J. Biochem. 2020;168(4):375–384 doi:10.1093/jb/mvaa054

Site-specific epitope insertion into recombinant proteins using
the MAP tag system
Received March 12, 2020; accepted April 24, 2020; published online May 9, 2020

Ayami Wakasa1, Mika K. Kaneko2,
Yukinari Kato2,3, Junichi Takagi1 and1,*Takao Arimori*Takao Arimori, Institute for Protein Research, Osaka University,
3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: þ81-6-68798608, Fax: þ81-6-6879-8609, email: arimori@protein.osaka-u.ac.jp

The MAP tag system comprises a 14-residue peptide
derived from mouse podoplanin and its high-afﬁnity
monoclonal antibody PMab-1. We determined the
crystal structure of PMab-1 complexed with the
MAP tag peptide and found that the recognition
required only the N-terminal 8 residues of MAP tag
sequence, enabling the shortening of the tag length
without losing the afﬁnity for PMab-1. Furthermore,
the structure illustrated that the MAP tag adopts a
U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume
conformation compatible with the PMab-1 binding.
We inserted the 8-residue MAP tag into multiple
loop regions in various proteins including ﬁbronectin
type III domain and G-protein-coupled receptors andtested if they maintain PMab-1 reactivity. Despite
the conformational restraints forced by the insertion
position, all MAP-inserted mutants were expressed
well in mammalian cells at levels comparable to the
non-tagged proteins. Furthermore, the binding by
PMab-1 was fully maintained even for the mutant
where MAP tag was inserted at a structurally
restricted b-hairpin, indicating that the MAP tag system has unique feature that allows placement in the
middle of protein domain at desired locations. Our
results indicate the versatile utility of the MAP tag
system in ‘site-speciﬁc epitope insertion’ application.
Keywords: antibody; epitope tag system; ﬂow
cytometry; G-protein-coupled receptor; X-ray
crystallography.
Abbreviations: CDR, complementarity-determining
region; cryo-EM, cryo-electron microscopy; PCR,
polymerase chain reaction; TBS, Tris-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco’s
Modiﬁed Eagle Medium; FCS, foetal calf serum;
GPCR, G-protein-coupled receptor; PAR, proteaseactivated receptor; PDB, Protein Data Bank.

C The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society.
V

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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1
Laboratory of Protein Synthesis and Expression, Institute forProtein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka
565-0871, Japan; 2Department of Antibody Drug Development,
Tohoku University Graduate School of Medicine and 3New
Industry Creation Hatchery Center, Tohoku University, 2-1
Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanAs recombinant DNA technology evolved, a numberof protein tagging systems have been developed forvarious purposes such as puriﬁcation, detection andquantiﬁcation of recombinant proteins (1). Amongthe tag systems, ‘epitope tag systems’ that utilize highafﬁnity and high-speciﬁcity binding between a peptide
tag and an anti-peptide monoclonal antibody are used
in a wide range of ﬁeld from cell biology to structural
biology. Since a short amino-acid sequence generally<20 residues is used as a tag sequence in epitope tag
systems, fusion of epitope tags have much smaller effect on the function and the folding of target molecules when compared with fusion of ‘protein tags’such as GST tag (26 kDa), MBP tag (43 kDa) andThioredoxin tag (12 kDa). However, attachment position of even a very short epitope tag is generally limited to either end of the target proteins (i.e. N- or Cterminus), since a loss of the reactivity by the monoclonal antibody or a disruption of a local conformation of a target molecule are expected when inserted
in the middle of folded domain. This is because mostepitope tags are recognized by their antibodies in an
extended conformation and thus difﬁcult to assume
antibody-reactive conformation when positions ofboth ends are restrained due to the insertion topology(2–4).
The PA tag system developed by our group is oneof the rare tag systems that are compatible with the insertion application. It utilizes a dodecapeptide (PAtag; GVAMPGAEDDVV) derived from human podoplanin, a type I transmembrane protein and a rat
monoclonal antibody (NZ-1) against it. NZ-1 binds to
the PA tag with extremely high afﬁnity (KD value:
4.0  1010 M) and high speciﬁcity, and the PA tag
system is applicable to various experiments including
afﬁnity puriﬁcation, Western blot and ﬂow cytometry(5). The crystal structure of the NZ-1 Fab in complex
with the PA tag peptide illustrated that the peptide
forms a type II b-turn in the antigen binding pocket,
where both the N- and C-termini of the peptide areprojecting outward from the antibody. This structuralfeature makes it possible to insert the PA tag into aturn-forming region of proteins without disrupting the
local conformation with its afﬁnity against NZ-1
maintained quite high (6).
The insertion compatibility of epitope tags offers
several advantages over other tag systems. One beneﬁt
is that they are applicable to proteins that do notallow fusing tags to either terminal ends. The terminal
regions of proteins are sometimes involved in the molecular functions or have limited accessibility fromantibodies. In such cases, the ‘internal tagging’ willoffer an alternative way of tag-based puriﬁcation anddetection. Further to that, internal tagging offers

