Protein Expression and Purification 147 (2018) 94–99

Contents lists available at ScienceDirectProtein Expression and Puriﬁcation
journal homepage: www.elsevier.com/locate/yprep

An anti-peptide monoclonal antibody recognizing the tobacco etch virus
protease-cleavage sequence and its application to a tandem tagging system

T

Sanae Tabataa,1,2, Yu Kitagoa,2, Yuki Fujiia,2,3, Emiko Miharaa, Keiko Tamura-Kawakamia,
Naoko Noriokaa, Katsu Takahashib, Mika K. Kanekoc, Yukinari Katoc,d, Junichi Takagia,∗aInstitute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan
Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japanc
Graduate School of Medicine, Tohoku University, Sendai, Miyagi, 980-8575, Japand
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, 980-8575, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Aﬃnity tag
Tandem tag
Tag removal
Tobacco etch virus protease
Monoclonal antibody
Sandwich ELISA

Peptide-based aﬃnity tags are commonly used in recombinant production/puriﬁcation of proteins, and are often
preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal
antibody 2H5 recognizing an undecapeptide tag called “eTev”, which contains a recognition sequence for
Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes
compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture
allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various
proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system
during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse
Wise protein.

Recombinant production and puriﬁcation of proteins for theirfunctional and structural analyses has become a routine practice in thebiomedical research ﬁeld. In most cases, the protein of interest is genetically fused with artiﬁcial sequences that code for “aﬃnity tag” ateither end, enabling detection, labeling, and puriﬁcation of the target
protein. Peptide-based epitope tag is particularly convenient because of
the small size of the tag, and many excellent epitope tag systems
comprising tag peptide and anti-peptide antibody are available [reviewed in Ref. [1]]. In certain applications it is desired to attach two or
more epitope tags to one protein so that each aﬃnity puriﬁcation/detection procedure can be done in a sequential manner [2]. “Double
tagging” also enables a sandwich ELISA, where one antibody is used tocapture a protein to a solid support and the other antibody is used for
detection [3]. This is particularly useful when there is no good antibody
available for the target protein, but the two tags must be well separated
in space to ensure the simultaneous binding of the two antibodies.
Even though the epitope tags are short and generally do not interfere with the function of the target protein, it is often necessary to remove the artiﬁcially added tag sequence after the puriﬁcation,∗

particularly when the protein is to be used in a crystallographic projects[4]. Inclusion of a protease-recognition sequence in between the protein
and the tag portion is the strategy routinely employed in such cases [5].
When multiple tagging is desired, however, putting the protease site ineach tag portion will require more complex construct design and may
result in unexpected eﬀect on the function of the protein due to thelonger artiﬁcial sequence to be attached. One potential way to getaround this problem is to use the protease-recognition sequence itself asa tag portion. In fact, the FLAG tag (DYKDDDDK) originally developedby Hopp and colleagues contains a recognition sequence for a protease
enterokinase and potentially can be used is such application [6].
However, the commercially available enterokinase is relatively expensive and does not have highly strict recognition speciﬁcity toward
the tag sequence, making the FLAG tag rarely used for a cleavage
purpose despite its wide usage in the general tagging purposes. In
contrast to enterokinase, tobacco etch virus (TEV) protease can be easily produced recombinantly in E. coli, has very high recognition speciﬁcity toward 7-residue sequence (ENLYFQG), and the cleavage reaction can proceed at 4° [7,8]. In this paper, we succeeded in establishing

Corresponding author. Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail address: takagi@protein.osaka-u.ac.jp (J. Takagi).
Present address: CURED Inc.
2
These authors contributed equally.
3
Present address: Ono Pharmaceutical Co., Ltd.
1

https://doi.org/10.1016/j.pep.2018.03.004
Received 12 March 2018; Accepted 13 March 2018
Available online 14 March 2018
1046-5928/ © 2018 Elsevier Inc. All rights reserved.

