Báo cáo khoa học: Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ pdf

Efficient synthesis of a disulfide-containing protein through a batch
cell-free system from wheat germ
Takayasu Kawasaki
1
, Mudeppa D. Gouda
1
, Tatsuya Sawasaki
1,2
, Kazuyuki Takai
1,2
and Yaeta Endo
1,2
1
Cell-free Science and Technology Research Center (CSTRC), and
2
Department of Applied Chemistry, Faculty of Engineering,
Ehime University, Matsuyama, Japan
We have developed a highly productive cell-free protein
synthesis system from wheat germ, which is expected to
become an important tool for postgenomic research.
However, this system has not been optimized for the
synthesis of disulfide-containing proteins. Thus, we sear-
ched here for translation conditions under which a model
protein, a single-chain antibody variable fragment (scFv),
could be synthesized into its active form. Before the start
of translation, the reducing agent dithiothreitol, which
normally is added to the wheat germ extract but which
inhibits disulfide formation during translation, was
removed by gel filtration. When the scFv mRNA was
incubated with this dithiothreitol-deficient extract, more
than half of the synthesized polypeptide was recovered in

the soluble fraction. By addition of protein disulfide
isomerase in the translation solution, the solubility of the
product was further improved, and nearly half of the
soluble polypeptides strongly bound to the antigen
immobilized on an agarose support. This strong binding
component had a high affinity as shown by surface-plas-
mon resonance analysis. These results show that the
wheat germ cell-free system can produce a functional scFv
with a simple change of the reaction ingredients. We also
discuss protein folding in this system and suggest that the
disulfide bridges are formed cotranslationally. Finally, we
show that biotinylated scFv could be synthesized in simi-
lar fashion and immobilized on a solid surface to which
streptavidin is bound. SPR measurements for detection of
antigens were also possible with the use of this immobi-
lized surface.
Keywords: cell-free synthesis; scFv; disulfide; wheat germ;
biotinylation.
As a consequence of the successes of the large-scale genome
sequencing projects, structural and functional analyses of
proteins are growing more and more important. We have
developed a highly productive cell-free protein synthesis
system from wheat germ, which enables the production of
milligram quantities of cDNA-encoded proteins and highly
parallel synthesis of many different proteins under unified
reaction conditions [1,2]. The expression of a cDNA clone in
this cell-free method can be performed with the 5¢-and
3¢-untranslated regions (UTRs) that were devised for
efficient mRNA translation [3]. It has been pointed out
that the parallel procedure, including cDNA amplification,

transcription and translation, could be operated by a
programmable liquid-handling machine. Therefore, the
wheat germ protein synthesis system is expected to become
a powerful tool for genome-wide high-throughput analyses
of proteins, proteome-based diagnosis and other post-
genomic applications [3].
On the other hand, it is clear that many proteins encoded
in the genomes have disulfide bonds that play important
roles in their function. However, no data have been reported
that demonstrate the successful production by a wheat germ
cell-free system of a disulfide-containing protein in active
form. In fact, disulfide bonds are unlikely to form during
cell-free synthesis because the synthesized polypeptides
normally are released into a buffer containing dithiothreitol
(DTT). DTT, or some other reducing agent, is required for
preserving the protein synthesis activity of the wheat germ
extract during storage and the translation reaction [1]. In
addition, the cell-free system may lack those enzymes that
would catalyze the correct disulfide formation in the lumen
of endoplasmic reticulum in vivo, such as protein disulfide
isomerase (PDI) [4,5].
In the present study, we searched for translation condi-
tions under which a disulfide-containing protein could be
efficiently produced in an active form. The model protein we
chose for the study was a single-chain antibody variable
fragment (scFv) against Salmonella O-antigen [6]. The scFvs
are engineered proteins that contain the heavy chain and
light chain variable domains (V
H
and V