A. Wakasa et al.

Materials and Methods
Preparation of PMab-1 Fv-clasp samples
Plasmids for bacterial expression of PMab-1 in the Fv-clasp(v2) format (non-tagged version), i.e. PMab-1VH(S112C)-SARAH and
PMab-1VL-SARAH(S37C), were prepared as previously describedusing cDNA clones of rat anti-MAP monoclonal antibody PMab-1(IgG2a, j) isolated previously (12, 13). N43Q and N54Q mutations
were introduced in the VH domain of the PMab-1VH(S112C)SARAH construct by a QuikChange mutagenesis, and the resultant

376

plasmid was used for production of the PMab-1(QQ) Fv-clasp mutant
in combination with the PMab-1VL-SARAH(S37C). Expression andpuriﬁcation of the Fv-clasp samples were carried out essentiallyaccording to the previously established sample preparation protocol(12), but with minor modiﬁcations. Namely, the denatured samples
after the cell lysis by sonication were diluted 20-fold in modiﬁed
refolding buffer (50 mM MES, 9.6 mM NaCl, 0.4 mM KCl, 2 mM
MgCl2, 0.5 M L-arginine, 1 mM reduced glutathione and 0.1 mM oxidized glutathione) in one step. After the refolding procedure, the samples were puriﬁed using a HiLoad 16/600 Superdex 200 pg column(GE Healthcare) and a Mono Q 5/50 GL column (GE Healthcare) as
previously described. To conﬁrm the antigen binding activities of the
puriﬁed samples, each sample with the amount shown in
Supplementary Fig. S1C was mixed with MAP14 peptide-conjugatedSepharose and incubated for 1 h at room temperature. After washing
three times with 20 mM Tris, pH 7.5 and 150 mM NaCl (Tris-bufferedsaline, TBS), bound proteins were eluted with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS–PAGE) sample buffer, subjected to 15% SDS–PAGE and stained with Coomassie Brilliant
Blue.
Structural analysis of PMab-1(QQ) Fv-clasp complexed with
the MAP peptide
PMab-1(QQ) Fv-clasp was concentrated to 10 mg/ml by ultraﬁltration using Amicon Ultra (Merck KGaA) and mixed with synthetic
MAP14 peptide to be the ﬁnal concentrations of 9 mg/ml PMab1(QQ) Fv-clasp (0.24 mM) and 1 mg/ml MAP14 peptide (0.7 mM).
Crystallization screening was carried out using JCSG-plus and
ProPlex (Molecular Dimensions) crystallization reagents by using thesitting-drop vapour diffusion method at 20 C, and crystals appeared
within a few days under 10 conditions in total. The crystals were subjected to X-ray diffraction experiment at beamline BL44XU ofSPring-8 (Hyogo, Japan). One crystal grown under the condition of
0.2 M potassium formate, 20% (w/v) PEG3350 (JCSG-plus, Tube 110) was especially diffracted well, and diffraction data were collected
at 100 K. The data were processed and scaled using X-ray Detector
Software (14). Initial phase was determined by molecular replacement with PHAER (15) from the CCP4 package (16) using the crystal structures deposited in Protein Data Bank (PDB) with IDs of
5w5z, 1lk3 and 5xct as search models for VH, VL and SARAH
domains, respectively. The structural models were modiﬁed with
COOT software (17) with model reﬁnement cycle with PHENIX(18). Data collection statistics and reﬁnement parameters are summarized in Table I.
Pull-down assays
The expression construct for Fn10-Fc contained DNA segments
coding for residues 1,417–1,509 of human ﬁbronectin followed by
Tobacco Etch Virus protease cleavage sequence and human IgG1
hinge-Fc and was made in pcDNA3.1 (Thermo Fisher Scientiﬁc)based vector containing a bovine prolactin signal sequence.
Placements of the MAP tag sequence at sites shown in Fig. 2B wereconducted by extension polymerase chain reaction (PCR). Alaninesubstituted mutants of Fn10-Fc were prepared by QuikChange
strategy. Each plasmid was transiently transfected into Expi293F
cells (Thermo Fisher Scientiﬁc) according to the manufacturer’s
protocol. Culture supernatants were harvested 4 days after the transfection and subjected to the pull-down assays using PMab-1immobilized Sepharose or rProtein A Sepharose (GE Healthcare).
After washing three times with TBS, bound proteins were eluted
from the beads with SDS–PAGE sample buffer, subjected to 12.5%
SDS–PAGE and stained with Coomassie Brilliant Blue.
Flow cytometry
DNA segments coding for protease activated receptors (PARs), i.e.
residues 36–426 of PAR1 and residues 25–385 of PAR4, were cloned
into a phCMV3 (Genlantis)-based vector containing a bovine prolactin signal sequence and a N-terminal FLAG tag. The MAP tag
was inserted into sites shown in Fig. 3B by inverse PCR. Expi293F
cells were transiently transfected with each plasmid. Cells were harvested after 48 h of transfection and incubated with the anti-FLAG
M2 antibody (Sigma-Aldrich, F3165) diluted at 1:500 in Dulbecco’s
Modiﬁed Eagle Medium (DMEM) with 10% foetal calf serum(FCS) or 1.0 lg/ml PMab-1 in DMEM/FCS for 2 h on ice. The cells
were then washed twice with DMEM/FCS and resuspended in
DMEM/FCS containing Alexa-Fluor-488-conjugated goat anti-