Protein Expression and Purification 147 (2018) 94–99

S. Tabata et al.

eTev peptide (30 μg/ml) and blocked with 5% skim milk in Tris-buffered saline (TBS). Hybridoma supernatants or puriﬁed IgG seriallydiluted in TBS containing 1% BSA were then added and incubated for
1 h at room temperature, and wells were washed 3 times with 200 μL
TBS. To detect bound antibody, horse radish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (Southern Biotech) diluted to
1:1000 in TBS was added and incubated for 30 min at room temperature, and wells were washed 4 times with TBS. Color was developed by
the addition of 100 μL of ABTS substrate solution (KPL) and absorbanceat 405 nm was recorded using a multiplate reader (Emax, Molecular
Devices).
For the sandwich ELISA, microtiter plates were coated overnight at
4 °C with anti-PA tag antibody NZ-1 (Wako Pure Chemical Co., 20 μg/ml), followed by blocking with 5% skim milk in TBS. The test samples
containing the proteins tagged with either nPAW or cPAW peptides
were then added to the wells and incubated for > 3 h at 4 °C to allow
the completion of the capture reaction. After washing the wells 3 times
with 200 μL TBS, biotinylated 2H5 IgG (10 μg/ml in TBS containing
0.1% BSA) was added and incubated for 1 h at room temperature. To
detect bound 2H5, HRP-conjugated streptavidin (VECTOR) diluted to
1:1000 in TBS containing 0.1% BSA was added and incubated for
30 min at room temperature, followed by the TBS wash and color development as described above.

an anti-peptide monoclonal antibody whose epitope includes the TEVprotease recognition sequence. This tag, dubbed eTev tag, was combined with another peptide (PA tag) [9] to make a single stretch ofpolypeptide carrying two closely positioned epitopes in tandem. Crystal
structure of the tag-antibody complex was used to optimize the linker
length between the PA-eTev or eTev-PA tandem that allows simultaneous binding of two antibodies, resulting in a development of a single
cleavable tag compatible with semi-quantitative sandwich ELISA
without the need for any antibodies speciﬁc for the target protein.
1. Materials and methods
1.1. Production of a monoclonal antibody
An 11-amino-acid peptide (RENLYFQGKDC) having the TEV protease recognition sequence (underlined) and charged amino acids atboth ends was synthesized using Fmoc chemistry, followed by a conjugation to keyhole limpet hemocyanin (KLH) or bovine serum albumin(BSA) through the cysteine located at the C-terminus. Immunization
was performed by intracutaneous administration of the peptide-KLH
complex together with an adjuvant (100 μL per foot, 200 μL/animal in
total) into the soles of rear feet of a Sprague Dawley rat (female, 8
weeks old). Two weeks after the immunization, the antibody titer wasmeasured by ELISA. The rat iliac lymphocytes were fused with mouse
myeloma cells (SP2/0 cell line) by the polyethylene glycol method.
Hybridomas were selected in complete RPMI1640-10% fetal calf serum(FCS) with HAT supplement and subjected to limiting dilution culture
in 96-well plates, followed by culture in HT medium. Supernatants were
initially screened for antibodies with ELISA using peptide-BSA conjugate as antigen, and later using a fusion protein described below.
Resultant hybridoma clone (2H5) produces IgG2a (κ), and the IgG was
puriﬁed from hybridoma culture supernatants using recombinantProtein G-Sepharose (GE Healthcare). N-terminal as well as several
internal amino acid sequences were obtained by Edman sequencing of
the puriﬁed antibody using a Shimadzu Protein Sequencer PPSQ-51A/
53A (Shimadzu Corp.), and the heavy and light chain genes were cloned
as described previously [10].

1.4. TEV cleavage assay
Various mutant eTev-Fn (400 μg/ml) was incubated with TEV protease at an enzyme/substrate ratio of 1:10 (wt/wt) in TBS for 16 h at
20 °C. The reaction was terminated by the addition of SDS-PAGE sample
buﬀer and analyzed by SDS-PAGE. TEV cleavage was judged by theconversion of intact eTev-Fn band (22 kDa) to a fast-migrating 19 kDaband on a 12% polyacrylamide gel.

1.5. Binding analysis using surface plasmon resonance (SPR)
Kinetic binding analysis was performed using BIACORE 2000 (GEHealthcare) as described previously [9]. First, puriﬁed 2H5 IgG was
immobilized on a CM5 sensor chip using amino coupling chemistry at
about 1000 RU according to the method provided by the manufacturer.
The eTev-Fn proteins were diluted in a running buﬀer (TBS containing
0.005% Tween 20, TBST) and injected at a ﬂow rate of 20 μl/min. Kinetic binding parameters were derived from the sensorgram curves by
global ﬁtting to a 1:1 Langmuir model using the BIAevaluation software(GE Healthcare).

