280 Protein & Peptide Letters, 2008, 15, 280-285

0929-8665/08 $55.00+.00 2008 Bentham Science Publishers Ltd.

T and B-cell Epitopes Prediction of Iranian Saffron (Crocus sativus) Pro-
filin by Bioinformatics Tools
Babak Saffari, Hassan Mohabatkar* and Sasan Mohsenzadeh
Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
Abstract: Plant profilins form a well-known panallergen family responsible for cross-sensitization between plant foods
and pollens. We sought to map T and B-cell epitopes on the Iranian Crocus sativus profilin by bioinformatics tools. The
predicted peptides are useful for further vaccine development.
Keywords: Epitope, Profilin, Crocus sativus, Bioinformatics
INTRODUCTION
Type I hypersensitivities, characterized by rhinitis, con-
junctivitis, and bronchial asthma affect 15 % of the popula-
tion of industrialized countries [1-3]. An allergic response is
initiated when polyvalent allergen molecules direct the clus-
tering of surface bound IgE receptors. This clustering is ac-
companied by the activation of one or more tyrosine kinases
and subsequent release of anaphylactic mediators including
histamine, leukotrienes, prostaglandins and cytokines. Profil-
ins were first described by Carlsson et al. [4] as low-
molecular-weight proteins in calf spleen; which inhibited the
growth of actin filaments in vitro. As a result of the forma-
tion of profilamentous complexes with actin, they were
named Profilins. The functions that have been associated
with the action of profilin, and may be temporally the spa-
tially correlated, include: (1) actin monomer and filament
end binding [5, 6]; (2) positive or negative control of actin
nucleation and polymerization [7-10]; (3) promotion of the
exchange of ATP or ADP bound to actin for ATP [11-13];
(4) participation in the phosphoinsitide secondary messenger
signaling pathway [14-18]. Profilins also bind poly-L-proline
(PLP) and phosphatidylinositol-4, 5-bisphosphate (PIP2)
[19-22]. The localization and binding properties of profilin
thus suggest that it acts at a critical control point in signaling
pathways initiated by events at the plasma membrane and
plays an important role in regulating the activity in the mi-
crofilament system and intracellular calcium levels. The
presence of profilin as an allergen was first reported by Va-
lenta et al. [1], during their research on birch pollen aller-
genic extracts. Patients suffering from multivalent allergies
exhibit extensive IgE cross-reactivity towards a broad range
of profilins extending from distantly related plants to human
profilin. This wide spread cross-reactivity has led to the des-
ignation of profilins as pan-allergens [8]. It is assumed that
allergic symptoms in profilin-sensitized patients are trig-
gered and maintained by the ubiquitous occurrence of these
proteins.
Based on the advance in bioinformatics, the immunomics
becomes a new alternative in vaccine development [23]. Ad-

*Address correspondence to this author at the Department of Biology, Col-
lege of Sciences, Shiraz University, Shiraz 71454, Iran; Tel: +98711-
2280916; Fax: +98711-2280926; E-mail: mohabatkar@susc.ac.ir
vanced technologies for vaccine development, such as ge-
nome sequence analysis, microarrays, proteomics approach,
high-throughput cloning, bioinformatics database tools and
computational vaccinology can be applied for vaccine devel-
opment of several diseases including emerging diseases. In
the case of T-cell epitopes prediction of peptide binding to
major histocompatibility complex (MHC) molecules is the
basis for epitope discovery-driven vaccine development.
