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

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.. H

derived from Bacillus thuringiensis Cry4Aa

Tohru Hayakawa1, Shinya Sato1,2, Shigehisa Iwamoto2, Shigeo Sudo2, Yoshiki Sakamoto1,

Takaaki Yamashita1, Motoaki Uchida1, Kenji Matsushima1, Yohko Kashino1and Hiroshi Sakai1

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: sakahrsh@biotech.okayama-u.ac.jp

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.

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 vivoproteinase and may also simplify the purification

steps

Cry4Aa is a dipteran-specific Cry toxin produced by

B thuringiensissubspecies 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

Cry4Aa

Insecticidal N-terminal half

C-terminal half

Mutants GST-4AaCter 852–1180 GST A 852 E 1180

I 801 F P T Y I F Q K I D E S K L K P Y T R Y L V R G F V G S S K D V E L - P 851

GST-4AaCter 801–834

GST-4AaCter 801–829

Block 7

R 824

GST GST-4AaCter 812–824

T 819

E 812

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.

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 NaHCO3, NaOH, pH 11) for 1 h at 37C,

852–1180

210 90 49 37

29

21

9

(kDa)

696–851 696–799 801–851 801–834 801–829 807–824 807–819 812–824 GST

GST-4AaCter

210 90 49 37

29

21

9

(kDa)

852–1180696–851696–799801–851801–834801–829 807–824 807–819812–824 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 insolusolu-ble 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

812–824 807–824

GST

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.

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)1Æ

nmol)1) (Figs 3B and 4) This value was a bit higher

than that of purified GST (319 ± 63 lmolÆmin)1Æ

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)1Ænmol)1, respectively), and GST–4AaCter 807–824 had a very low GST activity (24 ± 18 lmolÆ min)1Ænmol)1) (Fig 4) The GST fusion proteins that formed inclusions with lower efficiencies had moderate GST activity ranging from 65 to 110 lmolÆmin)1Æ 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 696–851 696–799 801–851 801–834 801–829 807–824 807–819 812–824

GST

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.

210 90 49 37 29 (kDa)

21 9

S I S I S I S I S I S I

4AaCter

4AaCter-TpN15

ATG

TpN17

TpN47

4AaCter-TpN17

4AaCter-TpN47

6xHis PP

4AaCter 4AaCter

Ptac

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.

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)1culture

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-posireac-tive 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 was removed (Tables 1 and 2) This result suggests that the human serum did not

Table 1 Reactivity of recombinant TpNs against Tp-positive human serums.

Table 2 Reactivity of recombinant TpNs against Tp-negative human serums.

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 lepilepi-dopteran-

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 difbuf-ferences 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

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 801–834 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

PRISM 310 genetic analyser (Applied Biosystems,

Foster City, CA, USA)

Characterization of GST–4AaCters

0.06 mm isopropyl b-d-1-thiogalactopyranoside at 37C for

3 h The E coli cells were disrupted by sonication, and

sol-uble and insolsol-uble proteins were separated by

centrifuga-tion (12 000 g, 5 min, 4C) 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 NaHCO3, NaOH, pH 11)

for 1 h at 37C 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)1Ænmol)1 protein by using the molar extinction coefficient e340= 9.8 mm)1Æcm)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 OsO4by 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)

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

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 NaHCO3, NaOH, pH

11) for 1 h at 37C The 4AaCter–TpNs were purified

using a Ni-NTA column (Invitrogen, Carlsbad, CA, USA)

and incubated with PreScission protease at a concentration

of 1 UÆ100 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 lgÆmL)1) per well was

incubated overnight in a 96-well microplate at 4C Native

T pallidumwas 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) AccurunSeries InfectrolD-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 H2O2

(2 lLÆmL)1) for 20 min at room temperature, the reaction

was stopped by adding 100 lL 1 m 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

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