Báo cáo khoa học: Novel strategy for protein production using a peptide tag derived from Bacillus thuringiensis Cry4Aa pptx

Novel strategy for protein production using a peptide tag
derived from Bacillus thuringiensis Cry4Aa
Tohru Hayakawa
1
, Shinya Sato
1,2
, Shigehisa Iwamoto
2
, Shigeo Sudo
2
, Yoshiki Sakamoto
1
,
Takaaki Yamashita
1
, Motoaki Uchida
1
, Kenji Matsushima
1
, Yohko Kashino
1
and Hiroshi Sakai
1
1 Graduate School of Natural Science and Technology, Okayama University, Japan
2 Department of Bioscience, Japan Lamb Co. Ltd, Development Division, Okayama, Japan
Introduction
Cry toxin, a specific insecticidal protein against insect
larvae, has been used as an insect pest control agent
throughout the world [1]. Cry toxin is usually pro-
duced intensively during sporulation and accumulates
in the form of protein crystals in Bacillus thuringiensis

cells. Hyperexpression of Cry toxin may be supported
by factors such as strong promoters, stable mRNAs
and protein crystallization [2]. In particular, crystal
formation is a characteristic of B. thuringiensis and
may be useful as an aid in the overproduction of
recombinant protein. Protein crystallization allows a
large amount of protein to be packed into the limited
intracellular space and protects protein from proteo-
lytic degradation in the environment. Indeed, Cry
toxin accumulates as a protein crystal that can account
for up to 25% of the dry weight of the cell [2].
The mechanism of protein crystallization may vary
with the type of Cry toxin. The larger-sized Cry pro-
toxin ( 130 kDa) contains a C-terminal extension in
addition to the insecticidal N-terminal region and
probably crystallizes by self-assembly. When Cry1,
a member of the large protoxin group, is expressed in
Keywords
4AaCter peptide tag; Bacillus thuringiensis;
Cry4Aa; Escherichia coli; TpN syphilis
antigen
Correspondence
H. Sakai, Graduate School of Natural
Science and Technology, Okayama
University, 3-1-1 Tsushima-Naka, Okayama
700-8530, Japan
Fax ⁄ Tel: +81 86 251 8203
E-mail:
Database
The nucleotide sequence of the synthetic

gene cry4Aa-S2 is available in the
EMBL ⁄ GenBank ⁄ DDBJ databases under
accession number AB513706
(Received 26 February 2010, revised
11 April 2010, accepted 3 May 2010)
doi:10.1111/j.1742-4658.2010.07704.x
Numerous proteins cannot be sufficiently prepared by ordinary recombi-
nant DNA techniques because they are unstable or have deleterious effects
on the host cell. One idea to prepare such proteins is to produce them as
protein inclusions. Here we developed a novel system to effectively prepare
proteins by using peptide tags derived from the insecticidal Cry toxin of a
soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, desig-
nated 4AaCter, facilitates the formation of protein inclusions of glutathione
S-transferase in Escherichia coli without losing the enzyme activity. Appli-
cation of 4AaCter to the production of syphilis antigens TpN15, TpN17
and TpN47 from Treponema pallidum yielded excellent results, including a
dramatic increase in the production level, simplification of the product
purification and high reactivity with syphilis antibody. The use of 4AaCter
may provide an innovational strategy for the efficient production of
proteins.
Abbreviations
CBB, Coomassie brilliant blue; CDNB, 1-chloro-2,4-dinitrobenzene; GST, glutathione S-transferase; PBST, phosphate-buffered saline
containing 0.05% Tween 20.
FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS 2883
Escherichia coli, protein inclusions containing biologi-
cally active toxin are formed [3,4]. Furthermore, the
bipyramidal crystals denatured in 8 m urea revert to
their original crystal shape when the urea is removed
by dialysis [5]. The C-terminal extension is not directly
related to the insecticidal activity and is usually

removed upon proteolytic activation. The amino acid
sequences of some segments in the C-terminal exten-
sion are highly conserved among the large protoxin
group and are believed to be important for protein
crystallization. The smaller-sized Cry protoxin
( 70 kDa or less) consists of an insecticidal N-termi-
nal region only and requires an accessory protein (such
as P20) for crystallization [2].
The protein crystallization mechanism of the Cry
toxin may prove beneficial for the expression of heterol-
ogous proteins in general. Crystallization of the large
protoxin seems to be dependent on some special
protein structure in the C-terminal extension; therefore,
this extension can be utilized as a peptide tag to facili-
tate the crystallization of fused proteins. The crystalli-
zation may protect the protein from degradation by
in vivo proteinase and may also simplify the purification
steps.
Cry4Aa is a dipteran-specific Cry toxin produced by
B. thuringiensis subspecies israelensis, and is a member of
the larger-sized protoxin group. In the midgut of a sus-
ceptible insect, Cry4Aa is processed into protease-resis-
tant segments of 20 and 45 kDa via a 60 kDa
intermediate generated by the removal of the C-terminal
extension [6]. In the present study, we developed a
Cry4Aa-derived peptide tag that facilitates the formation
of protein inclusions of fused proteins. We demonstrated
the usefulness of the peptide tag by efficiently producing
TpN antigens from Treponema pallidum, which is a spi-
rochetal bacterium causing syphilis. In general, because