Current developments in computational vaccinology mainly
support the analysis of antigen processing presentation and
the characterization of targets of immune response. But pro-
filins as allergens induce humoral branch and more specifi-
cally IgEs of immune system and therefore knowing their B-
cell epitopes or IgE binding sites is more valuable. There is
considerable evidence in favor of conformational instead of
linear IgE epitopes in profilins allergens [8, 23-27] so, the
study of these properties on the structures of plant profilins
can help to uncover both specific and cross-reactive IgE epi-
topes because profilin allergens are highly conserved (>70%
of amino acid sequence identity in most pair comparisons)
and therefore induce cross-sensitization between plant aller-
genic sources. These theoretical predictions can then be
tested by using experimental and complementary methods as
the analysis of IgE-binding properties of synthetic peptides
covering the amino acid sequence of the allergen. In the pre-
sent work, with the aim of identifying candidate antigenic
regions for vaccine design, we have carried out bioinformat-
ics prediction of potential B-cell and T-cell epitopes of Ira-
nian saffron (Crocus sativus) profilin by some servers. Util-
izing bioinformatics servers for prediction of proteins func-
tion [28, 29], localization [30-32], epitope regions, distinc-
tion of various segments of a putative protein such as do-
mains, motifs, secondary elements, signal peptides [33, 34]
and etc is a time saving approach which could significantly
help to increase our information about role and the structure
of hypothetical proteins, i.e. proteins predicted from nucleic
acid sequences only and protein sequences with unknown
function [reviewed in 28 and 31].
Identification of a variety of MHC-binding motifs and
experimental characterization of thousands of allele-specific
and promiscuous MHC binders and T-cell and B-cell epi-
topes provide a solid information base for computational
T and B-cell Epitopes Prediction of Iranian Saffron Protein & Peptide Letters, 2008, Vol. 15, No. 3 281
prediction of binding peptides and develop the corresponding
data bases in future.
MATERIALS AND METHODS
Amino Acid Sequences
The amino acid sequences of Iranian saffron profilin
(Crocus sativus) (accession number AAW81034.1) and other
profilins that appear in alignment analysis were fetched from
National Center for Biotechnology Information (NCBI).
Protein Secondary Structure Prediction
To find the probable secondary structure of the profilin of
Crocus sativus the PROFsec method was used (available
from the URL: http://www.predictprotein.org/) [35, 36]. The
PROFsec combines the information from multiple sequence
alignments with the optimization strength of the neural net-
work formalism. The method showed an overall prediction
accuracy of 76% of all residues.
T-cell Epitope Prediction
Potential T-cell epitopes of the Crocus sativus profilin
was predicted by Average Relative Binding (ARB) matrix
method (available at IEDB (The Immune Epitope Database)
server URL: http://www.immuneepitope.org) [37]. ARB
matrix method is based on the assumption that each residue
along the peptide molecule independently contributes to
binding affinity. The effect of each of the 20 possible amino
acids at each position along the peptide sequence is esti-
mated by a matrix of coefficients. The overall binding pro-
pensity of each peptide is then estimated by a simple poly-
nomial function and expressed as an algorithm score. The
ability of proteins to act as immunogens stems from the
binding of peptides, derived from antigen processing, to
MHC molecules. Peptides derived from exogenous proteins
are presented mainly by MHC class II molecules to TCRs on
CD4
+
T-cells. We selected HLA-DR alleles most frequent in
the Caucasian population (HLA DRB1*0101, HLA DRB1
*0301, HLA DRB1*0401, HLA DRB1*0404, HLA DRB1
*0405, HLA DRB1*0701, HLA DRB1*0802, HLA DRB1
*0901, HLA DRB1*1101, HLA DRB1*1302, HLA*DRB1
1501, HLA DRB3*0101, HLA DRB4*0101, HLA DRB5
*0101).
B-cell Epitope Prediction
For prediction of sequential B-cell epitopes BepiPred
server (available from the URL: http://www.cbs.dtu.dk/ serv-
ices/BepiPred) was used [38]. In this approach the hidden
Markov model (HMM) was combined with Parker propen-
sity scale method [39]. Parameters such as hydrophilicity,
flexibility, accessibility, turns, exposed surface, polarity and
antigenic propensity of polypeptides chains have been corre-
lated with the location of continuous epitopes. This has led to
a search for empirical rules that would allow the position of
continuous epitopes to be predicted from certain features of
the protein sequence. So for the examining the results of
BepiPred method, Chou and Fasman beta turn prediction
[40], Karplus and Schulz flexibility scale [41] and Parker
Hydrophilicity Prediction [39] based B-cell epitope predic-
tions were performed (available from the URL: http://tools.
immuneepitope.org/tools/bcell/iedb_input). Furthermore the
charts of the Abraham hydrophobicity [42], J anin accessibil-
ity [43], Hopp and Woods hydrophilicity [44] and Bhaskaran
average flexibility [45] scales were drawn. All prediction
calculations and charts are based on propensity scales for
each of the 20 amino acids. Each scale consists of 20 values
assigned to each of the amino acid residues on the basis of
their relative propensity to possess the property described by
the scale.
