gliadin gene to obtain the IgE-reactive
homogeneous recombinant x-5 gliadin protein that

nucleotide sequences extracted from a database of wheat
expressed sequence tags (ESTs). A high-fidelity DNA
polymerase was used to reduce the risk of introducing
errors into the sequence.
Amplification of genes from wheat genomic DNA
produced two products designated x-5a and x-5b,of
1.4 and 1.2 kb, respectively (Fig. 1). Both genes were
cloned into Escherichia coli XL-10 Gold and the
nucleotide sequence was determined. The x-5a gene
consists of 1413 bp and has an ORF throughout the
Table 1. IgE-binding epitope sequences for patients with WDEIA.
Unfilled circles indicate the IgE-binding epitopes detected in this
study.
Epitope sequence
Patient
123
1 QQIPQQQ
a

2 QQLPQQQ
a

3 QQFPQQQ
a

4 QQSPQQQ
a

5 QQSPEQQ
a

and AB181301. The protein encoded by the x-5a gene
is found to have 439 amino acid residues with a puta-
tive signal peptide of 19 amino acids. The molecular
mass of the protein without the signal sequence was
calculated to be 50 900.
Expression in E. coli and purification of
recombinant x-5 gliadin
As DNA encoding the full-length x-5a gliadin protein
could not subcloned into the E. coli expression vector
because of plasmid instability we tried to produce half
of the protein: the C-terminal 178 amino acids, at posi-
tion 813–1346 in x-5a gene in Fig. 2, includes all of
the detected IgE-binding epitope sequences. After
amplification of the DNA encoding this half of the
x-5a gliadin protein by PCR, the DNA fragment was
inserted into the expression vector pET-21a. E. coli
Rosetta (DE3) was used as a host strain as the x-5a
gene has a lot of rare E. coli codons. As shown in
Fig. 3 lane 2 a high level of expression of recombinant
protein, designated rO5GC, was obtained. The x-5
gliadin purified from wheat flour is slightly soluble in
water and soluble in 70% ethanol, whereas the rOG5C
protein is soluble in both water and 70% ethanol.
Therefore the recombinant protein was extracted with
TBS buffer and then 70% ethanol, and was separated
to homogeneity by RP-HPLC (Fig. 3, lane 4). The
apparent molecular mass (27.2 kDa) of the rOG5C
determined by SDS ⁄ PAGE was higher than the
molecular mass (21.7 kDa) calculated from the amino
acid sequence. It was confirmed that the first 10 amino

topes in x-5 gliadin and described the gene cloning,
expression in E. coli, purification, and immunological
characterization of the recombinant x-5 gliadin.
In our previous study we showed that QQIPQQQ,
QQFPQQQ, QQLPQQQ, QQSPQQQ, QQSPEQQ,
QQYPQQQ and PYPP sequences in x-5 gliadin are
IgE-binding epitopes in patients with WDEIA and that
four of these sequences, QQIPQQQ, QQFPQQQ,
QQSPQQQ and QQSPEQQ, are dominant [6]. In the
present study we carried out an additional IgE epitope
analysis in three patients with WDEIA. Two of the
three patients have IgE antibodies that react with the
four dominant epitope sequences but IgE antibodies in
Fig. 3. SDS ⁄ PAGE analysis of the proteins at various purification
steps. Lane 1, molecular mass size marker; lane 2, cell extract from
E. coli (pETO5C) grown in the presence of isopropyl thio-b-
D-gal-
actoside; lane 3, crude proteins extracted by 70% ethanol; lane 4,
purified recombinant protein.
Cloning and expression of wheat x-5 gliadin H. Matsuo et al.
4434 FEBS Journal 272 (2005) 4431–4438 ª 2005 FEBS
the serum of patient three reacted only with peptide
QQFPQQQ (Table 1). In addition, IgE antibodies of
patients two and three did not react with QQYPQQQ
but did react with YQQYPQQ. The two epitopes,
QSPEQQQ and QQFHQQQ, were detected only in
patient three, and similarly QQPPQQ was detected
only in patient one. These results indicate that the four
newly detected IgE-binding epitopes, QQPPQQ,
YQQYPQQ, QSPEQQQ and QQFHQQQ, are not

membrane. The membrane was blocked and incubated with 10% of the patient’s serum previously incubated with different concentrations
of purified native or recombinant x-5 gliadin.
H. Matsuo et al. Cloning and expression of wheat x-5 gliadin
FEBS Journal 272 (2005) 4431–4438 ª 2005 FEBS 4435
QQQQXP where X is F, I or L and the lack of a cys-
teine residue in x-5a gliadin are compatible with the
structural features of x-5 gliadin. Kasarda et al. repor-
ted that the N-terminal amino acid sequence of
x-5 gliadin from wheat (T. aestivum ‘Justin’) is
SRLLSPRGKELHTPQQQFPQQ [17]. DuPont et al.
showed that x-5 gliadin from wheat (T. aestivum
‘Butte’) was separated into two fractions, 1B1 and
1B2, and the N-terminal amino acid sequences are
SRLLSPRGKELHTPQEFQFPQQQ and SRLLSPRG
KELHTPQEQFPQQQ, respectively [9]. The deduced
N-terminal amino acid sequence of x-5a is identical
with 1B2 x-5 gliadin. The 1B2 x-5 gliadin fraction
from T. aestuvum Butte was resolved into three peaks
of molecular mass 49 085, 50 300, and 51 500 by
MALDI-TOF MS [9]. However the calculated mole-
cular mass (50 900) of x-5a gliadin did not coincide
with any of these molecular masses. The differences in
mass between the three 1B2 x-5 gliadins and x-5a glia-
din may be accounted for by a difference of the num-
ber of repeat sequences.
In wheat allergy, sensitization to inhaled wheat flour
leads to baker’s asthma [18], whereas sensitization to
ingested wheat develops into a common food allergy
or WDEIA. In addition, the causative allergen is dif-
ferent in various clinical manifestations, for instance

