MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 6, 2016
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0039

MAP Tag:
A Novel Tagging System for Protein
Purification and Detection
Yuki Fujii, Mika K. Kaneko, and Yukinari Kato

Protein purification is an essential procedure in fields such as biochemistry, molecular biology, and biophysics.
Acquiring target proteins with high quality and purity is still difficult, although several tag systems have been
established for protein purification. Affinity tag systems are excellent because they possess high affinity and specificity for acquiring the target proteins. Nevertheless, further affinity tag systems are needed to compensate for several
disadvantages of the presently available affinity tag systems. Herein, we developed a novel affinity tag systemdesignated as the MAP tag system. This system is composed of a rat anti-mouse podoplanin monoclonal antibody(clone PMab-1) and MAP tag (GDGMVPPGIEDK) derived from the platelet aggregation-stimulating domain ofmouse podoplanin. PMab-1 possesses high affinity and specificity for the MAP tag, and the PMab-1/MAP tag
complex dissociates in the presence of the epitope peptide, indicating that the MAP tag system is suitable for protein
purification. We successfully purified several proteins, including a nuclear protein, soluble proteins, and a membraneprotein using the MAP tag system. The MAP tag system is very useful not only for protein purification but also inprotein detection systems such as western blot and flow cytometric analyses. Taken together, these findings indicatethat the MAP tag system could be a powerful tool for protein purification and detection.
Keywords: monoclonal antibody, MAP tag, affinity tag, protein purification

IntroductionA

ffinity tag systems are highly useful for protein purification, detection, and analysis. Affinity tag systems areclassified broadly as ‘‘peptide tags’’ and ‘‘protein tags.’’ For
example, GST tag(1) and MBP tag(2) are protein tags. While thesetags are useful for soluble fraction proteins, they are large (over25 kDa) and thus can affect target protein function, requiring
their removal before protein analysis. In contrast, peptide
tags are less likely to affect the structure and function oftarget proteins because they are small (typically 1–2 kDa).
Thus, removal of the tag portion is not always required
before protein analysis. The most appropriate peptide tag
system can be chosen from a variety of options, including
the His tag,(3) FLAG tag,(4) TARGET tag,(5) PA tag,(6,7)
AGIA tag,(8) and many others.(9,10) The fusion peptides arecompatible with virtually all expression host cells, including yeast, insect, and mammalian cells, and Escherichia coli
and can be expressed extracellularly or intracellularly.
An excellent affinity tag system should have high affinity
and high specificity. However, not all peptide-based tag sys-

tems meet these criteria. For example, the purification ofoligohistidine-tagged proteins using metal chelate affinity resin
often results in the co-purification of metal-binding proteinspresent in the starting material, necessitating further purification steps.(11) Generally, epitope tag systems that utilize peptide
tags and anti-peptide monoclonal antibodies (mAbs) are highly
specific. However, we often encounter nonspecific binding ofmAbs to endogenous proteins in certain cell types,(12,13) evenwhen using the most popular tag system, FLAG tag/anti-FLAG
M2 antibody.(14) Importantly, the most suitable tag systems
must be chosen based on the target protein, expression host, andmany other variables. Therefore, the development of further
affinity tag systems is needed to overcome the disadvantages ofavailable affinity tag systems.
We previously established a useful rat mAb (clone
PMab-1) against a 14-residue peptide in the platelet
aggregation-stimulating (PLAG) domain of mouse podoplanin.(15) Podoplanin is a type I transmembrane protein that is
highly expressed in malignant cancer cells and is implicated intumor-induced platelet aggregation.(16) In our another study,
we developed the PA tag system with high affinity andDepartment of Regional Innovation, Tohoku University Graduate School of Medicine, Miyagi, Japan.
ª Yuki Fujii et al., 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly credited.

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specificity.(6) The PA tag is derived from the human podoplanin PLAG domain. Three tandem repeats of the PLAG
domain are conserved in podoplanin orthologs of therat, hamster, dog, cow, human, and mouse.(17) Additionally,
PMab-1 possesses high affinity and specificity toward mouse
podoplanin.(18) Therefore, it was predicted that PMab-1
would have characteristics suitable for an anti-tag antibody.
Here, we report the development of a novel affinity tag
system comprising PMab-1 and its epitope peptide MAP tag.
Materials and Methods
Cell lines