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more options in choosing the tagging sites on a protein, especially when special tag location is desired dueto the experimental requirement. For example, theinsertion-compatible PA tag has been introduced into
multiple locations of a polymorphic cell adhesion receptor aIIbb3 integrin, enabling the detailed analysis
of its conformational states during the afﬁnity modulation (6). PA tag was also used to achieve domain/
subunit identiﬁcation of multi-module proteins by visualizing the inserted tag location with Fab fragment
of NZ-1 antibody via electron microscopy (7, 8).
Although the ‘epitope insertion’ would lead to a signiﬁcant reduction in labour and cost to obtain amonoclonal antibody required for achieving speciﬁc
purposes, we do not have much options for the tag
systems compatible with this application other than
the PA tag. Furthermore, the PA tag system has one
serious drawback that precludes its application in certain experiments; due to the reactivity of the NZ-1
antibody against human podoplanin which is widely
expressed on many human cell lines, it cannot be usedin detecting tagged proteins expressed on human cellsurface.
In the present study, we discovered that the MAP
tag system is also compatible with the insertion application. The MAP tag system has been developed using
a 14-residue peptide (MAP tag; GDGMVPPGIEDKIT) derived from mouse podoplanin and a rat
IgG2a antibody against it (PMab-1) (9, 10). PMab-1
shows high speciﬁcity and high afﬁnity towards the
MAP tag with a KD value of 3.7  109 M. Similar to
the PA tag system, the MAP tag system can beapplied to a wide range of experiments. Althoughboth the MAP tag and the PA tag are derived frompodoplanin molecules, PMab-1 and NZ-1 show species speciﬁc podoplanin recognition and do not crossreact with podoplanin from different animals (11).
This makes the MAP tag system an ideal tool to detect tagged proteins on human cell surfaces.
Here, we conducted a structural analysis of PMab-1
complexed with the MAP tag peptide by utilizing a
hyper-crystallizable ‘Fv-clasp’ format developed recently in our group (12). The 2.49 Å resolution crystal
structure of PMab-1 Fv-clasp revealed recognition
mechanism of MAP tag peptide, suggesting that the
tag sequence can be shortened to 8 residues from its
original 14 residues and is compatible with the insertion into loop regions. We further demonstrated thatthe 8-residue MAP tag was successfully used to label
cell surface G-protein-coupled receptors (GPCRs)
that cannot be tagged at either termini due to thefunctional and topological requirements, by inserting
into two short extracellular loops.