L
, respectively) of
an antibody connected by a linker [7]. Each domain has
an intradomain disulfide bond. The anti-(O-antigen) scFv
(26 kDa) has been produced successfully through an
Correspondence to Y. Endo, Department of Applied Chemistry,
Faculty of Engineering, Ehime University, Matsuyama 790–8577,
Japan. Fax: + 81 899279941, Tel.: + 10 81 899279936,
E-mail:
Enzymes: biotin ligase from E. coli (EC 6.3.4.15); protein disulfide
isomerase (EC 5.3.41).
Abbreviations: DTT, dithiothreitol; PBT, phosphate-buffered
Tween 20; PDI, protein disulfide isomerase; scFv, single-chain anti-
body variable fragment; SPR, surface plasmon resonance; V
H
,heavy
chain variable domain; V
L
, light chain variable domain;
UTR, untranslated region.
(Received 4 August 2003, revised 30 September 2003,
accepted 20 October 2003)
Eur. J. Biochem. 270, 4780–4786 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03880.x
Escherichia coli secretory expression system, and its three-
dimensional structure has been solved [8]. We describe here
suitable conditions for the cell-free synthesis of the active
disulfide-containing scFv.
Materials and methods
Materials
The gene encoding the scFv fragment of the Se155–4 IgG

antibody, in the orientation VL-linker-VH (scFvLH) was a
generous gift from M. N. Young [6]. LATaq PCR kit, RNA
LA PCR kit, and protein disulfide isomerase (EC 5.3.41)
from bovine liver were obtained from Takara Shuzo, Japan.
pGEM-T Easy cloning kit, DNA ligase, RNasin, SP6 RNA
polymerase and streptavidin magnetic beads were obtained
from Promega. NICK columns, Sephadex G25 spin col-
umn and epoxy-activated Sepharose 6B gel were from
Amersham Pharmacia. Biotin ligase from E. coli (EC
6.3.4.15) was purchased from Avidity LLC. Restriction
enzymes were obtained from New England Biolabs, Inc.
Lipopolysaccharides from Salmonella typhimurium and
E. coli O111, biotin and cycloheximide were purchased
from Sigma. Amine-terminated magnetic beads were pur-
chased from Polysciences, Inc.
Construction of plasmids
The DNA fragment coding the scFvLH (10 pg) was
amplified with 0.4 l
M
each of the primers s1: 5¢-CTACC
AGATCTGCCATGCAGATCGTTGTTACCCAGG-3¢
and a1: 5¢-GGCTAAGAGCTCACGGTCAGGCTCG-3¢
by using a LATaq PCR kit (50 lL). The sequences
underlined are the BglII restriction site and initiation codon,
and the bold sequence is an anti-stop codon. The PCR
product was subcloned into pGEM-T Easy, and the
resultant plasmid DNA was digested with BglII and NotI.
This fragment was inserted into pEU [3] at the same
restriction sites. The obtained plasmid pEU–scFvLH was
purified by a standard cesium chloride density-gradient

centrifugation method. pEU-scFvLBH, which had a biotin
tag sequence coding 15 amino acids (GLNDIFEAQKI
EWHE) [9] at the linker region was constructed by ampli-
fying the whole plasmid pEU-scFvLH by inverse PCR
with the primers s2: 5¢-
CAAAAAATTGAATGGCATG
AACCGCCGAGCTCCAAC-3¢ and a2: 5¢-AGCTTCAA
AAATATCATTTAAACCCGACGGGCTGCTTTT-3¢
(the sequences for the biotin tag are underlined) followed by
circularization with DNA ligase.
Preparation of mRNAs
The pEU plasmids were transcribed in vitro (400 lL) at
37 °Cfor2hwith80m
M
Hepes-KOH (pH 7.6), 16 m
M
Mg(OAc)
2
,2m
M
spermidine, 10 m
M
DTT, 2.5 m
M
of each
nucleotide triphosphate (ATP, UTP, GTP and CTP), 0.8
unitÆlL
)1
RNasin (ribonuclease inhibitor), 20 lg plasmid
DNA, and 1.0 unitÆlL