Alignment
Sequences were aligned using ClustalW program [46]
from the Bioedit v5.0.9 package [47].
RESULTS
T-cell Epitopes
Peptides with the best predicted binding affinities are
presented in Table 1. Among all alleles, the results for DRB1
0101, DRB1 0701 and DRB1 0901 showed significant lower
IC50 than other alleles. Their IC50 values were 0.08, 0.23
and 0.88 respectively. It is very important to identify par-
tially self peptides since they could mount an autoimmune
response. So, these three peptides were analyzed for homol-
ogy with each of 45,513 proteins of human proteome using
the BLAST algorithm, but none of them seems to be com-
plete or partial self peptides. According to this work, 16-
DGHVLTSAAILGHDG-30 corresponding to DRB0101
allele is the peptide with the best binding affinity.
Table 1. Peptides with the Best Predicted Binding Affinities
for Each Allele
Alleles Peptides IC50 value
DRB1*0101 16-DGHVLTSAAILGHDG-30 0.08
DRB1*0301 95-KKSNMALIFGLYDEP-109 2.90
DRB1*0401 51-LNDFNEPGSLAPTGM-65 2.04
DRB1*0404 17-VDEHLMCDMDGHVLT-21 36.46
DRB1*0405 60-LAPTGMYINGAKYMV-74 5.37
DRB1*0701 92-VTIKKSNMALIFGLY-106 0.23
DRB1*0802 29-DGSVWAQSAGFPELK-43 37.85
DRB1*0901 63-TGMYINGAKYMVIQG-77 0.88
DRB1*1101 112-PGQCNLVVERLGDYL-126 20.12
DRB1*1302 23-AAILGHDGSVWAQSA-37 64.97
DRB1*1501 8-DEHLMCDMDGHVLTS-22 100.91
DRB3*0101 9-EHLMCDMDGHVLTSA-23 5.80
DRB4*0101 14-DMDGHVLTSAAILGH-28 58.21
DRB5*0101 91-GVTIKKSNMALIFGL-105 6.40
B-cell Epitopes
By BepiPred method four potential linear B-cell epitopes
were calculated which were I: 31- SVWAQSAGFPELKPA-
282 Protein & Peptide Letters, 2008, Vol. 15, No. 3 Saffari et al.
45, II: 54- FNEPGSLAP-62, III: 80-GVVIRGKKGSGG
VTI-94 and IV: 108- EPMTPG-113. The third one seems to
be the most probable epitope. Because Chou and Fasman
[40], Karplus and Schulz [41] and Parker [39] methods all
determine a segment of this peptide, 85-GKKGSGG-91, to
be the potential epitope by maximum score of 1.389, 1.126
and 5.814 respectively. Furthermore in the charts of the
Abraham hydrophobicity [42], J anin accessibility [43], Hopp
and Woods hydrophilicity [44] and Bhaskaran average flexi-
bility [45] scales which are shown in Fig. (1), this is the best
epitope in comparison with other ones (I, II and IV epitopes).
In the case of hydrophilicity, accessibility and flexibility
as the propensity scale increases the probability of epitopic-
ity increases too but for the hydrophobicity, the more hydro-
phobic the less epitopic. Because in a protein, antigenic de-
terminants lie in regions which are hydrophilic, and accessi-
bility and flexibility of these segments are always high.
Fig. (2) shows alignment of C. sativus profilin with two
human and one Arabidopsis profilins which their tertiary
structure and so their PLP and actin binding sites are known.