LN229, HEK293T, COS-7, and Chinese hamster ovary(CHO)-K1 cell lines were purchased from the American
Type Culture Collection (ATCC, Manassas, VA). LN229
was transfected with epidermal growth factor receptor(EGFR), the entire ectodomain of human EGFR (EGFRec),the entire ectodomain of human HER2 (HER2ec), and CD133
plasmids (LN229/EGFR, LN229/EGFRec, LN229/HER2ec,
and LN229/CD133, respectively) using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA).
CHO-K1 was transfected with CD133 plasmid (CHO/
CD133) using Lipofectamine LTX (Thermo Fisher Scientific,Inc.). HEK293T, COS-7, and CHO-K1 cells were transiently
transfected with the hPDPNdN55 plasmid (HEK293T/
hPDPNdN55, COS-7/hPDPNdN55, and CHO/hPDPNdN55,
respectively) using Lipofectamine LTX. LN229, HEK293T,
COS-7, LN229/EGFR, LN229/EGFRec, LN229/HER2ec, and
LN229/CD133 cells were cultured in Dulbecco’s modified
Eagle’s medium including 2 mM l-glutamine (Nacalai Tesque, Inc., Kyoto, Japan). CHO-K1 and CHO/CD133 were
cultured in RPMI 1640 medium including 2 mM l-glutamine(Nacalai Tesque, Inc.), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL
penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified atmosphereof 5% CO2.
Plasmid preparation

Human ATRX cDNA encoding amino acids 2273–2413
was obtained by polymerase chain reaction (PCR) using
a cDNA derived from human lung as a template.(19) DNA
encoding the PA tag (GVAMPGAEDDVV), RAP tag(DMVNPGLEDRIE), and MAP tag (GDGMVPPGIEDK)
was inserted into the NdeI-XhoI site of pET21b vector (Novagen; EMD Millipore Corp., Billerica, MA) using the
In-Fusion PCR cloning kit (Takara Bio, Inc., Shiga, Japan)(PA-RAP-MAP/pET21b vector). The expression construct
for recombinant ATRX (amino acids 2273–2413) was cloned
into the EcoRI site of PA-RAP-MAP/pET21b vector (PAATRXepi-RAP-MAP/pET21b). The DNAs encoding human
EGFR, human HER2, and human CD133 were obtained by
PCR using cDNAs derived from A431, A172, and HCT116
as a template, respectively. RAP tag and MAP tag were inserted into the BamHI-NotI sites of pCAG PA tag-N vector(Wako Pure Chemical Industries Ltd., Osaka, Japan) using
the In-Fusion PCR cloning kit (PA-RAP-MAP/pCAG vector). DNAs encoding EGFR, EGFRec, HER2ec, and CD133
were cloned into PA-RAP-MAP/pCAG vector (PA-EGFRRAP-MAP/pCAG, PA-EGFRec-RAP-MAP/pCAG, PA-HER2ec-

FUJII ET AL.

RAP-MAP/pCAG, and PA-CD133-RAP-MAP/pCAG,
respectively). The MAP-tagged deletion mutant of human
podoplanin without the PLAG domain (MAP-hPDPNdN55/
pCAG) was cloned into the pCAG vector as described previously.(20) All constructs were verified by DNA sequencing.
Binding kinetics analysis using surface
plasmon resonance

Rat anti-MAP mAb PMab-1 (IgG2a, k) was described
previously.(15) PMab-1 was immobilized on CM5 sensor chips
using amino coupling chemistry according to the method
provided by the manufacturer. The MAP-tagged ATRXepi
proteins were diluted in PBST (phosphate-buffered saline, pH
7.4, containing 0.005% Tween 20) and injected at a flow rate of
30 mL/min. The ATRXepi was diluted in PBST (12.5, 25, 50,
100, and 200 nM) and passed over the biosensor chip. The
binding was monitored for 60 seconds, followed by dissociation in running buffer for 120 seconds using the Single-cycle
kinetics method. Binding curves were analyzed using BIAcore
X100 Evaluation Software (GE Healthcare, Piscataway, NJ)
with curve fitting using a 1:1 binding model.
Western blot analyses

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). The proteins were
electrophoresed on 5%–20% polyacrylamide gels (Wako Pure
Chemical Industries Ltd.) and transferred onto a PVDF membrane (EMD Millipore Corp.). After blocking with 4% skim
milk (Nacalai Tesque, Inc.), the membrane was first incubatedwith 1 mg/mL PMab-1, anti-b-actin (AC-15; Sigma-Aldrich, St.
Louis, MO), anti-IDH1 (RcMab-1),(21) or an anti-RAP tag(PMab-2), and then with 1:1000 diluted peroxidase-conjugatedanti-rat or mouse IgG (Dako; Agilent Technologies, Inc.,
Glostrup, Denmark), followed by development in Pierce
Western Blotting Substrate Plus (Thermo Fisher Scientific,Inc.) using a Sayaca Imager (DRC Co. Ltd., Tokyo, Japan).
Flow cytometry