)1
SP6 RNA polymerase. The
reaction mixture was extracted with phenol/water, then
with chloroform/water. These mRNAs were purified by gel
filtration with NICK columns and finally by ethanol
precipitation.
Cell-free translation
The wheat-germ extract was prepared by the reported
method [1]. DTT in the extract (4 m
M
) was excluded just
prior to translation by gel-filtration with a Sephadex G25
spin column pre-equilibrated by a DTT-deficient buffer
[40 m
M
Hepes/KOH (pH 7.8), 100 m
M
KOAc, 5 m
M
Mg(OAc)
2
and 0.3 m
M
each of the 20 amino acids]. The
translation mixture (38 lL) containing 1.2 m
M
ATP,
0.25 m
M
GTP, 15 m

M
creatine phosphate, 0.4 m
M
spermi-
dine, 28 m
M
Hepes/KOH (pH 7.8), 0.23 m
M
each of
20 amino acids (including leucine), 1.8 mgÆmL
)1
creatine
kinase, 53 m
M
KOAc, 1.6 m
M
Mg(OAc)
2
,0.6m
M
CaCl
2
,
0.4 unitÆlL
)1
RNasin, 200 lgÆmL
)1
mRNA, and the DTT-
deficient wheat germ extract (A
260

¼ 42) was incubated at
26 °C for 4 h. To label the synthesized proteins, 4 lCiÆmL
)1
[
14
C]Leu was also included (nonradioactive leucine was not
omitted, and the final concentration of leucine thus adds up
to 0.24 m
M
). For biotinylation, the mRNA from pEU–
scFvLBH was translated in the same way, except that
19.5 l
M
biotin and 19.5 lgÆmL
)1
biotin ligase were added.
The amounts of the synthesized polypeptides were deter-
mined from the
14
C radioactivity of the trichloroacetic
acid-precipitated materials. The soluble fractions of the
synthesized polypeptides were obtained by centrifugation
(20 000 g,10min),andtheÔsolubilityÕ was determined as
the amount of the soluble fraction divided by the total
amount. The concentration of the scFv synthesized without
[
14
C]Leu was estimated from that of the polypeptide
synthesized with the radioisotope in parallel.
Affinity chromatography

The antigen-coupled agarose gel was prepared by a reported
method [10]. Lipopolysaccharide (15 mg) from Salmonella
typhimurium was deacetylated, oxidized, and aminated to
be approximately 10 mg of freeze-dried powder containing
25 lmol amino group, determined with picrylsulfonic acid.
The aminated polysaccharide was mixed with the epoxy-
activated Sepharose 6B gel (1.5 g) in 20 mL of 0.2
M
NaOH/
KCl (pH 12.5). The coupling yield was 1.3 lmol as deter-
mined by a standard phenol/sulfuric acid procedure. The
soluble fraction containing an scFv polypeptide (20 lL) was
diluted by the same volume of 50 m
M
Tris/HCl, pH 8.0, and
loaded on the antigen column (120 lL) pre-equilibrated
with the same buffer. The column was washed with 280 lL
of the buffer (fractions 2–8) and then 200 lL of the buffer
containing 0.15
M
NaCl (9–13). The antigen-bound poly-
peptide was eluted (14–22) with 360 lLof4%(w/v)SDS
(for analysis) or 0.1
M
glycine/HCl, pH 2.3 (for purifica-
tion). The amount of polypeptide in each 40 lLfractionand
the ratio of the antigen-binding fraction to the total loaded
polypeptide were determined by measuring the radioactivity.
SPR analysis
For the quantitative binding studies, an IAsys biosensor