T. pallidum cannot be cultured continuously in vitro,
establishment of the efficient production system for TpN
antigens that are used to diagnose syphilis is eagerly
desired. Our newly isolated peptide tag may contribute to
the establishment of novel and efficient expression sys-
tems for proteins.
Results
Glutathione S-transferase (GST) fused with
Cry4Aa peptides
To determine the peptide stretch involved in protein
crystallization, nine polypeptides from the C-terminal
region of Cry4Aa were fused, as peptide tags, to GST.
The resulting fusion proteins were named GST–4AaCt-
ers 852–1180, 696–851, 696–799, 801–851, 801–834,
801–829, 807–824, 807–819 and 812–824, based on the
amino acid number from the N-terminal end of the
Cry4Aa protoxin (Fig. 1). All GST fusion proteins
were successfully expressed in E. coli, and their sizes
estimated by SDS ⁄ PAGE were 64 kDa (GST–4AaCter
852–1180), 44 kDa (GST–4AaCter 696–851), 39 kDa
(GST–4AaCter 696–799), 32 kDa (GST–4AaCter 801–
851), 31 kDa (GST–4AaCter 801–834), 30 kDa (GST–
4AaCter 801–829), 29 kDa (GST–4AaCter 807–824),
28 kDa (GST–4AaCter 807–819) and 28 kDa (GST–
4AaCter 812–824) (Fig. 2A). These sizes correspond to
those predicted from the deduced amino acid
sequences of each GST fusion protein.
More than 95% of GST–4AaCters 696–851, 801–
851, 801–834 and 801–829, which contain the highly
C

ry
4Aa
Ins
e
cticidal N-te
r
minal h
a
lf
G
58
Q
695
I
6
96
E
1
1
80
C-
ter
m
in
al h
alf
Mu
ta
nts
GST

A
85
2
E
1
1
80
GST-4
Aa
Cter 852–1180
GST-
4
AaCte
r
696–851
GST
I
69
6
P
851
B
l
ock
s12 3
456
7
8
GST-4AaCter 696–799
GST

I
696
G
79
9
G
S
T-4AaCter 801–851
G
S
T
I
8
0
1
P
851
I
80
1
FPT
YI
FQK
ID
ES
K
LKP
Y
TRYL
V

R
G
FV
GS
SK
D
VEL-
-
-
P
851
GST
I
8
0
1
E
834
G
S
T
I
8
01
S
8
29
GST-
4A
a

C
te
r
8
0
1–834
GS
T
-
4A
a
C
ter 8
0
1–829
B
l
o
c
k7
GST-4A
a
Ct
e
r8
0
7–819
GST
F
8

07
R
82
4
GS
T
GS
T
-
4
A
aC
t
e
r
812–824
T
81
9
E
8
1
2
GST-4AaCter 807–824
GS
T
F
8
0
7

R
8
2
4
Fig. 1. Design of GST–4AaCters. The
schematic structure of GST–4AaCters is
shown. Nine peptide segments from the
Cry4Aa C-terminal region are fused with
GST. The amino acid sequence of Cry4Aa
block 7 is underlined.
Efficient protein production using 4AaCter T. Hayakawa et al.
2884 FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS
conserved Block 7 sequence, were found in the insolu-
ble fraction (Fig. 2B). In contrast, GST–4AaCters
852–1180 and 696–799 were mostly found in the
soluble fraction (< 10% insoluble). In the case of
GST–4AaCters 807–824, 807–819 and 812–824, 20–
40% of the proteins were insoluble (Fig. 2B).
Morphological observation of the inclusions of
GST
Spherical inclusion in cells expressing GST–4AaCters
were visible by light microscopy (Fig. 3A). Most
E. coli cells expressing GST–4AaCters 696–851, 801–
851, 801–834 and 801–829 contained inclusions, but
the percentage of cells with inclusions was decreased in
cells expressing GST–4AaCters 807–824, 807–819 and
812–824 (Fig. 3A). Almost no inclusions were observed
in cells expressing GST–4AaCters 852–1180 and 696–
799 (Fig. 3A). The percentage of protein expressed as
the insoluble form seemed to be correlated with the

percentage of cells containing inclusions. Scanning
electron micrographs revealed that the inclusions of
GST–4AaCters 696–851, 801–834 and 801–829 were
approximately spherical, with a diameter of 0.5–
0.7 lm, but the surface of the inclusions appeared to
be rugged (Fig. 3B). Contrary to our expectation, the
morphology of the GST–4AaCter inclusions looked
different from protein crystal as imagined in general.
Enzyme activity of GST–4AaCters derived from
inclusions
Upon incubation of the inclusions in alkali buffer
(50 mm NaHCO
3
, NaOH, pH 11) for 1 h at 37 °C,
852–1180
Insoluble (%) 4 100 10 100 100 100 35 20 25 5
210
119
90
65
49
37
29
21
9
(kDa)
SIS
I
SISI
S