1.2. Fusion protein
To test the reactivity of 2H5 toward eTev sequence, fusion proteins
containing the eTev peptide were prepared. To this end, the antigen
sequence or its variants were fused to the N-terminus of a fragment of
human ﬁbronectin comprising of the 9th to 10th type III repeats [11].
The peptide segments were prepared by extension PCR and the entire
coding region was cloned into the NdeI-BamHI site of the pET-16b
vector (Novagen), resulting in the N-terminally 10xHis-tagged eTev-Fnproteins. To validate the “tandem double tag” application, a 161-residue model protein T4 lysozyme (T4L) was fused either N-terminallywith a 29-residue peptide with the following sequence; GVAMPGAEDDVVGGGGNSRENLYFQGKDG (dubbed nPAW tag for “n-terminal PAcontaining double(W) tag”) or C-terminally with a 30-residue peptide
with the following sequence; RENLYFQGKDGGGGGSHMGVAMPGAEDDVV (dubbed cPAW tag for “c-terminal PA-containing W tag”), andcloned into the NdeI-BamHI site of the pET16b vector as above. All
these recombinant proteins were expressed in E. coli using theBL21(DE3) strain as a host, and puriﬁed from the soluble fraction of thebacterial lysate by Ni-NTA chromatography. Two more proteins, mouseneuropilin-1 ectodomain (Nrp1ec) and human serum albumin (HSA),
were also appended with cPAW tag (Fig. 3A) and produced in mammalian expression system in a similar way described previously [9].

1.6. Preparation and crystallization of 2H5 Fab fragment
2H5 IgG was puriﬁed from hybridoma culture supernatants using
recombinant Protein G-Sepharose (GE Healthcare). Puriﬁed IgG was
dialyzed against PE buﬀer (20 mM Na/K phosphate, 10 mM EDTA, pH
7.0) and incubated with Immobilized Papain (Thermo Scientiﬁc)(0.1 ml per mg IgG) in the presence of 25 mM Cys-HCl (pH 7.0) for∼4 h at 37 °C under gentle agitation. After the removal of the
Immobilized Papain, the reaction mixture was dialyzed against TBS,and slowly added to eTev peptide-conjugated Sepharose. Bound Fabwas eluted with 0.1 M Gly-HCl, pH 2.3, dialyzed against TBS, and further puriﬁed by gel ﬁltration chromatography on a Superdex 200 16/60
column equilibrated with TBS. The concentrated Fab solution (10.5 mg/ml) was mixed with 1.0 mM eTev peptide, and the crystallization
screening of the Fab-peptide complex was carried out using the hanging
drop vapor-diﬀusion method, resulting in many hit conditions. Uponthe optimization, the diﬀraction quality crystals were grown under thecondition of 0.1 M Tris-HCl pH 7.0, 20% polyethylene glycol monomethyl ether 2,000, 0.5 M NaCl.

1.3. ELISA
For the direct ELISA assay, microtiter plates (NUNC Maxisorp) were
coated overnight at 4 °C with eTev-Fn fusion proteins (10 μg/ml) or
95

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S. Tabata et al.

Table 1
X-ray data diﬀraction statistics and Reﬁnement statistics.
Diﬀraction data statistics
Space group
Unit cell parameters
No. Molecule/ASU
Resolution [Å]No. Unique Reﬂections
Multiplicity
Completeness [%]Rsyma
Mean I/σ(I)Wilson B factor
Reﬁnement statistics
Resolution Range [Å]No. of nonhydrogen atoms
No. of water molecules
Rwork/Rfreeb
RMSD bond length [Å]RMSD bond angle [°]Ramachandran plot
In most favored regions [%]In disallowed regions [%]P3221
a = b = 217.8 Å, c = 52.2 Å
250.0–2.05 (2.12–2.05)
88376 (8730)
5.3 (5.1)
99.5 (98.9)
0.148 (0.491)
21.0 (5.2)
21.9
45.7–2.05 (2.10–2.05)
7201
591
0.189/0.221 (0.241/0.273)
0.012
1.511
97.7
0.00

Values in parentheses refer to the highest resolution shell.
aRsym = ∑|Ihkl - 〈Ihkl〉|/∑Ihkl.
b
Rwork = ∑||Fo| - |Fc||/∑|Fo|, Rfree was calculated from the test set (5% reﬂections of thetotal data).