J ust as expected, there is a modest homology between these
proteins that could be the cause of cross-reactivity toward
human profilin. As it is shown in Fig. (2) all of our predicted
B-cell epitopes on C. sativus profilin are located in regions
that according to the alignment are concordance with PLP
and actin binding sites on human profilins. Thorn et al. [25]
have demonstrated that plant-specific binding pocket is the
major immunogenic region of plant profilins and IgE epitope
which encompasses the adjacent actin binding site to this
pocket generates a cross-reacting response to human as well














Figure 1. Bhaskaran average flexibility, Hopp and Woods hydrophilicity, Abraham hydrophobicity and J anin accessibility scales are shown.
All charts are based on propensity scales for each of the 20 amino acids. Note that the propensity scales for all amino acids are normalized.
The corresponding region to the intense change includes 80-GVVIRGKKGSGGVTI-94. As expected for the third epitope (III: 80-
GVVIRGKKGSGGVTI-94) except the score of hydrophobicity, all other scores are high.
T and B-cell Epitopes Prediction of Iranian Saffron Protein & Peptide Letters, 2008, Vol. 15, No. 3 283
as plant profilins. In our alignment two predicted epitopes (II
and III) overlap with the Arabidopsis thaliana profilin 1
binding pocket and note that the mentioned pocket on A.
thaliana profilin1 has great identity with corresponding seg-
ment on C. sativus profilin (out of 17 residues 14 are consen-
sus). All of these, somehow, are indicating the accuracy of
predicted peptides as potential B-cell epitopes. In Fig. (2)
predicted secondary structure of the C. sativus profilin also
has been shown which has a great similarity to the secondary
structure of A. thaliana profilin1. Both of them contain three
alpha helices and seven beta strands. This prediction is com-
patible with the classification of protein folds of allergens
which was made by Aalberse [48]. According to this classi-
fication, profilins classified as allergens with antiparallel
beta strands intimately associated with one or more alpha
helices. According to this prediction, epitope I includes beta
strand 2, a part of the helix 2 and the sequences between
them while epitope II starts from the next residue to the last
amino acid of helix 2. In the other hand, epitopes III and IV
are located inside or in the vicinity of beta strands 4, 5 and 6
and helix 3 which constitute the major actin binding sites of
profilins [22].
DISCUSSION
T-cell epitopes are peptides first presented by MHC
molecules, then recognized by T-cells. Peptide binding to
MHC molecules is a prerequisite for T-cell recognition. T-
cell epitopes are therefore a subset of MHC-binding linear
peptides. Because of a high number of peptides that can be
derived from pathogens and the combinatorial nature of hu-
man HLA phenotypes, the systematic identification of can-
didate T-cell epitopes requires computational screening,
combined with experimental validation. Identification of
epitopes capable of binding multiple HLA types will signifi-
cantly rationalize the development of epitope-based vaccines
[49]. Computational tools can reduce the time and minimize
the total number of required tests to find the possible proper
epitopes, the target for vaccine development. In this study
results of the potential T-cell epitope analysis indicate that
16-DGHVLTSAAILGHDG-30 corresponding to DRB0101
allele is the peptide with the best binding affinity. In the next
step, in vitro synthesis of determined peptide and in vivo
experimental study to test the efficacies are essential for as-
surance of accuracy of the predicted segment.
B-cell epitopes are parts of proteins or other molecules
that antibodies bind. Continuous (or sequential) epitopes are
mainly composed of a single stretch of the polypeptide
chain. There are numerous observations that IgEs from aller-
gic patients cross-react with multiple plant profilins as well
as human profilins [1-3]. Also individuals hypersensitive to
plant profilins show allergic reactions to human profilin,
suggesting that continued sensitization, even in the absence
of further exposure to plant profilins, is maintained by an
autoimmune reaction to human profilin [25]. Furthermore,
potential epitopes on plant profilins have been predicted
to overlap with ligand (actin and PLP)binding sites [24-26]
or to be located near the plant-specific binding pocket [24].