A new application of the MAP tag; insertion into loops

A

MAP peptide

B

Gln54

VL

VH

Fv

VH

VL

Gln43

C

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SARAH

D

Gly8

His35(H)
Gly1

VL

VH

Gly8
Asn96(L)Trp99(H)MAP peptide

Asp2

Val5

Met4

Gly1
His34(L)E

F

MAP peptide
C
Gly1

VH
Met4

VL

N

13.5 ÅGly8

MAP peptide

Fig. 1. Crystal structure of the PMab-1(QQ) Fv-clasp in complex with the MAP tag peptide. (A) Overall structure of the PMab-1(QQ) Fvclasp-MAP tag peptide complex. The VH-SARAH and the VL-SARAH are shown as cartoon presentation. The bound MAP tag peptide isshown as a stick model. The two substituted Gln residues, Gln43 and Gln54, in the VH domain are shown as sphere models. (B)Comparison of the Fv structures between the two PMab-1(QQ) Fv-clasp molecules in the asymmetric unit. The Fv region of the Mol-2(grey) is superposed on that of the Mol-1 (coloured), and they are shown in Ca tracing. (C and D) The expanded views of the antigen binding pocket of PMab-1. PMab-1 is shown as a surface model, and the MAP tag peptide is shown as a stick model with a transparent spheremodel (C). PMab-1 residues involved in the MAP tag peptide recognition are shown as stick models, and hydrogen bonds are denoted bydashed lines (D). (E) Sliced-surface view at the antigen binding pocket. Note that the side chain of Met4 of the MAP tag peptide is deeply
inserted into a cavity formed between the VH and the VL. (F) U-shaped conformation of the MAP tag peptide bound by PMab-1. The MAP
tag peptide is presented as a worm model. The Ca atoms of Gly1 and Gly8 are shown as sphere models.

377

A. Wakasa et al.
Table I. Data collection and reﬁnement statistics
PMab-1 Fv-clasp/MAP (PDB ID: 6lz4)
P21212167.7, 85.1, 115.7
48.15–2.49 (2.64–2.49)a
0.13 (1.42)
11.55 (1.37)
0.998 (0.650)
99.3 (96.0)
8.7 (9.0)
42.5–2.49
23,78724.5/26.9
5,081
94
79.8
71.4
0.005
0.830

Note: A single crystal was used for the structure.
Values in parentheses are statistics of the highest-resolution
shell.
amouse IgG (Thermo Fisher Scientiﬁc, A11029) diluted at 1:500 (forM2) or Alexa-Fluor-488-conjugated goat anti-rat IgG (Thermo
Fisher Scientiﬁc, A11006) diluted at 1:500 (for PMab-1). After
30 min incubation on ice, the cells were washed twice and suspended
in phosphate-buffered saline (pH 7.4) and analysed on a ﬂow
cytometer EC800 system (Sony). The data were analysed with
FlowJo software (BD).

Results
Crystallographic analysis of the PMab-1 Fv-clasp incomplex with the MAP tag peptide

In order to facilitate the crystallization of PMab-1, we
ﬁrst converted it to a small antibody fragment format‘Fv-clasp’ developed recently (12). Fv-clasp is a fusionof an anti-parallel coiled-coil structure derived fromthe human Mst1 SARAH domain to the antibody
variable region (i.e. VH and VL domains) and known
to exhibit very high crystallization tendency comparedto the corresponding Fab fragment. Additionally, wemutated two asparagine residues (Asn43 and Asn54)
in the VH domain to glutamine [PMab-1(QQ) Fvclasp] because they constitute ‘Asn-Gly’ sequences.
The asparagine residue in this sequence is prone to thespontaneous deamidation reaction under high-pH andhigh-temperature conditions, introducing a chemical
heterogeneity in the crystallization sample (19).
We conﬁrmed that the mutation eliminated the appearance of the deamidated protein peak in the anion
exchange chromatography, without affecting the apparent antigen binding activity (Supplementary
Fig. S1).
Crystallization screening of the PMab-1(QQ) Fvclasp was carried out in the presence of excess amountof the 14-residue MAP tag peptide, and crystals
appeared under many conditions. The crystals
obtained in this initial screening were directly
378

Optimization of the MAP tag sequence in length

The fact that the C-terminal half of the MAP tag peptide was disordered and not involved in the recognition by PMab-1 points to the possibility that the
MAP tag sequence can be shortened without reducing
the afﬁnity towards PMab-1. From the structure, theﬁrst 7 residues are expected to be essential for therecognition. As to the Gly8, corresponding electron
density was of poor quality (in Mol-1) or invisible (inMol-2), so it is possible that Gly8 has little or no