(Affinity Sensors) was used. The oxidized Salmonella
polysaccharide (0.3 lmol) was dissolved in 50 lLof
20 m
M
sodium borate buffer, pH 9.0, and then added onto
the amino cuvette supplied with the instrument at the level
of 233 arcÆsecond (6.2 ng). To avoid the nonspecific binding
Ó FEBS 2003 Cell-free synthesis of a disulfide-containing protein (Eur. J. Biochem. 270) 4781
of protein, the surface of the cuvette was prewashed with the
same volume of the wheat-germ extract. The scFv was
synthesized under the DTT-deficient condition in a 76 lL
reaction without a radioisotope and purified by the above
method. The resulting solution (50 lL) was neutralized
by 200 m
M
phosphate-buffered Tween 20 (PBT), pH 8.0,
desalted by G25 spin column pre-equilibrated with 10 m
M
PBT, pH 8.0, and then added to the antigen-coated cuvette
at various concentrations. The antigen–antibody association
curves were measured for 5 min at 25 °C, and the bound
polypeptide was dissociated from the surface by the addition
of 10 m
M
PBT, pH 8.0. The dissociation rate constant k
dissoc
(s
)1
) and the association rate constant k
assoc

(
M
)1
Æs
)1
)were
obtained by using the
FASTFIT
software supplied with the
instrument. The dissociation constant K
D
(
M
) was calculated
according to the equation: K
D
¼ k
dissoc
/k
assoc
.
Affinity capture of the nascent polypeptide–mRNA
complex
The linear DNA fragment (910 bp) including the scFvLH
sequence was amplified on the pEU–scFvLH using SP6
primer (5¢-ATTTAGGTGACACTATAG-3¢)andanti-pri-
mer (5¢-ATGGCGCCAGCTGCAGGCTA-3¢, anti-stop
codon in bold), and transcribed in the same way as above.
Translation was carried out with the purified mRNA
(75 lgÆmL

)1
)for30minat26°C as mentioned above,
and the reaction was stopped by addition of cycloheximide
(2.3 l
M
). The soluble fraction was diluted two-fold with
50 m
M
Tris/HCl, pH 7.5, containing 10 m
M
MgCl
2
and gel-
filtrated on a G25 spin column pre-equilibrated with the
same buffer. Antigen-coated magnetic beads were prepared
by mixing the oxidized polysaccharide with the amine
terminated particles. The antigen from Salmonella typhimu-
rium or E. coli O111 (0.32 lmol and 0.42 lmol, respectively)
was reacted with 100 lL of the beads in 20 m
M
sodium
borate buffer, pH 9.0, at room temperature for 6 h (total
200 lL). The coupling yield was 0.14 lmol for either antigen
as determined by a standard phenol–sulfuric acid procedure.
The filtrated solution (30 lL) containing the mRNA–
polypeptide complex was mixed with the antigen beads
(5 lL) for 10 min at room temperature. The beads were
washed five times with 30 lLof50m
M
Tris/HCl (pH 7.5)

containing 10 m
M
MgCl
2
and 0.15
M
NaCl, then finally
mixed with the same volume of 0.1
M
glycine/HCl (pH 2.3)
for 15 min. The eluted mRNA was recovered by precipita-
tion with ethanol and reversely transcribed and amplified by
using RNA LA PCR kit with the above primers.
Results
Plasmid construction
The cDNA for the scFv was first inserted into the pEU
vector [3] that was designed for the specific purpose of
protein expression in the cell-free system. pEU contains the
sequences for the SP6 promoter, translation enhancer
region independent of the cap structure at the 5¢-end,
restriction enzyme sites, and a 3¢-untranslated region
required for the high translation efficiencies (Fig. 1A). The
transcript from a cDNA inserted into this plasmid was used
directly for the cell-free translation.
Synthetic condition allowing efficient disulfide formation
The reducing agent DTT has been conventionally inclu-
dedinthewheat-germextractatahighconcentrationfor
the sake of stability during storage [1]. Thus, in order to
reduce the DTT concentration for the translation reaction,
Fig. 1. Syntheses of the scFv. (A) Transcription unit in the pEU