This region is a solvent filled pocket located near the actin
binding surface and is specific for plants profilins. The best
single method for predicting linear B-cell epitopes is the
hidden Markov model [38]. BepiPred method which was
used in this work for prediction of continuous B-cell epitopes
is based on this model and when tested on the validation data
set, performs better than any of other methods significantly
[15]. Epitope mapping of birch pollen profilin (PDB-ID:
1CQA) that shows 73% identity with C. sativus profilin,
indicates a clustering of reactive epitopes at the N- and C-
terminal regions of this protein [24]. Due to the high similar-
ity between these proteins and high degree of cross reactivity
in profilin family, it was expected that the same regions in C.
sativus profilin have IgE binding capability. Out of 4 con-
tinuous predicted B-cell epitopes two epitopes (I and IV epi-
topes) are located at similar segments in C. sativus profilin
but the other two (II and III epitopes) are not. First of all, it





Figure 2. Alignment of C. sativus profilin (CsPRO) with Arabidopsis thalina profilin 1 (AtPRO1) (NP_179566.1) and two human profilins
(HsPRO1 and HsPRO2) (P07737, P35080). Secondary structures of A. thaliana profilin 1 and C. sativus profilin (predicted) are indicated
below their primary sequences: cylinders for alpha helices and arrows for beta strands. Shaded segments refer to peptides which are predicted
to be potential B-cell epitopes on C. sativus profilin and amino acids which have been supposed [25] to be actin binding sites are shown in
boxes. ClustalW consensus residues (Clustal Con) are shown with star and two stars indicate that the corresponding conserved amino acid is
a PLP binding site [25]. Dark lines above the sequences indicate corresponding residues of the Arabidopsis profilin which constitute its spe-
cific binding pocket.
284 Protein & Peptide Letters, 2008, Vol. 15, No. 3 Saffari et al.
must be noted that our predicted epitopes are not limited to
IgE class of antibodies and include all probable B-cell epi-
topes. Second, except the third epitope (III: 80-G
VVIRGKKGSGGVTI-94) flexibility, accessibility, hydro-
philicity and hydrophobicity features of other epitopes do not
emphasize on their potential epitopicity. Third, however II
and III epitopes do not conform to be IgE epitopes on birch
pollen profilin, but their sequences overlap with the plant
specific binding pocket and adjacent actin binding site which
supposed to be the major immunogenic segments [25].
Fourth, these are sequential epitopes and prediction of con-
formational epitopes is more complicated. Additionally for
that prediction information about 3-dimensional structure of
protein (such as pure energetics, heuristic and homology
modeling approach) is necessary [50]. Finally results of the
present study are only predicted ones and confirmation is
required.
Furthermore as mentioned before, BepiPred algorithm
calculate the average amino acid propensity value over a
sliding window along a query protein sequence. Unfortu-
nately, to ones disappointment, a recent study by Blythe and
Flower [51] has led to the conclusion that single-scale
amino acid propensity profiles cannot be used to predict epi-
tope location reliably, as reflected by the fact that even the
best amino acid propensity scales could only yield a success
rate marginally better than that by randomly using ROC (re-
ceiver operating characteristics). In this manner Chou et al.
[52] have indicated that the use of amino acid pairs as a se-
quence coupling mode evidently increases the accuracy of a
predicted segment as a potential epitope. Their "amino acid
pair antigenicity" scale approach can give much better results
than various amino acid propensity scale approaches which
are used in the existing B-cell epitope prediction algorithms.
Results of sequence alignment show predicted epitopes
overlap with ligand (PLP and actin) binding site and plant
specific binding pocket which is in agreement with previous
observations [24, 25, and 27]. For determining the ligand
binding site and functional importance of specific residues,
structure based alignment is more accurate and therefore
more informative than a sequence based alignment. But since
in our alignment PLP binding residues -which are very con-
served over all organisms from viruses to mammals- and
plant specific binding pocket have demonstrated accurate
alignment of C. sativus and H. sapiens and A. thaliana pro-
filins, we can claim that the results of the alignment are
valid.
CONCLUSION
Profilin has been identified as an important cross-reactive
allergen for patients suffering from multivalent type I al-
lergy. So the determined peptides are useful for further vac-
cine development because they can reduce the time and
minimize the total number of required tests to find the possi-
ble proper epitopes, the target for vaccine development.
ACKNOWLEDGEMENT
Support of this study by Shiraz University is acknowl-
edged.