1.7. X-ray data collection and structural determination
The diﬀraction data sets were collected at BL32XU beamline,
SPring-8 (Hyogo, Japan) with the helical data collection mode [12].
The diﬀraction images were processed with HKL2000 [13], and theinitial phases were calculated by molecular replacement method with
PHASER [14] using a Fab structure of IgG from rat (PDID: 1BFO) as asearch model. The resultant model was reﬁned using REFMAC5 [15]with manual editing on COOT [16]. The model validation was carriedout with MOLPROBITY [17]. Diﬀraction data statistics and ﬁnal reﬁnement statistics are shown in Table 1. All structural ﬁgures wereprepared with the program PyMOL (Schrödinger, LLC). Atomic coordinates and structural factors have been deposited in the Protein Data
Bank under an accession number 5AUM.

Fig. 1. Reactivity of 2H5 antibody toward eTev sequence. (A) Direct ELISA. Serially diluted 2H5 hybridoma culture supernatants were incubated with wells coated with indicated proteins. Bindings were evaluated by measuring the absorbance at 405 nm after
addition of chromogenic substrate to the wells. (B) Surface plasmon resonance analysis.
Serially diluted eTev-Fn proteins (31.25, 62.5, 125, 250, and 500 nM) were injected over
the CM5 sensor chip immobilized with 2H5 for 60 s, followed by dissociation in TBST for
120 s at a ﬂow rate of 20 μl/min.

however, we suspected that seven residues may not be long enough toelicit speciﬁc and strong immune reaction when immunized in rats. So
we extended the peptide at both sides with charged residues, and used a
decapeptide (RENLYFQGKD, dubbed eTev for extended TEV recognition sequence) as an immunogen after conjugation with KLH throughan extra cysteine placed at the C-terminus. Using a standard hybridoma
technology, a rat-mouse hybridoma clone 2H5 secreting rat IgG2a/kappa antibody was isolated. The culture supernatants from 2H5
showed strong reactivity in ELISA assays with the eTev peptide used in
the immunization as well as the eTev-Fn fusion protein directly coatedonto the microtiter wells, with virtually no binding toward control BSAcoated surface (Fig. 1A). We also analyzed the kinetic binding behaviorof puriﬁed 2H5 antibody toward eTev-Fn by using surface plasmon
resonance, and determined the kinetic parameters as kon = 5.02 × 105
M−1s−1 and koﬀ = 0.0359 s−1, resulting in the KD value of 71.4 nM(Fig. 1B). Due to the rather fast dissociation rate, eTev-2H5 system may
not be ideal for the protein puriﬁcation purpose. However, the aﬃnity
is high enough to be applied in an ELISA-based detection/quantiﬁcation
procedures, as will be discussed in the later sections. In order to deﬁne
the sequence speciﬁcity of the recognition, we performed alaninescanning experiment using the eTev-Fn mutants. Fig. 2A shows theconcentration dependent binding of puriﬁed 2H5 IgG toward various
Ala mutant eTev-Fn proteins. Ala substitutions at R1, L4, Y5, G8, K9,
and D10 had strong negative eﬀect on the 2H5-reactivity resulting in apartial to complete elimination of the binding, indicating the criticalinvolvement of these residues in the recognition. Ala substitutions at

1.8. Establishment of stable cell line expressing mouse Wise
A plasmid DNA encoding nPAW-tagged mouse Wnt modulator insurface ectoderm (Wise) in the pcDNA3.1 backbone was co-transfectedwith a plasmid containing puromycin resistance gene into Expi293F
cells (Thermo Fisher Scientiﬁc) and plated into multiple 96-well plates.
Cells were cultured for 5 weeks in a medium containing 1 mg/mL geneticin (G418) and 10 μg/mL puromycin to select for stable integrants.
Culture supernatants from wells containing single colony were assessed
for the presence of nPAW-Wise using the sandwich ELISA describedabove, and positive clones were expanded and further evaluated for the
expression level after several passages. Several cell clones stably
maintaining the high expression level over a long culture period wereobtained.
2. Results and discussion
2.1. Isolation of anti-eTev tag antibody 2H5 and its epitope mapping
The TEV protease is known to recognize a stretch of 7 amino acids(ENLYFQG) and cleaves between Gln and Gly [8]. The unusually longrecognition sequence makes this endopeptidase highly unique anduseful because the chance of unwanted cleavage within the target
protein at positions other than the engineered site is very low. For the
production of monoclonal antibodies recognizing this sequence,96Protein Expression and Purification 147 (2018) 94–99