ISIS
I
SISIS
I
696–851
696–799
801–851
801–834
801–829
807–824
807–819
812–824
GST
GST-4AaCter
210
119
90
65
49
37
29
21
9
(kDa)
852–1180
696–851
696–799
801–851
801–834
801–829

807–819
812–824
807–824
GST
GST-4AaCter
B
A
Fig. 2. Expression of GST–4AaCters in E. coli. (A) GST–4AaCters expressed in E. coli were separated by SDS ⁄ PAGE (15%) and stained with
CBB. GST–4AaCters are indicated by arrowheads. (B) Cells expressing GST–4AaCters were disrupted by sonication and separated into solu-
ble and insoluble proteins by centrifugation. Proteins were analysed by SDS ⁄ PAGE (15%) and visualized with CBB. GST–4AaCters are indi-
cated by arrowheads. The percentages of GST–4AaCters in the insoluble fraction are shown. S, soluble proteins; I, insoluble proteins.
852–1180
696–851
696–799
801–851
801–834
801–829 807–819
812–824
807–824
GST
696–851 801–834 801–829
A
B
Fig. 3. Micrograph of the inclusions of
GST–4AaCters. (A) Micrograph of the E. coli
cells expressing GST–4AaCters. Spherical
inclusions of GST–4AaCters are indicated by
arrows. Bar, 1 lm. (B) Scanning electron
micrograph of the inclusion bodies of
GST–4AaCters 696–851, 801–834 and

801–829. Bar, 600 nm.
T. Hayakawa et al. Efficient protein production using 4AaCter
FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS 2885
GST–4AaCters were solubilized almost completely.
This suggested that the inclusion of GST–4AaCters
shared some characteristics with the protein crystal
produced by B. thuringiensis and was different from
the inclusion of denatured proteins frequently observed
in heterologous protein expression systems. GST-
4AaCters recovered in soluble fraction were subjected
to a 1-chloro-2,4-dinitrobenzene (CDNB) assay to
measure their GST activities. GST–4AaCter 696–851,
which formed inclusions with almost 100% efficiency,
had the highest GST activity (459 ± 103 lmolÆmin
)1
Æ
nmol
)1
) (Figs 3B and 4). This value was a bit higher
than that of purified GST (319 ± 63 lmolÆmin
)1
Æ
nmol
)1
). Although GST–4AaCters 801–851, 801–834
and 807–824 formed inclusions with almost 100% effi-
ciency, GST–4AaCters 801–851 and 801–834 had
decreased GST activities (135 ± 26 and 141 ± 59
lmolÆmin
)1

Ænmol
)1
, respectively), and GST–4AaCter
807–824 had a very low GST activity (24 ± 18 lmolÆ
min
)1
Ænmol
)1
) (Fig. 4). The GST fusion proteins that
formed inclusions with lower efficiencies had moderate
GST activity ranging from 65 to 110 lmolÆmin
)1
Æ
nmol
)1
(Fig. 4). Thus, because relatively high GST
activity was observed, the inclusion formed by 4AaCt-
ers seemed to be different from the inclusion formed
by denatured proteins.
Production of TpN antigens using 4AaCter
GST fused with the 4AaCter 696–851 efficiently
formed protein inclusions in E. coli, and the fusion
protein solubilized from the inclusions had the highest
GST activity among the tested samples. Therefore, it
was most probable that 4AaCter 696–851 could be
used as a peptide tag for the efficient production of
heterologous proteins in E. coli. TpNs are surface anti-
gens of T. pallidum and are highly demanded to diag-
nose syphilis. We anticipated that the efficient
production system of TpN antigens would be con-

structed by using 4AaCter as a peptide tag. We used
modified 4AaCter 696–851 that was fused with a
6 · His oligopeptide at the N-terminal end and an
oligopeptide containing the cleavage site for Pre-
Scission protease at the C-terminal end (Fig. 5A). The
fusions of TpNs and modified 4AaCter tag were
expressed using pGEX–DGST vector.
0
100
200
300
400
500
600
852–1180
696–851
696–799
801–851
801–834
801–829
807–819
812–824
807–824
GST
GST activity
(µmol·min
–1
·nmol
–1
)

GST–4AaCters
Fig. 4. Enzyme activities of GST–4AaCters. Alkali-solubilized GST–
4AaCters from the inclusions were subjected to CDNB assay. Error
bars represent the standard deviation of three or more replicate
experiments.
21
0
119
90
65
4
9
3
7
2
9
(
kDa
)
21
9
S
I
S
ISISI
S
I
S
I
4

AaCter
TpN15
TpN17
T
pN47
4
A
aCt
e
r
-
TpN15
ATG
4
A
aCte
r
TpN15
T
pN17
TpN47
4
Aa
C
ter-
T
pN
1
7
4