SDS ⁄ PAGE. This difference in mass is accounted for
by the behaviour of native x-5 gliadin as published
previously [9].
It is vital to compare immunological properties of
a recombinant protein with those of the native form
before using the recombinant for diagnosis or treat-
ment of food allergies. Western blot analysis of nOG5
and rOG5C showed that the IgE antibodies in sera of
patients with WDEIA react to nOG5 rather than to
rOG5C. Dot blot inhibition assays indicate that the
IgE-binding capacity of nOG5 is larger than that of
rOG5C due to a lack of N-terminal half of rOG5C.
However, rOG5C had sufficient ability to detect the
specific IgE to x-5 gliadin because rOG5C completely
inhibited the IgE binding to nOG5. Thus the recom-
binant x-5 gliadin produced in this study provides rea-
gent quantities of protein that would be useful in the
serologic diagnosis of WDEIA.
Experimental procedures
Identification of IgE-binding epitope
in x-5 gliadin
Overlapping peptides of x-5 gliadin were synthesized on
SPOTs membranes (Sigma-Genosys, The Woodlands, TX,
USA); sera from three patients with WDEIA and a positive
provocation test result were used to probe the membrane
as described previously [4].
Purification of x-5 gliadin from wheat flour
Gliadin mixture purchased from Tokyo Kasei Kogyo
(Tokyo, Japan), dissolved in 70% (v ⁄ v) ethanol and puri-
fied by HPLC on a Jasco model 880 (Tokyo, Japan) and a

Cloning and sequencing of PCR products
The PCR product was analysed by electrophoresis through
1% agarose gel and purified using MinElute
TM
Gel Extrac-
tion Kit (Qiagen, Valencia, CA, USA). The purified PCR
product was cloned into a pPCR-Script Amp cloning vector
(Stratagene, La Jolla, CA, USA) and then the ligated plasmid
was transformed into E. coli XL10-GoldÒ Ultracompetent
cells (Stratagene). Five clones containing the wheat DNA
fragment were selected. Then the EcoRI digested DNA frag-
ments were subcloned into pUC18 and sequenced by the
dideoxy chain termination method using a BigDye termina-
tion sequencing kit and the ABI 3100 DNA sequencer
(Applied Biosystems, Foster City, CA, USA).
Expression and purification of recombinant
protein
Sense (5¢-ATTTCATATGCAACAACAATTCCCCCAGC
AACAATCA-3¢) and antisense (5¢-TCTCGGATCCTCA
TAGGCCACTGATACTTATAACGTCGCTCCC-3¢) oligo-
nucleotide primers having an initiation codon and an NdeI
site at the 5¢- and a BamHI site at the 3¢-adjacent region,
were designed and synthesized based on the determined
nucleotide sequences of x-5a gliadin gene. PCR was per-
formed using the conditions described above using plasmid
DNA containing the cloned x-5 gliadin gene as template.
PCR product was digested with NdeI and BamHI and
ligated to an expression vector, pET-21a, digested with
same enzymes to generate pETO5C. E. coli Rosetta (DE3)
cells harbouring pETO5C was grown in terrific broth (Dif-

serum. After washing with TBST, the membrane was incu-
bated with horseradish peroxidase-conjugated goat antihu-
man IgE (BioSource, Camarillo, CA, USA). To detect
human IgE binding, ECL Plus Western blotting detection
reagents (Amersham Biosciences, London, UK) was used.
The resulting light was detected on autoradiography film.
Dot blot immunoassay for inhibition test
Dot blots were performed by applying 2 lg of the native x-5
gliadin onto a polyvinylidene difluoride membrane
(Immobilon-P) using a dot-blot manifold. After blocking
with 5% skim milk in TBST the blots were washed three
times with TBST for 10 min. The membrane was then incu-
bated with a 1 : 10 dilution of the patients’ serum that had
been previously incubated with different concentrations of
purified recombinant or native x-5 gliadin overnight at 4 °C.
After washing with TBST, the bound IgE antibodies were
detected as described above. After scanning the film, the spot
intensities were measured using the Gel-Pro Analyzer soft-
ware (Media Cybernetics Inc., Silver Spring, MD, USA).
Acknowledgements
We thank Dr Yuji Yamaguchi from the Shimane Agri-
cultural Experiment Station for providing us with
wheat plant. This study was supported by a grant from
the Iijima Memorial Foundation for the Promotion of
Food Sciences and Technology.
H. Matsuo et al. Cloning and expression of wheat x-5 gliadin
FEBS Journal 272 (2005) 4431–4438 ª 2005 FEBS 4437
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