S. Tabata et al.

Fig. 3. Structural mechanism of eTev peptide recognition by 2H5. (A) Overall crystal
structure of eTev tag peptide bound by 2H5 Fab. Structure is presented in a cartoon modelwith each chain diﬀerently colored in magenta (eTev), gold (2H5 heavy chain), and silver(2H5 light chain). Side chains for the eTev peptide are also shown in stick models. (B)Close-up view of the eTev-2H5 interface. eTev peptide is shown as in (A), and the residues
critical for the recognition are labeled. (C) Simulated model of the tandem double tag"cPAW″ simultaneously bound by 2H5 and NZ-1 Fab. The model was obtained bymanually aligning eTev-2H5 complex structure (this study) and previously determined
PA-NZ-1 complex structure (PDB ID:4YO0) side-by-side and the intervening heptapeptide
segment (GGGGSHM) was arbitrarily modeled as extended peptide. Note that a turnforming PA tag peptide (red) is bound at the interface between heavy (lightblue) and light(wheat) chains of NZ-1 Fab with the C-terminus exposed to solvent. The N-terminus of thecPAW would be fused to a protein of interest (POI) through a short linker segment (dottedline). (For interpretation of the references to color in this ﬁgure legend, the reader isreferred to the Web version of this article.)

Fig. 2. Alanine scanning mutagenesis. (A) Sequence speciﬁcity of the recognition by 2H5.
Mutant eTev-Fn proteins containing Ala-substitution at the indicated amino acid position
was coated onto the microtiter wells, and concentration-dependent binding of 2H5 IgG
was evaluated by direct ELISA as in Fig.1A. BSA-coated wells were used as background(BG) control. (B) Sequence speciﬁcity of recognition by TEV protease. The Ala-substituted
mutant eTev-Fn proteins were incubated with TEV protease at enzyme:substrate ratio of
1:10 for 16 h. Cleavage reaction was monitored by SDS-PAGE as the conversion of theeTev-Fn band migrating at 22 kDa (uncleaved) into a 19-kDa band (cleaved).

E2, N3, F6, and Q7 did not have any eﬀect, suggesting that the side
chains of these residues do not contribute to the binding. Ala substitution at M0, which was not present in the immunized peptide, did
not have any eﬀect, as expected. To our surprise, Ala substitution at
G11 in the context of eTev-Fn signiﬁcantly attenuated the binding, eventhough the immunized peptide had Cys at this position which was used
for the conjugation with KLH. Although the mechanism of the acquisition of G11 speciﬁcity by 2H5 is unclear, the above result deﬁned the
epitope structure required for the full reactivity with 2H5 as an 11residue segment containing the following sequence: RxxLYxxGKDG.
When we analyzed the susceptibility of each Ala mutant to the TEVprotease cleavage, L4, Y5, and Q7 were found to be important for therecognition by the enzyme (Fig. 2B). This result is in general agreementwith the TEV substrate speciﬁcity reported earlier [8] but is clearly
diﬀerent from the recognition speciﬁcity of the 2H5 antibody shown in
Fig. 2A, suggesting that the recognition mode for the antibody and the
enzyme are diﬀerent. Collectively, we succeeded in isolating a monoclonal antibody recognizing an 11-residue peptide including the TEVprotease recognition motif with a modest solution aﬃnity.

entire peptide was accommodated in the deep groove formed by the
CDR loops of both chains (Fig. 3B). The helical conformation of thebound eTev peptide explains why the recognition motif spans as long as
11-residue portion, with critical amino acids appearing in a periodical
fashion. In fact, all the residues determined as critical for the interaction by the alanine scanning experiments (i.e., R1, L4, Y5, G8, K9, andD10) are in direct contact with the antibody, while side chains of the
non-critical residues are all pointing away from the 2H5 surface(Fig. 3B). Although the N-terminus of the eTev peptide is well exposed
above the pocket and pointing outward, C-terminal end (residue 11) islocated at the bottom of the deep binding groove near the exit point,
explaining why glycine is preferred at this position. Nevertheless, bothends of the binding groove are open to the solution, suggesting thateTev tag can be placed in between two other components, namely, a
protein of interest and a second epitope tag.
2.3. Design of tandem double tag