LIST OF ABBREVIATIONS
PLP = Poly L-Proline
ARB = Average Relative Binding
HMM = Hidden Markov Model
MHC = Major Histocompatibility Complex
PIP2 = Phosphatidylinositol-4,5-bisphosphate
REFERENCES
[1] Valenta, R., Duchene, M., Pettenburger, K., Sillaber, C., Valent, P.,
Bettelheim, P., Breitenbach, M., Rumpold, H., Kraft, D. and
Scheiner, O. (1991) Science, 253, 557-560.
[2] Valenta, R., Duchene, M., Ebner, C., Valent, P., Sillaber, C., Devil-
ler, P., Ferreira, F., Tejkl, M., Edelmann, H. and Kraft, D. (1992) J.
Exp. Med., 175, 377-385.
[3] Valenta, R., Duchene, M., Sperr, W., Valent, P., Vrtala, S., Hirsch-
wehr, R., Ferreira, F., Kraft, D. and Scheine, O. (1992) Int. Arch.
Allergy Immunol., 99, 271-273.
[4] Carlsson, L., Nystn, L.E., Sunkvist, I., Markey, F. and Lindberg,
U. (1977) J. Mol. Biol., 115, 465-483.
[5] Stossel, T.P., Chaponier, C., Ezzell, R.M., Hartwig, J .H., J anmey,
P.A., Kwiatkowski, D.J ., Lind, S.E., Smith, D.B., Southwick, F.S.
and Yin, H.L. (1985) Ann. Rev. Cell. Biol., 1, 353-402.
[6] Staiger, C.J ., Goodbody, K.C., Hussey, P.J ., Valenta, R., Drbak,
B.K. and Lloyd, C.W. (1993) Plant J., 4, 631-641.
[7] Tilney, L.G., Bonder, E.M., Coluccio, L.M. and Mooseker, M.S.
(1983) J. Cell. Biol., 97, 112-124.
[8] Buss, F., TemmGrove, C., Henning, S. and J ockusch, B.M. (1992)
Cell. Motil. Cytoskeleton, 22, 51-61.
[9] Cooley, L., Verheyen, E. and Ayers, K. (1992) Cell. 69, 173-184.
[10] Grote, M., Vrtala, S. and Valenta, R. (1993) J. Histochem. Cyto-
chem., 41, 745-750.
[11] Mockrin, S.C. and Korn, E.D. (1980) Biochemistry, 19, 5539-5362.
[12] J anmey, P.A., Hvidt, S., Oster, G.F., Lamb, J ., Stossel, T.P. and
Hartwig, J .H. (1990) Nature, 347, 95-98.
[13] Goldschmidt-Clermont, P.J ., Furman, M.I., Wachsstock, D., Safer,
D., Nachmias, V.T. and Pollard, T.D. (1992) Mol. Biol. Cell., 3,
1015-1024.
[14] Lassing, I. and Lindberg, U. (1985) Nature, 314, 472-474.
[15] Machesky, L.M., Goldschmidt-Clermont, P.J . and Pollard, T.D.
(1990) Cell. Regul., 1, 937-950.
[16] Goldschmidt-Clermont, P.J . and J anmey, P.A. (1991) Cell, 113,
1081-1089.
[17] Vojtek, A., Haarer, B., Field, J ., Gerst, J ., Pollard, T.D., Brown, S.
and Wigler, M. (1991) Cell, 66, 497-505.
[18] Aderem, A. (1992) Trends. Biochem. Sci., 17, 438-443.
[19] Tanaka, M. and Shibata, H. (1985) Eur. J. Biochem. 151, 291-297.
[20] Lindberg, U., Schutt, C.E., Hellsten, E., Tjder, A.C. and Hult, T.
(1988) Biochim. Biophys. Acta, 967, 391-400.
[21] Berridge, M.J . (1993) Nature, 361, 315-325.
[22] Schutt, C.E., Myslik, J .C., Rozycki, M.D., Goonesekere, N.C. and
Lindberg, U. (1993) Nature, 365, 810-816.
[23] Gsell, O. (1993) Zentralbl. Bakteriol., 273, 412-427.