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Data collection
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym
I/rI
CC1/2
Completeness (%)
Redundancy
Reﬁnement
Resolution (Å)
No. of reﬂections
Rwork/Rfree (%)
No. of atoms
Protein
PeptidesB-factors
Proteins
PeptidesR.m.s. deviations
Bond lengths (Å)
Bond angles ( )subjected to X-ray diffraction experiments without
further optimization, and one of the crystals diffracted
to 2.49 Å resolution (Table I). The crystal contained
two PMab-1(QQ) Fv-clasp molecules in the asymmetric unit (Mol-1 and Mol-2), and extra electron densities were found in the antigen binding pocket ofboth molecules after model building of the Fv-clasp
region. We carefully analysed the electron densities
and ﬁnally assigned the peptide sequence from the Nterminal 8 and 7 residues into the densities of Mol-1
and Mol-2, respectively (Fig. 1A and Supplementary
Fig. S2). The Fv region structure of the two moleculesin the asymmetric unit is nearly identical, with rootmean-square deviation value of 0.47 Å for superposed
210 Ca atoms, and notable difference was notobserved even in the antigen binding sites (Fig. 1B).
Because the overall electron density of Mol-1 is slightly better than that of Mol-2, we will use the Mol-1
model in the following discussion unless otherwise
speciﬁed.
The MAP tag peptide ﬁts into the PMab-1 antigenbinding groove formed between the VH and VL
domains (Fig. 1C). The substituted glutamine residues(Gln43 and Gln54) are located far from the antigen
binding site, consistent with the lack of functional effect of these mutations (Fig. 1A and Supplementary
Fig. S1C). Unlike typical antigen binding mode ofsequence-speciﬁc anti-peptide antibodies, the peptide
recognition by residues in the complementaritydetermining region (CDR) of PMab-1 involved many
interactions with the main chain portion of the MAP
tag peptide (Fig. 1D), except for the side chains of justtwo residues, Asp2 and Met4. The side chain of Asp2
forms an intermolecular hydrogen bond with the side
chain of His35 in the CDR-H1 of PMab-1 (Fig. 1D).
The side chain of Met4 is completely buried in the
antibody molecule by inserting it into the hydrophobic
cave at the centre of the antigen-binding groove(Fig. 1E). Furthermore, Asp2 side chain makes anintramolecular hydrogen bond with the main chain
amide of Met4, stabilizing the compact conformationof the Asp-Gly-Met tripeptide motif to ensure its accommodation in the small binding pocket. Theseobservations strongly suggest that Asp2 and Met4 but
not other residues in the MAP tag play critical rolesin the sequence-speciﬁc binding by PMab-1. Anotherintriguing observation in the crystal structure is that
the MAP tag peptide adopts a U-shaped conformation where the both terminal ends are exposed tosolvent (Fig. 1F), suggesting that the MAP tag is compatible with the insertion into loop regions as in thecase of the PA tag.

A new application of the MAP tag; insertion into loops

contribution to the PMab-1 binding (Supplementary
Fig. S2). Nevertheless, we chose the 8-residue portion
as the new shortened version of MAP tag (MAP8 tag;
GDGMVPPG), hoping that the last Gly may contribute favourably to the recognition by functioning as aspacer. First, we assessed the function of the MAP8
tag by attaching it at the N-terminal of a model

protein, the tenth ﬁbronectin type III domain ofhuman ﬁbronectin (Fn10). Fn10 is a well-folded small
protein (10 kDa) with a b-sandwich structure composed of seven strands and is frequently used as a
scaffold for studies on protein engineering, biophysics
and protein folding (Fig. 2A) (20, 21). For efﬁcient expression in mammalian cells and easy detection by the379

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Fig. 2. The MAP tag can be inserted into the long protruded loop of Fn10. (A) Fn10 structure extracted from the crystal structure of humanﬁbronectin fragment (PDB ID: 1fnf) is shown as a cartoon model. The N- and C-termini of Fn10 are shown as spheres. The seven strands
forming the b-sandwich structure of Fn10 are labelled with A–G. The expanded view of the FG loop shown as stick models is provided in
the inset. (B) The amino-acid sequences for N-terminally MAP-tagged or MAP-inserted Fn10-Fc mutants near the MAP tag-fused portions.
The MAP tag-derived sequences and linker residues are underlined. Residue numbers of original Fn10 are provided above the sequences. (Cand D) Pull-down assay of the MAP-tagged Fn10-Fc by PMab-1-immobilized Sepharose (upper panel) and rProtein A Sepharose (lower
panel). The Fn10-Fc samples were expressed in Expi293F cells and precipitated with each resin, followed by 12.5% SDS–PAGE under nonreducing conditions and stained with Coomassie Brilliant Blue. All mutants were expressed well in Expi293F cells as conﬁrmed by therProtein A Sepharose pull-down assay (C, D, lower panels). Note that the nMAP8, iFG and iFG-G samples were captured by PMab-1 withequivalent efﬁciency with the nMAP14 sample (C, top panel) and that the D2A and M4A mutants showed severe decrease in binding afﬁnity
for PMab-1 (D, top panel).