AaCt
e
r-TpN47
6xH
is
PP
4
A
aC
te
r
4
A
a
Ct
e
r
4AaCter
4AaCter
pG
E
X-
Δ
GST
P
t
ac
AB
Fig. 5. Design and expression of 4AaCter–TpNs. (A) Schematic structure of 4AaCter–TpNs. TpN15, 17 and 47 were fused with a 6 · His
tag, 4AaCter 696–851 and the recognition site of PreScission protease. The 4AaCter–TpNs were expressed using pGEX–DGST. 4AaCter,

4AaCter 696–851 (peptide tag); PP, recognition site of PreScission protease; Ptac, tac promoter. (B) Expression of 4AaCter–TpNs in E. coli.
TpNs with or without the 4AaCter peptide tag were expressed in E. coli. Upon disruption by sonication, soluble and insoluble proteins were
separated by SDS ⁄ PAGE (15%) and visualized with CBB. The 4AaCter–TpNs are indicated by arrowheads. S, soluble proteins; I, insoluble
proteins.
Efficient protein production using 4AaCter T. Hayakawa et al.
2886 FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS
SDS ⁄ PAGE analysis of TpN antigens fused with
4AaCter tag at the N-terminal end revealed proteins
with the expected size in the insoluble fraction
(Fig. 5B). The expression levels of 4AaCter–TpNs were
significantly higher than the levels of TpNs without
4AaCter, as estimated by densitometric scanning of the
protein bands. On the other hand, when 4AaCter tag
was fused at the C-terminal end of TpNs, no enhance-
ment was observed in the expression level compared
with that of TpNs without 4AaCter (data not shown).
Cells were disrupted by sonication, and the inclusions
of 4AaCter–TpNs were isolated by centrifugation. The
yields of 4AaCter–TpN15, –TpN17 and –TpN47 were
0.31, 0.31 and 0.45 mgÆmL
)1
culture, respectively.
4AaCter–TpNs solubilized in alkali buffer were already
relatively pure, and their purities were estimated as
70%, 78% and 85%, respectively (Fig. 5B). 4AaCter–
TpNs were further purified using a Ni-NTA column
and were then treated with PreScission protease. The
released 4AaCter tag and uncleaved 4AaCter–TpNs
were removed using the Ni-NTA column. The purities
of the final products of TpN15, TpN17 and TpN47

were 88%, 94% and 85%, respectively. Thus, to obtain
highly purified TpNs, complicated purification steps
were not required in this system. The estimated final
yields of purified TpN15, TpN17 and TpN47 were
0.09, 0.07 and 0.12 mgÆmL
)1
culture.
Reactivity of recombinant TpN antigens
The reactivity of the 4AaCter–TpNs against human
serums was evaluated by ELISA and compared with
that of native TpN antigens. The TpNs produced by
removing 4AaCter peptides from the corresponding
4AaCter–TpNs were also used in this assay. The
recombinant TpNs prepared by our method showed
similar reactivity to that of the native TpN antigens
(Tables 1 and 2). Among the recombinant TpNs,
4AaCter–TpN17 and the TpN17 were the most reac-
tive with Tp-positive human serum (Table 1). A similar
observation was reported for a recombinant TpN17
constructed in another experiment [7]. On the other
hand, the reactivity of recombinant TpN15 and -47
varied among different human serum samples
(Tables 1 and 2). There was no significant difference in
reactivity between the 4AaCter–TpNs and the TpNs in
which the 4AaCter peptide wa sremoved(Tables1and2).
This result suggests that the human serum did not
Table 1. Reactivity of recombinant TpNs against Tp-positive human serums.
Sample name 4AaCter–TpN15 TpN15 4AaCter–TpN17 TpN17 4AaCter–TpN47 TpN47 Native Tp antigen
10175535 0.20 0.22 1.73 1.79 0.14 0.12 0.38
10175539 0.14 0.15 0.14 0.14 0.06 0.06 0.14

10175550 0.22 0.28 1.50 1.41 0.09 0.08 0.54
10175556 0.44 0.48 1.90 1.88 0.87 0.79 1.17
10175572 0.07 0.05 0.78 0.78 0.08 0.06 0.13
10175575 1.20 1.27 2.65 2.69 2.32 2.24 2.65
10175582 0.26 0.35 3.48 3.50 0.78 0.66 1.74
10175586 0.21 0.18 1.36 1.35 0.23 0.20 0.71
Infectrol D-00 0.20 0.21 1.07 0.99 0.40 0.36 0.60
Table 2. Reactivity of recombinant TpNs against Tp-negative human serums.
Sample name 4AaCter–TpN15 TpN15 4AaCter–TpN17 TpN17 4AaCter–TpN47 TpN47 Native Tp antigen
FH15079 0.08 0.06 0.07 0.07 0.08 0.06 0.11
FH15082 0.11 0.08 0.09 0.07 0.08 0.07 0.06
FH15772 0.09 0.05 0.08 0.05 0.09 0.05 0.06
FH15784 0.15 0.05 0.16 0.05 0.09 0.05 0.07
FH15788 0.07 0.05 0.08 0.05 0.09 0.05 0.06
FH15808 0.10 0.06 0.08 0.08 0.10 0.07 0.08
FH15821 0.08 0.05 0.05 0.06 0.05 0.04 0.23
FH15829 0.08 0.07 0.07 0.06 0.09 0.06 0.19
FH15833 0.31 0.28 0.25 0.27 0.21 0.31 0.17
FH15834 0.06 0.06 0.11 0.21 0.08 0.07 0.09
T. Hayakawa et al. Efficient protein production using 4AaCter
FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS 2887
contain antibodies against the 4AaCter peptide and
that the protein produced by our method can be used
for diagnostic purposes without removing the 4AaCter
peptide.
Discussion
The crystallization mechanism of Cry toxins is
not fully understood. Although 3D structures of the
N-terminal toxic region consisting of domains I, II and
III have been determined for several Cry toxins [8–13],