plasmid containing the scFvLH.
P
SP6, SP6 promoter; 5¢-UTR,
5¢-untranslated region; 3¢-UTR, 3¢-untranslated region. The initiation
codon and the stop codon were introduced by the PCR primers (s1 and
a1). (B) Incorporation of [
14
C]Leu into polypeptides during the
cell-free translation of the scFvLH mRNA with various additives.
(a) 2 m
M
external DTT (e); (b) 2 m
M
external DTT + 0.5 l
M
PDI
(h); (c) 0 m
M
external DTT (n); (d) 0 m
M
external DTT + 0.5 l
M
PDI (·); (e) translation under the conventional condition (2.5 m
M
DTT) with the untreated extract (s). (C) Autoradiogram of SDS/
PAGE separating the scFvLH polypeptides synthesized under the
same conditions as indicated in (B). Total (t) and soluble (s) fractions
were denatured without 2-mercaptoethanol before loading onto
the gel.
4782 T. Kawasaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003

the extract was passed through a gel filtration column just
before the start of reaction. With the use of this low-DTT
extract, the scFvLH mRNA was translated in the
presence of [
14
C]Leu, and the productivity was monitored
by measuring the radioactivity of the trichloroacetic acid-
insoluble precipitates (Fig. 1B). The polypeptide produc-
tion under the DTT-deficient condition (Fig. 1Bc) was
more than half that under the DTT-rich condition
(Fig. 1Ba). The translation activity of the wheat-germ
extract was not affected by the gel filtration, as the
productivity under the DTT-rich condition using the gel-
filtrated extract (Fig. 1Ba) was almost the same as that
under the conventional condition with the untreated
extract (Fig. 1Be). We also tested if PDI would affect
the productivity. Although the synthesis efficiency was
slightly less in the presence of PDI (Fig. 1Bb,d) than in
the absence (Fig. 1Ba,c), which might be due to the
phosphate that buffers the PDI stock solution, the enzyme
did not largely interfere with the translation machinery at
the low level (0.5 l
M
). The polypeptide productivity of the
DTT-deficient, PDI-containing reaction was approxi-
mately 40 lgÆmL
)1
.
Then, in order to estimate the extent of disulfide
formation, the synthesized polypeptide was analyzed on a

nonreducing SDS gel after 4 h translation (Fig. 1C). In this
gel, the oxidized form runs faster than the reduced form [11].
The result showed that the polypeptides from the DTT-
deficient reactions (Fig. 1Cc,d) migrated faster than those
from the DTT-rich reactions (Fig. 1Ca,b) around 30 kDa.
Both sets of samples had the same mobility on a reducing
gel (data not shown). The solubility of the synthesized scFv
polypeptide was calculated to be 50% in (Fig. 1Ca), 50% in
(Fig. 1Cb), 65% in (Fig. 1Cc), and 85% in (Fig. 1Cd),
respectively. Therefore, the DTT-deficient condition in the
presence of PDI was effective for the disulfide formation
and increased the soluble polypeptides. Although we have
not determined the exact concentration of DTT in the
translation mixture, the above results were highly reprodu-
cible.
Fig. 2. Functional analyses. (A) Elution profiles of the soluble scFvLH
polypeptides on the agarose-gel coupled with Salmonella polysaccha-
ride. Solvents: 50 m
M
Tris/HCl buffer, pH 8.0 (numbers 2–8); the same
buffer containing 0.15
M
NaCl (numbers 9–13); 4% SDS (numbers
14–22). e,2m
M
external DTT; h,2m
M
external DTT + 0.5 l
M
PDI; n,0m