2.2. Crystal structure of 2H5-eTev complex
If we are to use eTev tag in a “tandem double tagging” format
compatible with a sandwich ELISA, 2H5 and the other anti-tag antibody
have to bind to a stretch of peptide sequence simultaneously without
the steric occlusion by each other. Based on the structure of eTev-2H5complex described above, we predict that eTev tag and a neighboring
recognition sequence must be intervened by at least 6 (for N-terminalside) or 7 (for C-terminal side) residues of ﬂexible linker. As the second

In order to know the detail of the antigen recognition mechanism of
2H5 antibody, we crystallized the Fab fragment of 2H5 in complex withthe eTev peptide. The structure determined at 2.05 Å resolution revealed that the eTev peptide forms nearly 2-turns of α-helix in thebinding pocket between the H and L chains of 2H5 Fab (Fig. 3A). All of
the 11 residues can be clearly assigned in the electron density, and the97Protein Expression and Purification 147 (2018) 94–99

S. Tabata et al.

two tags in the linear sequence. We next went on to test if the tandem
double tagging can be applied to larger and more complex proteins. To
this end, cPAW tag was appended to human serum albumin (HSA,
70 kDa) or mouse neuropilin-1 ectodomain (Nrp-1ec, 95 kDa) (Fig. 4A).
These proteins were transiently expressed in HEK293 cells, puriﬁed
from the culture supernatants to homogeneity, and subjected to thesandwich ELISA as above. In spite of the large diﬀerence in their molecular property (size, multi-modular nature, and the presence of glycan
chains), all three exhibited quite similar concentration-dependency on
molar basis (Fig. 4D). This result not only conﬁrmed the universal applicability of the “tandem double tag” to a wide range of target molecules, but also suggested a possibility of using it for a quantitative
purpose in a standardized assay format.

functional unit that constitute tandem double tag along with eTev, wechose PA tag that had been developed in our group [9]. PA tag is adodecapeptide epitope tag with a sequence of GVAMPGAEDDVV, and isrecognized by an antibody NZ-1 with an extremely high aﬃnity. For an
N-terminal tagging, PA tag was followed by eTev tag with an intervening 6 residue linker, resulting in a 29-residue “nPAW” tag (GVAMPGAEDDVVGGGGNSRENLYFQGKDG). For a C-terminal tagging, PA tag
was preceded by eTev sequence with 7-residue linker, resulting in a 30residue “cPAW” tag (RENLYFQGKDGGGGGSHMGVAMPGAEDDVV).
Using the crystal structure of PA tag complexed with NZ-1 Fab that has
been determined in our group [18], we can simulate binding of both
2H5 and NZ-1 antibodies to the neighboring motifs presented in thecontext of either cPAW or nPAW tag. Resultant structural model revealed that the linker between PA and eTEV portions is predicted to belong enough to ensure wide separation of the two binding sites thatallows simultaneous binding of 2H5 and NZ-1 in cPAW (Fig. 3C) as wellas in nPAW (data not shown).

2.5. Application to a rapid screening of stable cell clone producing tagged
proteinPreparation of high-quality glycoprotein sample for structural analysis often demands establishment of stable cell line due to the low
production rate of mammalian cells as well as the low yield during the
puriﬁcation. As a “high producer cell line” can be obtained by chance, it
is important to perform large-scale screening of the transfected cells forthe expression level of the target protein. We applied the tandem
double tag strategy to our eﬀort to isolate stable cells expressing mouse
Wise protein. Wise (also known as USAG-1, Sostdc1, Ectodin) is an∼190-residue secreted glycoprotein known to antagonize BMP and Wnt
signaling pathway [19]. After stably transfecting Expi293F cells with
nPAW-Wise, it was possible to screen a large number of cell clones byusing the sandwich ELISA system established above in just one experiment (Fig. 5A). From the 21 cell clones shown highly positive in theﬁrst screening (shown red in Fig. 5A), four cell clones exhibited rapid
growth and sustained high expression of Wise (Fig. 5B). Finally, we
chose clone #29 for the large scale culture and protein production,