the structure of the C-terminal nontoxic region has not
yet been determined. Therefore, the crystal structure of
Cry protoxin is still unknown. The formation of the
crystals and their solubility characteristics presumably
depend on a variety of factors, but almost none of
them has been identified. The primary amino acid
sequences of the three segments designated as blocks 6,
7 and 8 in the C-terminal extension are highly con-
served among members of the large protoxin group [1].
Therefore, the C-terminal extension is thought to con-
tain important elements responsible for protein crystal-
lization. In addition, the cysteines located in the
C-terminal extension may form intermolecular disulfide
bridges in the crystal lattice [14], which may result in
the solubilization in the presence of reducing agents at
the high pH that is typical for the midgut juice of lepi-
dopteran and dipteran larvae [15]. In lepidopteran-
specific Cry1Ab, 14 of the 16 cysteines are localized to
the C-terminal extension, and the replacement of one
cysteine to a heterogeneous amino acid affects the for-
mation of the crystal [16]. These observations suggest
that the cysteine-rich C-terminal extension is involved
in the crystallization and stabilization of Cry toxin.
The C-terminal extension of Cry4Aa is structurally
similar to that of Cry1 [17]. Eight of the 13 cysteines
of Cry4Aa are located in the C-terminal extension.
These observations suggest that Cry1 and Cry4Aa
have similar crystallization mechanisms. In the present
study, we discovered that polypeptides containing the
conserved block 7 sequence of Cry4Aa facilitated the

formation of protein inclusions of fused heterologous
protein in E. coli. Therefore, the block 7 sequence may
be one of the factors responsible for the crystallization
of Cry toxins. Because no cysteines are located in
block 7 of Cry4Aa, the mechanism mediated by block
7 was independent of intermolecular disulfide bridges.
GST fused with 4AaCter 696–851, which contains
block 6 in addition to block 7, formed crystal-like
inclusion bodies with almost 100% efficiency and had
much greater enzyme activity than GST fused with
4AaCter 801–834, which contains only block 7. This
result suggests that some other sequence stretch in the
C-terminal extension, such as block 6, also had a role
in maintaining the structural stability of GST incorpo-
rated into the inclusions. The toxicity of the Cry1Ab
C-terminal extension against E. coli and Agrobacte-
rium tumefaciens has been reported previously [18].
The toxicity is reduced or neutralized when the
C-terminal region is fused with domain III of the
insecticidal N-terminal region. However, 4AaCter 696–
851 by itself was expressed and formed inclusions in
E. coli as efficiently as GST–4AaCter 696–851 (data
not shown). This observation suggests that the
C-terminal extensions of each Cry toxin may have dif-
ferent functions.
We used the segment 696–851 of the C-terminal
half of Cry4Aa as a peptide tag for the efficient pro-
duction of T. pallidum antigens (TpNs). This experi-
ment was a touchstone to evaluate the usability of
4AaCter 696–851 as a peptide tag for efficient protein

production. The use of this peptide tag resulted in
increased expression efficiency and simplified purifica-
tion steps. The reactivity of the recombinant TpNs
against human serum was similar to the reactivity of
native TpNs, as estimated by western blotting and
ELISA.
Although fusion of 4AaCter 696–851 at the
C-terminal end of TpNs conferred almost no positive
effect, fusion at the N-terminal end caused a dramatic
increase in the expression level and the formation of
the inclusions that were easily solubilized in alkali buf-
fer (pH 11). The reason why such differences were
observed between fusions at the N- and C-terminal
ends of TpNs remains to be resolved. The toxic
domain of TpNs may be in the N-terminal region and
4AaCter at the N-terminal end may neutralize the
toxicity of TpNs, but 4AaCter at the C-terminal end
may not.
In general, because some TpNs are hard to express
without the appropriate tag, TpNs have been produced
using a tag such as GST derived from Schistosoma ja-
ponicum [7]. However, S. japonicum is one of the
human parasites and there is a possibility that the prod-
ucts prepared using the GST tag react with the human
serum and cause objective background. On the other
hand, 4AaCter 696–851 is derived from Cry toxin
produced by the soil bacterium B. thurigiensis, and
may not react with the human serum. As expected, the
peptide of 4AaCter 696–851 did not show specific
reactivity against the human serum. This was a great