The mouse anti-human podoplanin mAb LpMab-17 (IgG1,k) was described previously.(22) Cell lines were harvested bybrief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai
Tesque, Inc.). After washing with PBS, the cells were treatedwith PMab-1 (1 mg/mL) or LpMab-17 (1 mg/mL) for 30
minutes at 4C, followed by treatment with 1:1000 diluted
Oregon Green 488 goat anti-rat or mouse IgG (Thermo Fisher
Scientific, Inc.). Fluorescence data were collected using a
Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Protein expression and purification

PMab-1 was coupled to CNBr-activated Sepharose 4B(GE Healthcare) according to the protocol provided by the
manufacturer, routinely yielding coupling levels of *2 mg
IgG/mL gel. Recombinant ATRXepi (PA-ATRXepi-RAPMAP) was expressed in transformed E. coli BL21 (DE3)
cultured for *3 hours at 37C in the presence of 1 mM IPTG,
and the bacterial pellet from a 100 mL culture was suspended
in 10 mL of Tris-buffered saline (TBS; pH 7.5). Bacteria werelysed by sonication, and the soluble fraction was obtained bycentrifugation at 15,000 g for 15 minutes. The lysates werepassed through a 0.45 mm filter to remove any trace amounts

A NOVEL TAGGING SYSTEM: MAP TAG

of insoluble materials. The cleared lysates were mixed with
PMab-1-Sepharose (1 mL bed volume) and incubated at 4Cfor 3 hours under gentle agitation. The beads were thentransferred to a column and washed with 20 mL of TBS. The
bound protein was eluted with TBS containing 0.1 mg/mL
epitope peptide (GDGMVPPGIEDKIT). The elution was
conducted at room temperature in a stepwise manner(1 mL · 10), with a dissociation time of 5 minutes allowed foreach elution step.
For the purification of soluble EGFRec and HER2ec secreted from mammalian cells, LN229/EGFRec, and LN229/
HER2ec were plated in 225 cm2 flask (Thermo Fisher Scientific, Inc.), and 1 L of culture supernatant was harvested.
Cleared supernatant was passed through PMab-1-Sepharose

295(4 mL bed volume), and the same process was repeated threetimes. The beads were then washed with 80 mL of TBS andeluted with the same solution containing 0.1 mg/mL epitope
peptide in a stepwise manner (4 mL · 10) as described above.
For the purification of EGFR from mammalian cell membranes, LN229/EGFR was plated onto hundred 10-cm culture
dishes. Cells were detached from the plates using PBS containing 1 mM EDTA. The pelleted cells were washed using
PBS and solubilized by adding 10 mL of PBS containing 1%(w/v) Triton X-100 and 0.05 mg/mL aprotinin. The solubilized cell lysates were incubated at 4C for 30 minutes andfurther centrifuged 15 minutes at 13,000 g at 4C. The pelleted cells were solubilized in 5 mL of the same solution, andthe process was repeated twice. The cleared lysates were

FIG. 1. Characterization of the MAP tag system. (A) Surface plasmon resonance kinetic analysis of PMab-1 binding
toward the MAP tag. Serially diluted, MAP-tagged ATRXepi (12.5, 25, 50, 100, and 200 nM) was injected over the CM5
sensor chip immobilized with PMab-1 for 60 seconds, followed by dissociation in PBST for 120 seconds at a flow rate of
30 mL/min. The binding curve was globally fitted to a 1:1 binding model to derive the equilibrium dissociation constant (KD)values shown. (B) Western blot analysis of MAP tagged CD133 with PMab-1. Total cell lysates (LN229, LN229/CD133,
CHO-K1, and CHO/CD133) were electrophoresed under reducing conditions on a 5%–20% SDS-PAGE gel and transferred
to a membrane. A membrane containing the same amount of lysate was immunoblotted with 1 mg/mL PMab-1, AC-15, or
RcMab-1 for 30 minutes and incubated with peroxidase-conjugated secondary antibody specific for rat (PMab-1 and RcMab1) or mouse (AC-15) IgG. (C) Flow cytometric analysis of MAP-tagged membrane protein. HEK293T, COS-7, and CHO-K1
cells and those transiently transfected with MAP-hPDPNdN55 were incubated with 1 mg/mL PMab-1 or LpMab-17 andstained with Oregon Green 488-labeled secondary antibody specific for rat or mouse IgG. CHO, Chinese hamster ovary;
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SS, signal sequence.