M
external DTT; ·,0m
M
external DTT + 0.5 l
M
PDI.
The inserted autoradiogram showed the nonreducing SDS/PAGE.
(B) SPR analysis using IAsys instrument. The concentrations of the
purified scFv polypeptides were 41, 21 and 11 n
M
(from top to bot-
tom). (C) Post-translational effect of PDI on the active fraction of the
scFv. The enzyme was added in the DTT-deficient synthesis solution
after 3 h translation, and the reaction was further continued for the
same number of hours. The soluble material of the product was sep-
arated on the affinity column described in (A). (D) Agarose gel (1.8%)
electrophoreses of cDNAs from the nascent polypeptides–mRNA
complexes. Translations were performed under the two different con-
ditions, and each solution was mixed with the cognate or noncognate
antigen beads. The captured mRNA was dissociated from the complex
with the acidic buffer, recovered with ethanol precipitation, reversely
transcribed, and amplified. Lane 0, 100 bp DNA marker; lane 1, DTT-
deficient condition (0 m
M
external DTT) with Salmonella antigen;
lane 2, DTT-deficient condition with E. coli antigen; lane 3, DTT-rich
condition (2 m
M
external DTT) with Salmonella antigen; lane 4, DTT-
rich condition with E. coli antigen.

Ó FEBS 2003 Cell-free synthesis of a disulfide-containing protein (Eur. J. Biochem. 270) 4783
Antigen-binding analyses
We then examined the antigen binding activity. The soluble
material from each reaction was loaded on an antigen
column and was separated into the unbound (numbers 2–8),
washed (numbers 9–13), and eluted (numbers 14–22)
fractions (Fig. 2A) according essentially to the method
described for the Se155–4 IgG antibody [10]. The third
fraction that eluted with SDS was judged to include the
strong-binding polypeptide. The results clearly showed
that the only products from the DTT-deficient media
(Fig. 2Ac,d) contained significant amounts of the active
molecules, whereas most of the products from the DTT-rich
media (Fig. 2Aa,b) were recovered in the inactive unbound
fractions. The total amount of the active fractions (numbers
16–18) in Fig. 2Ad was 37% of the loaded soluble
polypeptides. The sample from fraction number 17 gave a
radioactive band on the nonreducing SDS gel correspond-
ing to the oxidized form. Therefore, PDI increased the
fraction of functional scFv during translation under DTT-
deficient conditions.
Next, this active polypeptide was eluted by the acidic
buffer instead of SDS solution and applied to the SPR
analysis (Fig. 2B). The antigen–antibody association curves
were measured on addition of various concentrations of the
purified polypeptide onto the antigen-coated surface. K
D
was calculated by using the linear equation, k
on
¼

k
assoc
[ligate] + k
dissoc
(K
D
¼ k
dissoc
/k
assoc
), where k
on
(s
)1
)
is the on-rate constant defined as a slope of the exponential
association curve, and [ligate] is the concentration of the
added polypeptide [12]. The resulted K
D
for scFvLH was
found to be in the order of 10
)8
M
, which indicated that the
synthesized polypeptide had a high affinity for the immo-
bilized antigen (Table 1).
These results showed that approximately 13 lgofa
functional scFv was produced in a 1-mL batch reaction
under the DTT-deficient, PDI-containing conditions, a
simple alteration of the conventional translation condition.

Affinity capture of the nascent polypeptide
We also tested a post-translational effect of PDI on the
active fraction of the mature scFv polypeptide. After the 3 h
synthesis under the DTT-deficient conditions without PDI,
the translation solution was incubated with the enzyme for
the same number of hours. The soluble material was loaded
on the affinity column (Fig. 2C). The active fraction that
eluted with SDS made up only 12% of the total loaded
polypeptides. This might mean that the disulfide isomeriza-
tion proceeds more efficiently during elongation of the
polypeptide chain, rather than after the release of mature
polypeptide from the ribosomes. Thus, we examined
whether the complex of the growing polypeptide of the
correctly folded scFv and the mRNA connected via the
ribosomes could be isolated. The polypeptide elongation
was stopped by addition of cycloheximide, and the mRNA–
polypeptide complexes were captured on antigen-coated
magnetic beads. The isolated complex was dissociated with
an acidic buffer, and the recovered mRNA was reversely
transcribed and amplified (Fig. 2D). The cDNA band from
the DTT-deficient reaction (lane 1) was more clearly seen
than that from the DTT-rich reaction (lane 3). In addition,
the cDNA bands from the reactions with the noncognate
antigens (lane 2 and 4) were relatively weak. These results
indicate that a larger fraction of the nascent polypeptide
produced under the DTT-deficient conditions than under
the DTT-rich conditions recognized the antigen and thus
had the correct disulfide bonds.
Functional biotinylation
To show a possible application of the present method, we