A. Wakasa et al.

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Fig. 3. The MAP-inserted GPCRs expressing on human cells were efﬁciently stained with PMab-1 in ﬂow cytometry analysis. (A) Overall structure of PAR1. The crystal structure of PAR1 was determined as a T4 lysozyme-fused mutant (PDB ID: 3vw7), and the ﬁgure was drawn as
a cartoon model after eliminating the T4 lysozyme portion from the model. The expanded view at the MAP tag insertion regions is shownin the right. (B) The amino-acid sequences for MAP-inserted PAR1 and PAR4 mutants near the insertion portions. The MAP8 tag sequenceand linker residues are underlined. Residue numbers of the original receptors are provided above the sequences. (C) Flow cytometry analysisof MAP-inserted PAR1 and PAR4 mutants. PAR1 and PAR4 mutants were transiently expressed on Expi293F cells and incubated with
PMab-1 or anti-FLAG antibody M2, followed by staining with corresponding Alexa-Fluor-488-labelled secondary antibodies. Signals from
cells transfected with each sample and not treated with any antibodies (grey area) were used to deﬁne PMab-1- or FLAG-positive cell populations (regions including <1% of non-treated cells were indicated by the brackets and deﬁned as the positive region for each antibody).
Staining efﬁciencies are expressed as the % positive cells and provided in the each panel.

380

A new application of the MAP tag; insertion into loops

Insertion of the MAP8 tag into an extended loop
region of Fn10

Next, we tested if the MAP8 tag can be inserted into aloop region of a protein. The long and protruding FG
loop of Fn10 was chosen as an insertion point because
it is known to tolerate various sequence modiﬁcations(Fig. 2A) (22, 23). We designed a mutant of Fn10-Fc
by inserting the MAP8 tag between Gly1494 andSer1496 after removing Asp1495 located at the tip of
the FG loop (Fn10-iFG8, Fig. 2B). In the MAP–
PMab-1 complex structure, the distance between the
Gly1 and Gly8 is 13.5 Å (Fig. 1F). As the Ca atoms of
Gly1494 and Ser1496 are separated by only 5.5 Å in
the Fn10 structure (PDB ID: 1fnf; Fig. 2A), wethought that the MAP8 insertion may cause disruption of the local conformation of Fn10 and decrease
the binding afﬁnity of the inserted tag towards PMab1. Thus, we included another mutant where theinserted MAP8 was ﬂanked by extra Gly residues atboth ends (Fn10-iFG-G, Fig. 2B). Both Fn10-iFG
and Fn10-iFG-G were expressed well at levels equivalent to the non-tagged Fn10-Fc, indicating that the insertion of the MAP tag sequence did not affect the
Fn10 structure (Fig. 2C, bottom, lanes 2, 5 and 6).
Moreover, as judged by the pull-down assay using
PMab-1-immobilized Sepharose, bindings of both
MAP8-insertion mutants were indistinguishable fromthat of N-terminally MAP-tagged samples (Fig. 2C,
top, lanes 3–6), indicating that the MAP8 tag inserted
at a loop remains fully functional even without any
linker residues, as long as the loop is long enough.
Alanine scanning of the MAP tag sequence

For a detailed investigation of the MAP tag recognition mechanism by PMab-1, each residue in the
MAP8 tag sequence was individually mutated to alanine (G1A, D2A, G3A, M4A, V5A, P6A, P7A andG8A mutants) in the context of Fn10-iFG, and thebinding of the mutants to PMab-1 was evaluated by
the pull-down assay. As a result, the D2A mutant
showed a marked decrease in the binding activity to
PMab-1, and the M4A mutant completely lost the reactivity (Fig. 2D). On the other hand, none of othermutants showed appreciable decrease in the binding.
This result is fully consistent with the structural analysis of the complex described earlier, and agrees withthe previous study that evaluated the PMab-1 binding
towards Ala-substituted full-length mouse podoplanin
expressed on cell surface by ﬂow cytometry (24).
Thus, we concluded that Asp2 and Met4 in the MAP
tag sequence are irreplaceable residues in this tag
system.
MAP8 tag insertion into the surface-exposed loop of
GPCRs