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FUJII ET AL.

mixed with PMab-1-Sepharose (1 mL bed volume) and incubated at 4C for 3 hours under gentle agitation. The beads
were then transferred to a column and washed with 20 mL of
PBS. The bound protein was eluted with PBS containing
0.1 mg/mL epitope peptide in a stepwise manner (1 mL · 10)
as described above.
Ten peptide-elution fractions from the column chromatography were subjected to 5%–20% SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue
(CBB) or Oriole fluorescent protein stain (Bio-Rad Laboratories Inc., Berkeley, CA).
Results
Kinetic analysis of PMab-1/MAP tag interaction

First, we investigated whether PMab-1 maintains a highaffinity toward MAP tag ‘‘GDGMVPPGIEDK.’’ We analyzed the binding affinity between PMab-1 and MAP tag
using the BIAcore X100 system. Curve-fitting analysis
showed high-affinity PMab-1/MAP tag interaction (ka,
1.1 · 105 M-1s-1; kd, 4.2 · 10-4 s-1; KD, 3.7 · 10-9 M)(Fig. 1A).
Specificity analysis of PMab-1/MAP tag interaction

We next evaluated the specificity of PMab-1 for the MAP
tag. As shown in Figure 1B, PMab-1 produced a strong single
band corresponding to the expressed CD133 with MAP tag(*120 kDa) in western blot analysis of the cell lysate. PMab1 did not show any reactive bands in CHO-K1 cells, indicating that PMab-1 is very specific against the MAP tag.
The specificity was also analyzed using flow cytometry(Fig. 1C). PMab-1 reacted with the hPDPNdN55 plus Nterminal MAP tag (extracellular domain), which is expressed in HEK293T, COS-7, and CHO-K1 cells (Fig. 1C,
middle panel) and did not react with the corresponding
untransfected cells (Fig. 1C, upper panel). LpMab-17, amouse anti-human podoplanin mAb, recognized all transfected cell lines (Fig. 1C, lower panel).
Application to the protein purification system

We attempted to purify the four proteins with MAP tag
using the MAP tag system. ATRXepi (*25 kDa) was expressed in E. coli and purified from the soluble fraction of thebacterial lysate (Fig. 2). This protein eluted in fractions 2–10,
with virtually no contamination. The soluble ectodomain
fragments of EGFR (EGFRec) and HER2 (HER2ec) wereexpressed in LN229 and purified from the culture supernatant(Figs. 3A and 3B). Because EGFRec and HER2ec are highly
glycosylated, they electrophoresed more slowly than predicted by the theoretical molecular weights. These proteins
were eluted in elution fractions 2–10, with virtually no contamination. EGFR (*170 kDa) was expressed in LN229 andwas purified from the membrane fraction of LN229 (Fig. 4).
This protein eluted in fractions 2–10, and the single bands
were detected in elution fractions 4–10 (Fig. 4A). It was also
confirmed that the strong bands in elution fractions were
derived from EGFR in western blot analysis using an antiRAP tag mAb (clone: PMab-2) (Fig. 4B). All MAP-tagged
proteins were captured onto PMab-1-Sepharose and eluted
from the resin with a solution containing 0.1 mg/mL free
epitope peptide (GDGMVPPGIEDKIT). Despite the high

FIG. 2. Purification of the ATRX fragment (ATRXepi)
from transformed E. coli. The soluble fraction (5 mL) from thetransformed E. coli after PMab-1-Sepharose capture (flowthrough), and the fifth of five washes in 20 mL TBS (wash),and 10 peptide-elution fractions (lanes 1–10) from the column
chromatography were subjected to 5%–20% SDS-PAGE
under reducing conditions and stained with CBB. TBS, Trisbuffered saline; CBB, Coomassie Brilliant Blue.
affinity between PMab-1 and MAP tag (Fig. 1A), these antigens were eluted by the peptide. We performed the elution
at room temperature in a stepwise manner, with a dissociation
time of 5 minutes allowed for each elution step. These eluted
proteins showed high purity (Figs. 2–4).
Discussion

An excellent affinity tag system requires high affinity andspecificity. PMab-1, a rat anti-mouse podoplanin mAb, wasproduced by immunization with the synthetic peptide‘‘GDGMVPPGIEDKIT’’.(15) PMab-1 has high affinity andspecificity toward mouse podoplanin as described previously.(18) Therefore, we investigated whether PMab-1
maintains high affinity and specificity toward the MAP tag‘‘GDGMVPPGIEDK’’ that was attached to several proteins.
First, we analyzed the binding affinity between PMab-1
and the MAP tag. Curve-fitting analysis showed high-affinity