advantage of using the recombinant TpNs prepared by
our method for diagnostic purposes. Thus, the use of
peptide tag 4AaCter 696–851 is very simple and
applicable for the efficient production of heterologous
proteins in E. coli.
Efficient protein production using 4AaCter T. Hayakawa et al.
2888 FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS
Materials and methods
Construction of GST expression vectors
Nine peptide sequences derived from the Cry4Aa C-termi-
nal region were fused with GST and expressed in E. coli
strain BL21. Briefly, the DNA fragment encoding the poly-
peptide from I696 to P851 of Cry4Aa was amplified by
PCR. A synthetic cry4Aa gene, cry4Aa-S2 (AB513706), was
used as a template. The amplified fragment was inserted in
frame into the XhoI site of the expression vector pGEX-
6P-1 (GE Healthcare, Little Chalfont, UK) to generate
pGST–4AaCter 696–851. Similarly, to construct pGST–
4AaCter 852–1180, the DNA fragment encoding the poly-
peptide from A852 to E1180 of Cry4Aa was excised from
cry4Aa-S2 by NaeI cleavage and inserted into the SmaI
site of pGEX-6P-1. The recombinant plasmids pGST–
4AaCter 801–851 and 696–799 were constructed by
removing the EcoRI–KpnI and KpnI–NaeI segments,
respectively, from pGST–4AaCter 696–851. pGST–
4AaCter 801–834 and 801–829 were constructed through
a 1 day mutagenesis procedure [19] using pGST–4AaCter
801–851 as a template. To construct pGST–4AaCter 807–
824, 807–819 and 812–824, the DNA fragments encoding
the polypeptides F807 to R824, F807 to T819 and E812

to R824 of Cry4Aa, respectively, were synthesized by
using a recursive PCR procedure [20,21], in which over-
lapping oligonucleotides matching parts of the sense and
antisense strands of the DNA sequence were used. The
synthesized fragment was inserted between the BamHI
and XhoI sites of pGEX-6P-1. Nucleotide sequences of
the recombinant plasmids were confirmed with an ABI
PRISMÔ 310 genetic analyser (Applied Biosystems,
Foster City, CA, USA).
Characterization of GST–4AaCters
Expression of the GST–4AaCters was induced with
0.06 mm isopropyl b-d-1-thiogalactopyranoside at 37 °C for
3 h. The E. coli cells were disrupted by sonication, and sol-
uble and insoluble proteins were separated by centrifuga-
tion (12 000 g, 5 min, 4 °C). Proteins were separated by
SDS ⁄ PAGE (15%) and then visualized by Coomassie bril-
liant blue (CBB) staining. Protein concentrations were esti-
mated by using a protein assay kit (Bio-Rad Laboratories,
Hercules, CA, USA) with bovine serum albumin as a stan-
dard or by using the densitometric scanning method with a
cooled CCD camera system (Ez-Capture; ATTO, Tokyo,
Japan) and image analysis software (CS analyser ver. 3.0;
ATTO, Tokyo, Japan).
The insoluble pellet was washed twice with water and sol-
ubilized in alkali buffer (50 mm NaHCO
3
, NaOH, pH 11)
for 1 h at 37 °C. GST activities of GST–4AaCters were
analysed by using glutathione and CDNB, as described pre-
viously [22]. GST purified by using glutathione sepharose,

as described previously [21], was used as a positive control
for the assay. The reaction was performed in a well of a
96-well microtitre plate in a final volume of 200 lL, which
contained 100 mm phosphate buffer (pH 7.4), 1 mm gluta-
thione, 1 mm CDNB and an appropriate amount of alkali-
soluble recombinant protein. After incubation for 1 min at
room temperature, the absorbance was read once every
minute at 340 nm for 5 min. The changes in absorbance
per minute were converted into mol substrate conju-
gatedÆmin
)1
Ænmol
)1
protein by using the molar extinction
coefficient e
340
= 9.8 mm
)1
Æcm
)1
.
Microscopic observation
Escherichia coli cells expressing GST–4AaCters were
observed under a light microscope (Axio Observer A1; Carl
Zeiss, Go
¨
ttingen, Germany). In addition, the bacterial cells
were disrupted, and crystal-like inclusion bodies were har-
vested by centrifugation and fixed with phosphate-buffered
saline containing 2% glutaraldehyde and 2% formaldehyde