The successful insertion of the MAP8 tag into the protruding loop of Fn10 implies that the inserted MAP
tag formed the U-shaped conformation as observed in
the crystal structure without disrupting the conformation of Fn10. This may not be surprising, however,
because Fn10 is stable enough to withstand artiﬁcial
modiﬁcations at the FG loop. To demonstrate that
the MAP8 tag has broad utility in the loop-insertion
applications, we chose GPCRs, the largest family of
membrane receptors that present major challenges
during the expression, puriﬁcation and structural
analyses for drug discovery, as the insertion target.
GPCRs are seven-transmembrane receptors that havethree extracellular loops (ECL1–3) and three intracellular loops (ICL1–3) within the molecules. The C-terminus of GPCRs is located on the cytoplasmic side
where antibodies cannot access from outside the cell.
Therefore, a tag should be fused to the extracellular
N-terminal side when detection of cell surface GPCRs
is required. However, the N-terminal region of certain
GPCRs cannot be modiﬁed for functional reasons.
Such case is exempliﬁed by the GPCR subclass called
PARs. Two representative PARs, PAR1 and PAR4,
are activated by a proteolytic digestion at the N-terminal region (i.e. between Arg41 and Ser42 of PAR1and between Arg47 and Gly48 of PAR4) by thrombin,
and the newly exposed N-termini after the cleavage
behave as tethered ligands that activate the receptors
for the downstream signalling (Supplementary Fig.
S3) (25). Because of this nature, the N-terminal tagging cannot be accepted in PARs, especially when the
post-cleavage receptor is to be analysed. We decided
to test MAP8’s insertion utility by using PAR1 andPAR4 as the target proteins. The crystal structure ofPAR1 but not PAR4 is available (Fig. 3A) (26).
In PAR1, the ECL3 connecting the C-terminus of thetransmembrane helix 6 and the N-terminus of thetransmembrane helix 7 is rather long and largely
exposed, especially at the side chains of Ser341 and
His342 (Fig. 3A). We ﬁrst tested if the MAP8 tag canbe inserted into this region of PAR1 (PAR1-iECL3)
and the corresponding site of PAR4 (between
Ser311 and Pro312, PAR4-iECL3) (Fig. 3B and
Supplementary Fig. S3). Since the biological activitiesof the receptors were not to be evaluated in this experiment, we fused the FLAG tag to the N-terminusof all samples as a control tag to monitor the expression proﬁles. The receptors were expressed on the surface of Expi293F cells and analysed for the PMab-1
reactivity by ﬂow cytometry. As shown in Fig. 3C,
upper panels, expression proﬁles of the MAP-inserted
receptors were essentially the same as those of their respective control receptors that does not have the
MAP tag, suggesting that the MAP tag insertion did
381

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pull-down assays, we fused the human IgG1 Fc domain to the C-terminus of Fn10 (called Fn10-Fc hereafter). Either the MAP8 or the original 14-residue
MAP tags were fused to the N-terminus of Fn10-Fc(Fn10-nMAP8 and Fn10-nMAP14; Fig. 2B). The proteins were expressed in Expi293F cells, and their expression levels and PMab-1 reactivities were analysedby the pull-down assay from the conditioned mediausing rProtein A Sepharose and PMab-1-immobilized
Sepharose, respectively. Fn10-Fc forms a dimer via disulphide bonds and migrates as a single band of
90 kDa in non-reducing SDS–PAGE gel. As shownin Fig. 2C, lanes 2–4, Fn10-nMAP8 showed binding
ability comparable to Fn10-nMAP14 against PMab-1,
indicating that the 8-residue MAP tag sequence islong enough for the full PMab-1 binding.

A. Wakasa et al.

Discussion
In the present study, we showed that the MAP tag
shortened to 8 residues (i.e. MAP8 tag) is fully functional and Asp2 and Met4 of the MAP8 tag are critical for the binding. It is noteworthy that all residues
consisting the MAP8 tag except for Asp2 and Met4
are small amino acids. In our crystal structure, theMAP8 tag peptide ﬁts tightly in the small antigen
binding pocket of PMab-1, and there is no room toaccommodate larger amino acids in the pocket(Fig. 1C). Therefore, the MAP tag seems to achieve
its high sequence speciﬁcity by using many ‘featureless’ amino acids rather than an elaborate combination of large side chains, which is typical for manyanti-peptide antibodies. Although only a short segment of the MAP8 tag is recognized by PMab-1, allresidues are efﬁciently used to constitute the binding
epitope that spans relatively large contact area, which
warrants high afﬁnity. In fact, the contact area on the
7-residue MAP tag (excluding the Gly8 that does notseem to contribute much to the interface) is 661.0 Å2,
accounting for 75% of the total surface area of theentire peptide, which is comparable to that between
the 9-residue HA tag and its monoclonal antibody
12CA5 (675.7 Å2, which corresponds to 49% of thetotal surface area of the HA peptide). In addition, therelatively low total chemical complexity of the aminoacids found in the MAP8 tag sequence is expected tohave less deteriorating effects on the functional andphysical properties of target molecules.
The most important ﬁnding in this study is the factthat the MAP8 tag can be inserted into loop regions382