[24] Fedorov, A.A., Ball, T., Mahoney, N.M., Valenta, R. and Almo,
S.C. (1997) Structure, 5, 33-45.,
[25] Thorn, K.S., Christensen, H.E., Shigeta, R., Huddler, D., Shalaby,
L., Lindberg, U., Chua, N.H. and Schutt, C.E. (1997) Structure, 5,
19-32.
[26] Rihs, H.P., Chen, Z., Ruff, F., Petersen, A., Rozynek, P.,
Heimann, H. and Baur, X. (1999) J. Allergy. Clin. Immunol., 104,
1293-1301.
[27] Asturias, J .A., Gmez-Bayn, N., Arilla, M.C., Snchez-Pulido, L.,
Valencia, A. and Martnez, A. (2002) Int. Immunol., 14, 993-1001.,
[28] Lubec, G., Afjehi-Sadat, L., Yang, J .W. and J ohn, J .P. (2005) Prog.
Neurobiol., 77, 90-127.
[29] Shen, H.B. and Chou, K.C. (2007) Biochem. Biophys. Res. Com-
mun., 364, 53-59.
[30] Chou, K.C. and Shen, H.B. (2007) Biochem. Biophys. Res. Com-
mun., 360, 339-345.
[31] Chou, K.C. and Shen, H.B. (2007) Anal. Biochem., 370, 1-16.
[32] Shen, H.B. and Chou, K.C. (2007) Biopolymers, 85, 233-240.
T and B-cell Epitopes Prediction of Iranian Saffron Protein & Peptide Letters, 2008, Vol. 15, No. 3 285
[33] Chou, K.C. and Shen, H.B. (2007) Biochem. Biophys. Res. Com-
mun., 357, 633-640.
[34] Shen, H.B. and Chou, K.C. (2007) Biochem. Biophys. Res. Com-
mun., 363, 297-303.
[35] Rost, B. (2001) Struct. Biol., 134, 204-218.
[36] Rost, B. and J infeng, L. (2003) Nucleic. Acids. Res., 31, 3300-
3304.
[37] Bui H.H., Sidney, J ., Peters, B., Sathiamurthy, M., Sinichi, A.,
Purton, K.A., Moth, B.R., Chisari, F.V., Watkins, D.I. and Sette
A. (2005) Immunogenetics, 57, 304-314.
[38] J ens, J .E., Lund, L. and Nielsen, M. (2006) Immunome. Res., 2, 2.
[39] Parker, J .M., Guo, D. and Hodges, R.S. (1986) Biochemistry, 25,
5425-5432.
[40] Chou, P.Y. and Fasman, G.D. (1978) Adv. Enzymol. Relat. Areas.
Mol. Biol., 47, 45-148.,
[41] Karplus, P. and Schulz, G. (1985) Naturwissenschafren, 72,212-
213.
[42] Abraham, D. and Leo, A.J . (1987) Proteins, 2, 130-152.
[43] J anin, J . (1979) Nature, 277, 491-492.
[44] Hopp, T.P. and Woods, K.R. (1981) Proc. Natl. Acad. Sci. USA,
78, 3824-3828.
[45] Bhaskaran, R. and Ponnuswamy, P.K. (1984) Int. J. Pept. Protein
Res., 24, 180-191.
[46] Thompson, J .D., Higgins, D.G. and Gibson, T.J . (1994) Nucleic
Acids Res., 22, 4673-4680.
[47] Hall, T.A. (1999) Nucleic Acids Symp. Ser., 41, 95-98.
[48] Aalberse, R.C. (2000) J. Allergy Clin. Immunol., 106, 228-238,
[49] Doytchinova, I. and Flower, D. (2003) Org. Biomol. Chem., 1,
2648-2654.
[50] Chou, K.C. (2004) Curr. Med. Chem. 11, 2105-2134.
[51] Blythe, M.J . and Flower, D.R. (2005) Protein Sci., 14, 246-248.
[52] Chen, J ., Liu, H., Yang, J . and Chou, K.C. (2007) Amino Acids, 33,
423-428.



Received: November 21, 2007 Revised: December 13, 2007 Accepted: December 14, 2007