tested whether a biotinylated scFv could be synthesized by
a modification of this method. We used a biotin ligation
enzyme from E. coli for biotinylation [9]. A biotin tag
sequence was introduced in the middle of the original
linker of scFvLH by PCR (Fig. 3A), and the mRNA
from this plasmid was translated in the presence of biotin
and the enzyme. The efficiency of biotinylation was
evaluated by measuring the fraction that could be trapped
by the streptavidin-conjugated magnetic beads (Fig. 3B).
The figure shows the autoradiogram of SDS/polyacryl-
amide gel separating three materials [total solution before
mixing with the beads (t), unbound solution (u) and
bound solution (b) after mixing with the beads] of the
synthesized polypeptides. The results clearly showed that
much more polypeptide from the DTT-deficient reaction
bound to the beads than that from the DTT-rich reaction.
Therefore, the disulfide-formed polypeptides were prefer-
entially biotinylated in this system. The biotinylated scFv
was then purified with the use of the antigen column, and
the affinity was measured in the same way as above. The
antigen-binding activity was found to be as high as the
wild type (Table 1), which showed that the present system
was able to produce a biotinylated scFv without any loss
of its function.
Then, the crude synthesis solution containing the biotin-
ylated scFv was immobilized on a streptavidin-coated
cuvette for the IAsys instrument, and SPR measurements
were carried out (Fig. 3C). When the cognate antigen was
added (Fig. 3C
A

), the response values increased as the
concentrations increased. The affinity of the antigen to the
immobilized scFv was determined to be 4.4 · 10
)6
M
K
D
by
using the same equation as above. The control reaction with
noncognate antigen (Fig. 3C
B
) gave low response levels.
These results show that a sensitive antigen-detection system
Table 1. Affinity parameters.
K
D
(
M
)
k
dissoc
(s
)1
)
k
assoc
(
M
)1
Æs

)1
)
scFvLH 4.3 · 10
)8
(± 0.6) 0.7 · 10
)1
(± 0.07) 1.7 · 10
6
(± 0.06)
Biotinylated scFvLH 5.4 · 10
)8
(± 1.3) 0.8 · 10
)1
(± 0.1) 1.5 · 10
6
(± 0.1)
4784 T. Kawasaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003
can be conveniently constructed by using the crude cell-free
protein synthesis solution.
The present biotinylation technique may be useful for
preparation of biotin-labeled proteins and also for non-
radioisotope detection of cell-free synthesized proteins [13].
Discussion
This report is the first to describe the synthesis by the wheat-
germ cell-free system of a disulfide-containing protein. The
functional and structural analyses revealed that a consider-
able fraction of the synthesized polypeptides folded cor-
rectly under the DTT-deficient condition with PDI present
during peptide synthesis. The results show that the thiol/
disulfide interchange reaction proceeds efficiently in this cell-