2.4. Sandwich ELISA
The compatibility of the tandem double tag with the sandwich
ELISA detection was assessed by using a model protein T4 lysozyme(T4L). To this end, nPAW-T4L and T4L-cPAW are constructed to containN-terminal His tag and expressed in E. coli cytoplasm (Fig. 4A). They
were puriﬁed using Ni-NTA resin, and subjected to a sandwich ELISA
system where NZ-1 and 2H5 were used as capturing and detection
antibodies, respectively (Fig. 4B). As shown in Fig. 4C, both nPAW-T4L
and T4L-cPAW in the test solution could be detected at concentration as
low as 0.5 nM (12.5 ng/ml), suggesting a full compatibility of these tagsin this sandwich ELISA. More importantly, both tagged versions produced similar dose-dependent curves saturating at > 10 nM. It is clear
from this result that the two antibodies can indeed bind to a single
polypeptide without mutual competition, regardless of the order of theFig. 4. Tandem double tag and sandwich ELISA.
(A) Design of ﬁve diﬀerent expression constructs
containing either N-terminal (nPAW) or C-terminal (cPAW) tandem double tag. Theoretical molecular size for each recombinant protein is shownin the parenthesis along with the expression host.
SS, signal sequence. (B) Schematic outline of the
sandwich ELISA procedure. (C,D) Concentration
dependency of various PAW-tagged proteins inthe sandwich ELISA system shown in (B). Signals
are shown as endpoint (T = 40 min) absorbanceat 405 nm after the subtraction of the value obtained with blank wells. Results are representative of three independent experiments
performed in duplicate.

98Protein Expression and Purification 147 (2018) 94–99

S. Tabata et al.

Fig. 5. Rapid establishment of stable cell line producing mouse Wise protein. (A) Culture supernatants from the single-colony wells were screened for the presence of nPAW-Wise using
the sandwich ELISA. Out of 90 clones, 21 were considered positive (showing > 4-fold absorbance value compared with the mock control, red bars) and four clones indicated by arrows
were chosen for further consideration based on their growth ﬁtness. (B) Conﬂuent culture supernatants from the four stable clones as well as the 4-day old culture media from thetransient transfection were subjected to NZ-1 pull-down. Shown is a 15% polyacrylamide SDS-PAGE gel run under nonreducing condition to evaluate the presence of PA-tagged Wise
protein (arrowhead). (C) Purity of the ﬁnal Wise preparation obtained from the cell clone #29. Samples (1 μg/lane) were analyzed by SDS-PAGE using 15% gel under nonreducing (NR)and reducing (R) conditions. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

References

resulting in a successful preparation of highly pure recombinant Wise
protein at an yield of ∼1 mg/l culture medium (Fig. 5C).

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3. Conclusions
In a recombinant production and subsequent puriﬁcation of biologically important proteins, availability of antibody-based detection andisolation tools are vital for the success of the project. When the target of
the investigation is a membrane protein expressed on cell surface,having one good antibody, either against the protein itself or the genetically fused tag portion, is suﬃcient for rapid detection and (semi-)
quantitation by the use of FACS analysis. For soluble proteins, however,more than one antibody is required to perform quantitative analysis byusing sandwich ELISA or like methods. The dual-function eTev peptide
reported here can be used as detection and puriﬁcation tag when simply
attached to a protein of interest (POI), but it is most useful whencombined with another epitope tag, as represented by the successful
creation of “tandem double tag” (Fig. 4). We succeeded in placing eTev
and PA tags in a stretch of ∼30-residue peptide using the three dimensional structure information about the tag peptide-antibody complex as a guide. When singly attached to N- or C-terminus of POI, thenPAW or cPAW tags enables easy detection and (semi)quantiﬁcation of
the protein through a generalized sandwich ELISA protocol. We predictthat this will greatly facilitate large-scale and eﬃcient screening of
stable cell clones that secrete high level of valuable proteins for which
no good antibodies are available. The hybridoma cell line 2H5 will bedeposited in public cell bank for the distribution upon request.

Acknowledgements
We would like to thank Drs. Kunio Hirata and Masaki Yamamoto at
SPring-8 BL32XU for their help with X-ray data collection. This work
was supported in part by MEXT KAKENHI Grant Number JP17H01420
from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT), and by the Platform Project for
Supporting Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science Research[BINDS]) from Japan Agency for Medical Research and Development(AMED) under grant numbers JP17am0101075 (to J.T.) and
JP17am0101078 (to Y.K.). This work was performed under the
Cooperative Research Program of Institute for Protein Research, Osaka
University (CR17-05).

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