FIG. 3. Purification of the soluble ectodomain fragments of EGFRec (A) and HER2ec (B). PMab-1-Sepharose captured
these proteins in the culture supernatant from transfected LN229 and was washed with 20 mL TBS. Ten peptide-elution
fractions (10 mL) (lanes 1–10) from the column chromatography were subjected to 5%–20% SDS-PAGE under reducing conditions and stained with CBB. SS, signal sequence; EGFR, epidermal growth factor receptor; CBB, Coomassie
Brilliant Blue.

FIG. 4. Purification of EGFR from LN229 cells. PMab-1-Sepharose captured the proteins in the detergent-solubilized celllysates from transfected LN229 and was washed with 20 mL PBS. (A) Ten peptide-elution fractions (10 mL) (lanes 1–10)
from the column chromatography were subjected to 5%–20% SDS-PAGE under reducing conditions and stained with
Oriole fluorescent protein stain. (B) Ten peptide-elution fractions (5 mL) (lanes 1–10) from the column chromatography
were subjected to 5%–20% SDS-PAGE under reducing conditions and transferred to a membrane. The membrane was
immunoblotted with 1 mg/mL of PMab-2 (anti-RAP tag) for 30 minutes and incubated with peroxidase-conjugated secondary antibody specific for mouse IgG. PBS, phosphate-buffered saline; SS, signal sequence.
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PMab-1/MAP tag interaction (KD, 3.7 · 10-9 M) (Fig. 1A).
These data do not differ from those of previous studies
that analyzed PMab-1/mouse podoplanin interaction using
flow cytometry (KD, 3.2 · 10-9 M).(18) Next, we evaluatedthe specificity of PMab-1 against MAP tag. As shown in
Figures 1B and 1C, PMab-1 has high specificity toward MAP
tag in Western blot analysis and flow cytometry. Although
HEK293T cells are reported to express human podoplanin,(23) PMab-1 did not react with untransfected HEK293T
cells because PMab-1 does not cross-react with human podoplanin. In fact, the important amino acids for PMab-1/
MAP tag interaction are the first aspartic acid and the methionine in the MAP tag (data not shown); human podoplanin
does not have this sequence. These results indicate that
PMab-1 has a high affinity and specificity toward not only
mouse podoplanin but also the MAP tag. Therefore, we
presumed that the PMab-1/MAP tag system would be applicable to the protein purification system.
We attempted to purify MAP-tagged ATRXepi, EGFRec,
HER2ec, and EGFR using the MAP tag system (Figs. 2–4).
These purified proteins showed high purity and were readyfor use in the downstream experiments without further purification. Purifying membrane proteins is generally difficult
because membrane proteins require detergent to make themsoluble in an aqueous solution. In contrast, the MAP tag
system purified membrane proteins such as EGFR in thepresence of detergent (Fig. 4). These results indicate that the
MAP tag system is a powerful protein purification tool that
does not depend on the host or the type of protein.
In conclusion, we successfully developed a novel affinity
tagging system designated ‘‘MAP tag’’ that employs a uniquemAb PMab-1 against mouse podoplanin. Owing to its highaffinity and specificity toward linear dodecapeptide antigen,PMab-1 can be used to detect and purify tagged proteins invarious assay formats. In the near future, we will compare the
MAP tag to several other affinity tag systems against manytargets and determine the types of applications most suitable
for the MAP tag system.
Acknowledgments

We thank Takuro Nakamura, Noriko Saidoh, and Kanae
Yoshida for their excellent technical assistance. We also
thank Satoshi Ogasawara for their great suggestions and advice. This work was supported in part by the Platform for
Drug Discovery, Informatics, and Structural Life Science(PDIS) from Japan Agency for Medical Research and development, AMED (Y.K.), by the Regional Innovation
Strategy Support Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan(Y.K.), by JSPS KAKENHI [Grant No. 26440019 (M.K.K.)
and Grant No. 16K10748 (Y.K.)], and by the Basic Scienceand Platform Technology Program for Innovative Biological
Medicine from AMED (Y.K.). This work was performed inpart under the Cooperative Research Program of Institute forProtein Research, Osaka University, CR-15-05 and CR-1605 (Y.K.). The authors would like to thank Enago (www.enago.jp) for the English language review.
Author Disclosure Statement

No competing financial interests exist.

FUJII ET AL.
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Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 15, 2016
Accepted: October 6, 2016