for 8 h. The samples were sequentially immersed in 50–
99.5% ethanol and then soaked in 100% t-butanol. The
samples were allowed to dry and then coated with OsO
4
by
using an osmium coater HPC-1S (Vacuum Device Inc.,
Mito, Japan). The scanning electron micrographs were
taken on a S-900 scanning electron microscope (Hitachi
Kyowa Engineering Co., Ltd., Hitachi, Japan) at a voltage
of 20 kV.
Construction of TpNs expression vector
The synthetic TpN15, TpN17 and TpN47 genes were PCR
tailed with BamHI and XhoI cleavage sequences at the
upstream and downstream termini, respectively. The DNA
fragment encoding 4AaCter 696–851 was PCR amplified
and tailed with the BamHI or XhoI cleavage sequence at
both termini. In addition, the amplified DNA fragment was
designed to add a short peptide encoding 6 · His and the
recognition site of PreScission protease (GE Healthcare,
Little Chalfont, UK) upstream and downstream ends of
4AaCter, respectively.
pGEX–DGST was used for the expression of TpN anti-
gens. pGEX–DGST was constructed by removing the GST
coding region, except for the first ATG, from the expression
vector pGEX–4T-3 (GE Healthcare, Little Chalfont, UK)
using a 1 day mutagenesis procedure [19]. Specific primer
pairs, pDGST–4T-3MB-f (GGATCCCCGAATTCCCGG)
and pDGST–4T-3MB-r (CATGAATACTGTTTCCTG),
were used for PCR. The synthetic TpN genes were inserted
between the BamHI and XhoI sites of pGEX–DGST to gener-

ate pDGST–TpNs, and then the DNA fragment encoding
4AaCter was inserted into the BamHI or XhoI site of
pDGST–TpNs to generate pDGST–4AaCter–TpNs.
T. Hayakawa et al. Efficient protein production using 4AaCter
FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS 2889
Preparation of 4AaCter–TpNs
4AaCter–TpNs were expressed in E. coli BL21. The crystal-
like inclusion bodies were harvested by centrifugation and
solubilized in alkali buffer (50 mm NaHCO
3
, NaOH, pH
11) for 1 h at 37 °C. The 4AaCter–TpNs were purified
using a Ni-NTA column (Invitrogen, Carlsbad, CA, USA)
and incubated with PreScission protease at a concentration
of 1 U Æ100 lg
)1
protein to remove the 6 · His–4AaCter
tag. The released 6 · His–4AaCter tag and undigested
4AaCter–TpNs were removed using a Ni-NTA column.
The 4AaCter–TpNs and the purified recombinant TpNs
were analysed by western blotting with anti-TpN human
antiserum (ProMedDx, Norton, MA, USA).
ELISA
In total, 100 lL recombinant TpNs (1 lgÆ mL
)1
) per well was
incubated overnight in a 96-well microplate at 4 °C. Native
T. pallidum was prepared from rabbit testis, as described pre-
viously [23]. Native TpNs used as a positive control was
extracted in the extraction buffer (50 mm Tris ⁄ HCl, 5 mm

EDTA, 3% n-octyl-b-d-thioglucoside) and purified using
Superdex 200 (GE Healthcare, Little Chalfont, UK). The
wells were washed with phosphate-buffered saline containing
0.05% Tween 20 (PBST) and then treated with blocking
solution (PBST containing 4% Block Ace; DS Pharma Bio-
medical, Osaka, Japan) for 2 h at room temperature. The
reactivity of TpN antigens was evaluated with eight different
human serum samples that were reactive against T. pallidum
antigens (ProMedDx, Norton, MA, USA) and 10 healthy
donor serum samples (Uniglobe Research, Reseda, CA,
USA). Accurun
Ò
Series Infectrol
Ò
D-00, which is of human
serum origin and is reactive against T. pallidum antigens
(SeraCare Life Science, Norton, MA, USA), was also used
for the assay. Native T. pallidum antigens prepared from
rabbit testis were used as a positive control. After washing
the wells with PBST, 100 lL human antiserum diluted 200-
fold in blocking solution was added to each well for 1 h at
room temperature. The wells were washed with PBST and
diluted (·5000) goat anti-human Ig conjugated with horse-
radish peroxidase (Biosource, Camarillo, CA, USA) was
added to the wells and incubated for another 1 h at room
temperature. After incubation with phosphate citrate buffer
containing o-phenylenediamine (0.4 mgÆmL
)1
) and H
2

O
2
(2 lLÆmL
)1
) for 20 min at room temperature, the reaction
was stopped by adding 100 lL1m sulfuric acid, and the
absorbance of each well at 490 nm was measured.
Acknowledgement
This work was supported in part by a grant from the
Kurata Memorial Hitachi Science and Technology
Foundation.
References
1 Schnepf E, Crickmore N, Van RieJ, Lereclus D, Baum
J, Feitelson J, Zeigler DR & Dean DH (1998) Bacillus
thuringiensis and its pesticidal crystal proteins. Microbiol
Mol Biol Rev 62, 775–806.
2 Agaisse H & Lereclus D (1995) How Bacillus thuringien-
sis produce so much insecticidal crystal protein?
J Bacteriol 177, 6027–6032.
3 Chak K-F & Ellar DJ (1987) Cloning and expression in
Escherichia coli of an insecticidal crystal protein gene
from Bacillus thuringiensis var. aizawai HD-133. J Gen
Microbiol 133, 2921–2931.
4 Oeda K, Oshie K, Shimizu M, Nakamura K, Yamam-
oto H, Nakayama I & Ohkawa H (1987) Nucleotide
sequence of the insecticidal protein gene of Bacillus
thuringiensis strain aizawai IPL-7 and its high-level
expression in Escherichia coli. Gene 53, 113–119.
5 Lecadet M-M (1967) Action compare
´