free system. The fact that the growing scFv polypeptide was
captured by the affinity to the antigen means that the correct
folding of the polypeptide catalyzed in part by PDI proceeds
more or less before the release from the ribosomes. This
seems consistent with the suggestion that de novo proteins
fold cotranslationally during the eukaryotic protein synthesis
[14], which might make the system suitable for the synthesis
of multidomain proteins. In fact, our method does not
require the addition of the chaperone molecules such as
GroEL and GroES, whose addition appears to be required
to support correct formation of disulfide bonds in E. coli
cell-free protein synthesis systems [15,16]. The active frac-
tion of the scFv produced in the E. coli cell-free system with
chaperones was nearly half of the synthesized protein, which
was almost the same as in our case. Therefore, the wheat-
germ system might have a practical advantage over the
bacterial cell-free system. The scFv against Salmonella
O-antigen was for the first time prepared successfully
through an E. coli expression system, in which the produced
polypeptide was secreted into the periplasmic space of the
bacteria [6]. The result of the antigen-binding analysis
demonstrated that the scFv produced in the wheat-germ cell
free system has a higher affinity than that produced in the
E. coli system [8]. This difference in the activity of the
synthesized polypeptide might be due to the difference in
the intrinsic characteristics between the eukaryotic and
prokaryotic translation systems.
The synthesis method described in this paper may be
more convenient than the bacterial expression system if only
a small amount (1–20 lgÆmL

)1
) of the active protein is
required. To improve the protein yield in the cell-free
system, it might be necessary to add some reductive
potential buffer to prolong the protein synthesis. Although
we have already tried several other scFvs and observed that
the method worked as far as these scFvs are concerned, the
general applicability of this method for scFvs and other
disulfide-containing proteins is yet to be determined.
However, in the postgenomic researches, parallel produc-
tion of many different proteins will be also needed. A
protein synthesis machine that can perform PCR, tran-
scription and translation automatically with a highly
parallel operation is being developed in our laboratory.
Fig. 3. Biotinylation of scFv. (A) Transcription unit in the pEU plas-
mid containing the scFvLBH. The biotin-tag was introduced by PCR
primers (s2 and a2). (B) Autoradiogram of a reducing SDS gel sep-
arating the biotinylated scFv. The mRNA from pEU-scFvLBH was
translated (38 lL) under the DTT-deficient (–) or the DTT-rich (+)
condition in the presence of PDI for three hours. The soluble fractions
were twice diluted with 50 m
M
Tris/HCl, pH 8.0 and loaded onto G25
spin columns pre-equilibrated with the same buffer to remove free
biotin. The filtrate (30 lL) was mixed with 5 lL of streptavidin-
magnetic beads at room temperature for 15 min and the bound
polypeptide was eluted with 4% SDS. Three materials (filtrate before
mixing with the beads (t), unbound solution (u) and bound solution (b)
after mixing with the beads) were denatured with 2-mercaptoethanol
before loading onto the gel. (C) Binding responses of the antigens to

the biotinylated scFv on the streptavidin-coated cuvette of IAsys
instrument. The biotinylated scFv was synthesized under the DTT-
deficient condition (76 lL reaction without a radioisotope), and the
mixture was desalted through G25 spin column pre-equilibrated with
10 m
M
PBT (pH 8.0). The biotin cuvette was first coated by strept-
avidin (34 ng), then by the crude protein solution including the bio-
tinylated scFv (50 lL). The responses were measured by the addition
of free antigens A: Salmonella polysaccharide, at 9.7 l
M
,4.9l
M
and
1.2 l
M
(upper, middle and bottom responses, respectively); B: E. coli
O111 polysaccharide, 10 l
M
(upper), 5.0 and 2.5 l
M
(lower).
Ó FEBS 2003 Cell-free synthesis of a disulfide-containing protein (Eur. J. Biochem. 270) 4785
The present modification of the protein synthesis condition
should be amenable to automation, because it involves only
the alterations in the reaction ingredients.
Acknowledgements
We thank Dr N. M. Young for providing the scFvLH gene. This work
was supported by Japan Society for the Promotion of Science Grant
JSPS-RFTF 96100305 (to Y.E.).

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4786 T. Kawasaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003