of proteins. Since small difference in PMab-1 binding
efﬁciency was observed among the PAR1-iECL2
mutants depending on the linker length (Fig. 3C),
linker optimization may be sought for each insertion
design to achieve the maximum reactivity when inserting the MAP8 tag into loops. Nevertheless, it is also
notable that all MAP-inserted mutants tested in thisstudy showed sufﬁcient sensitivity to PMab-1 for detection even without any linker. As we did not see difference in the PMab-1 reactivity against terminallyadded MAP tag and inserted ones (Fig. 2C), the afﬁnity difference between the two different tag presentation modes seems to be negligible, as long as theyremain fully accessible to the antibody. This loopinsertion compatibility of the MAP tag system
expands its utility, especially in the structural biology
ﬁeld. In X-ray crystallography, antibody fragments
are often used as crystallization chaperones which facilitate the crystallization of target molecules byimproving structural homogeneity and providing lattice packing interactions (27–29). As monoclonal antibodies that reduce structural ﬂexibility upon binding
to the native three-dimensional epitope on the target
molecule must be used in the crystallization chaperone
application; however, it is generally necessary to
screen for large number of available antibodies or prepare new antibody for this purpose only. Recently,
Tamura et al. succeeded in utilizing the PA tag system
as a crystallization chaperone, by inserting the PA tag
into a b-hairpin region of a bacterial Site-2 proteaseand making complex with anti-PA NZ-1 antibody
Fab fragment. The resolution of the diffraction datafrom the NZ-1-bound PA-inserted sample was
improved to 2.0 Å compared to the native (i.e. nontagged) sample that diffracted to 2.8 Å resolution (30),suggesting that the bound Fab assisted the formationof the well-ordered crystals. Thus, we expect that the‘epitope insertion’ or ‘epitope grafting’ may become apopular method in the chaperone-assisted crystallography, because the establishment of a monoclonal
antibody for each target molecule is no longer necessary. Furthermore, this method can greatly simplify
the experimental workﬂow because both protein puriﬁcation and chaperone-assisted crystallization areachieved with a single tag system, and the enzymatic
treatment for removing the tag before crystallization
can be omitted.
The ‘epitope grafting’ would also be useful for highresolution structural analysis via cryo-electron microscopy (cryo-EM). Due to the recent advances in the
cryo-EM methodology, determination of near-atomic
resolution three-dimensional structures of macromolecules that are biologically important and difﬁcult tocrystallize is reported one after another. However,high resolution cryo-EM analysis of structurally ﬂexible, small in size (<100 kDa) and unstable samples isstill considered as a challenging task. To address these
problems, many researchers use antibody fragmentswith the hope of improving structural homogeneity,
increasing size and improving stability of target molecules (31–35). The bound antibody fragments also
serve as a ﬁducial marker to facilitate particle alignment. Therefore, PMab-1 Fv-clasp or Fab fragment

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not grossly affect the structural integrity of these
GPCRs. More importantly, MAP8 tag inserted into
the ECL3 was recognized well by PMab-1, as evident
from the strong staining of cells transfected with thesereceptors (Fig. 3C, lower panels). It is particularly
notable that even the MAP8 tag inserted into ECL3
of PAR4, which was designed without an experimental structure of PAR4, was efﬁciently recognized by
PMab-1, indicating that the precise structural information is not always necessary for designing MAP tag
insertion constructs.
We next investigated if the MAP tag can be inserted
into more structurally restricted region, a b-hairpin.
ECL2 of PAR1 assumes a b-hairpin structure with the
distance between the Ca atoms of Pro247 and Leu249
being 5.6 Å (Fig. 3A). In contrast to the long and mobile Fn10 FG loop, the b-hairpin of the ECL2 would
force close apposition of the termini of the MAP8
peptide when inserted, which may result in the conformation incompatible with the PMab-1. Therefore,
we inserted MAP8 tag between Pro247 and Leu249 ofPAR1 with 0, 1 and 2 spacer residues at both ends
(Fig. 3B). Again all mutants were expressed well regardless the linker length (Fig. 3C, upper panels) and,surprisingly, all insertion mutants including the plain
MAP8 insertion (i.e. no spacers) were recognized by
PMab-1 (Fig. 3C, lower panels), indicating that the
MAP8 tag retains high reactivity to PMab-1 evenwhen inserted into a very tight hairpin of GPCRs.

A new application of the MAP tag; insertion into loops

Supplementary Data
Supplementary Data are available at JB Online.

Acknowledgements
We would like to thank Rebecca Budde for performing preliminary alanine scanning experiment and the staff in BL44XU, SPring8 for their help with X-ray data collection.

Funding
This work was supported in part by the JSPS KAKENHI grant
number JP18H02389 from Japan Society for the Promotion of
Science to T.A. and by the Platform Project for Supporting Drug
Discovery and Life Science Research (Basis for Innovative Drug
Discovery and Life Science Research) from Japan Agency for
Medical Research and Development under grant numbers
JP19am0101078 to Y.K. and JP19am0101075 to J.T. This work
was performed under the Cooperative Research Program ofInstitute for Protein Research, Osaka University (CR18-05).

Conflict of Interest
The authors have no conflict of interest to declare.

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