e de l’ure
´
eetdu
thioglycolate sur la toxine figure
´
edeBacillus thuringien-
sis. C R Acad Sci Hebd Seances Acad Sci D 264, 2847–
2850.
6 Yamagiwa M, Esaki M, Otake K, Inagaki M, Komano
T, Amachi T & Sakai H (1999) Activation process of
dipteran-specific insecticidal protein produced by
Bacillus thuiringiensis subsp. israelensis. Appl Environ
Microbiol 65, 3464–3469.
7 Fujimura K, Ise N, Ueno E, Hori T, Fujii N & Okada
M (1997) Reactivity of recombinant Treponema palli-
dum (r-Tp) antigens with anti-Tp antibodies in human
syphilitic sera evaluated by ELISA. J Clin Lab Anal 11,
315–322.
8 Boonserm P, Davis P, Ellar DJ & Li J (2005) Crystal
structure of the mosquito-larvicidal toxin Cry4Ba and
its biological implications. J Mol Biol 348, 363–382.
9 Boonserm P, Mo M, Angsuthanasombat C & Lescar J
(2006) Structure of the functional form of the mosquito
larvicidal Cry4Aa toxin from Bacillus thuringiensis at a
2.8-angstrom resolution. J Bacteriol 188, 3391–3401.
10 Galitsky N, Cody V, Wojtczak A, Ghosh D, Luft JR,
Pangborn W & English L (2001) Structure of the insec-
ticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus
thuringiensis. Acta Crystallogr D Biol Crystallogr 57,
1101–1109.

11 Grochulski P, Masson L, Borisova S, Pusztai-Carey M,
Schwartz JL, Brousseau R & Cygler M (1995) Bacillus
thuringiensis CryIA(a) insecticidal toxin: crystal struc-
ture and channel formation. J Mol Biol 254, 447–464.
12 Li J, Carroll J & Ellar DJ (1991) Crystal structure of
insecticidal delta-endotoxin from Bacillus thuringiensis
at 2.5 A
˚
resolution. Nature 353, 815–821.
13 Morse RJ, Yamamoto T & Stroud RM (2001) Structure
of Cry2Aa suggests an unexpected receptor binding epi-
tope. Structure 9, 409–417.
Efficient protein production using 4AaCter T. Hayakawa et al.
2890 FEBS Journal 277 (2010) 2883–2891 ª 2010 The Authors Journal compilation ª 2010 FEBS
14 Nickerson KW (1980) Structure and function of the
Bacillus thuringiensis protein crystal. Biotechnol Bioeng
22, 1305–1333.
15 Bietlot HP, Vishnubhatla I, Carey PR, Pozsgay M &
Kaplan H (1990) Characterization of the cysteine
residues and disulphide linkages in the protein crystal
of Bacillus thuringiensis. Biochem J 267, 309–315.
16 Yu J, Xie R, Tan L, Xu W, Zeng S, Chen J, Tang M &
Pang Y (2002) Expression of the full-length and
3¢-spliced cry1Ab gene in the 135-kDa crystal protein
minus derivative of Bacillus thuringiensis subsp. kyushu-
ensis. Curr Microbiol 45, 133–138.
17 Ho
¨
fte H & Whiteley HR (1989) Insecticidal crystal pro-
teins of Bacillus thuringiensis. Microbiol Rev 53, 242–

255.
18 Vazquez-Padron RI, de la Riva G, Agu
¨
ero G, Silva Y,
Pham SM, Sobero
´
n M, Bravo A & Aı
¨
touche A (2004)
Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab
insecticidal crystal protein. FEBS Lett 570, 30–36.
19 Imai Y (1999) Wan dei mutajeneshisu. Jikkenigaku
(in Japanese) 17, 771–775.
20 Prodromou C & Pearl LH (1992) Recursive PCR: A
novel technique for total gene synthesis. Protein Eng 5,
827–829.
21 Hayakawa T, Howlader MTH, Yamagiwa M & Sakai
H (2008) Design and construction of a synthetic
Bacillus thuringiensis Cry4Aa gene: Hyper-expression
in Escherichia coli. Appl Microbiol Biotechnol 80,
1033–1037.
22 Habig WH, Pabst MJ & Jakoby WB (1974)
Glutathione S-transferases. The first enzymatic step in
mercapturic acid formation. J Biol Chem 249, 7130–
7139.
23 Hanff PA, Norris SJ, Lovett MA & Miller JN (1984)
Purification of Treponema pallidum, Nichols strain, by
Percoll density gradient centrifugation. Sex Transm Dis
11, 275–286.
T. Hayakawa et al. Efficient protein production using 4AaCter

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