MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 2, 2017
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0052

Short Communications

Development of RAP Tag, a Novel Tagging Systemfor Protein Detection and Purification
Yuki Fujii,1 Mika K. Kaneko,1 Satoshi Ogasawara,1 Shinji Yamada,1 Miyuki Yanaka,1 Takuro Nakamura,1Noriko Saidoh,1 Kanae Yoshida,1 Ryusuke Honma,1 and Yukinari Kato1,2Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification.
The most suitable tag for a particular purpose should be selected from many available affinity tag systems.
In this study, we developed a novel affinity tag called the ‘‘RAP tag’’ system, which comprises a mouse antirat
podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful
not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent
assay, but also for protein purification.
Keywords: monoclonal antibody, RAP tag, affinity tag, protein purification, podoplanin

which is highly expressed in many normal cells and cancercells, and is involved in tumor-induced platelet aggregation
by binding to CLEC-2 on platelets.(3,25–33) Because PMab-2
possesses high affinity and specificity against rat podoplanin,(24) it was expected to be useful as an antitag antibody.
Herein, we developed a novel affinity tag system, the ‘‘RAPtag’’ system, using PMab-2 mAb.

Introduction

Affinity tag systems, which are useful for protein purification and detection, are classified into ‘‘peptidetags’’ and ‘‘protein tags.’’ Protein tags, including glutathioneS-transferase tag,(1) maltose-binding protein tag,(2) Fc tag of
immunoglobulin,(3) and green fluorescent protein tag,(4) havenumerous advantages because they are useful for protein
expression in the soluble fraction or are easily detected using
monoclonal antibodies (mAbs). In contrast, protein tags
sometimes affect the character of the target proteins if they
are large in size (>25 kDa); therefore, their removal is necessary before any protein analysis. Peptide tags are less likely
to affect the structure and function of target proteins becauseof their small size (typically 1–2 kDa); therefore, it is not always necessary to remove the tag portion for protein analysis.
The most appropriate peptide tag system can be selectedfrom many available peptide tags, such as FLAG tag,(5)TARGET tag,(6) PA tag,(7–16) and MAP tag,(17–20) among
many others.(21–23) These systems sometimes have disadvantages, such as low specificity, low affinity, or difficulty inachieving protein elution. Therefore, we need to develop
further affinity tag systems to overcome these disadvantagesof established affinity tag systems.
We previously developed a mouse mAb (clone PMab-2)
against the platelet aggregation-stimulating domain of rat
podoplanin.(24) Podoplanin is a type I transmembrane protein,
12

Results and Discussion

We first investigated the binding affinity between PMab-2
mAb and RAP tag (DMVNPGLEDRIE) using the BIAcore
X100 system. Curve fitting showed the affinity for PMab-2–
RAP tag interaction: ka = 2.0 · 105 M-1 s-1, kd = 2.0 · 10-3 s-1,and KD = 9.7 · 10-9 M, indicating that PMab-2 showed moderate affinity toward the RAP tag. Next, we examined whether the RAP tag system is useful for several protein detection
systems. As depicted in Figure 1A, PMab-2 showed a strong
single band corresponding to the molecular weight of human epidermal growth factor receptor (EGFR) with RAP tag(*170 kDa) in Western blot analysis. Transfectants ofEGFR were established previously.(17) PMab-2 did not show
any nonspecific bands in CHO-K1 and LN229 cells, indicating that PMab-2 is very specific to the RAP tag. PMab-2
reacted with the EGFR with the N-terminal RAP tag, which
was expressed in CHO-K1 cells in flow cytometry (Fig. 1B,left). As a positive control, antihuman EGFR mAbs (EMab-Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

ª Yuki Fujii et al., 2017; 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 anymedium, provided the original work is properly credited.

68FIG. 1. Detection and purification of RAP-tagged EGFR by the RAP tag system. (A) Western blot analysis of RAP-tagged
EGFR using anti-RAP tag mAb, PMab-2. Total cell lysates (CHO-K1, CHO/EGFR, LN229, and LN229/EGFR) were
electrophoresed under reducing conditions using 5%–20% SDS-PAGE gel and transferred to a membrane. The membrane
containing the same amount of lysate was immunoblotted using 1 mg/mL PMab-2 (anti-RAP tag), AC-15 (anti-b-actin), orRMab-3 (anti-IDH1) for 30 minutes, and incubated with a peroxidase-conjugated secondary antibody specific for mouse
IgG. (B) Flow cytometric analysis of RAP-tagged membrane protein. Two antihuman EGFR mAbs (clones: EMab-51 andAY13) were used in this study. EMab-51 (IgG1, k) was established in our laboratory. AY13 (IgG1, k) was purchased from
BioLegend (San Diego, CA). CHO-K1 cells and CHO/RAP-EGFR were treated with 1 mg/mL PMab-2, EMab-51, or AY13for 30 minutes at 4C, followed by 1:1000 diluted Oregon Green 488 goat antimouse IgG (Thermo Fisher Scientific, Inc.,Waltham, MA). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan). (C) Sandwich
ELISA of RAP-tagged protein. PMab-2 was immobilized at a concentration of 10 mg/mL for 30 minutes. After blocking
with 1% bovine serum albumin in 0.05% Tween 20/phosphate-buffered saline (pH 7.4), recombinant PA-EGFRec-RAPMAP was added at a concentration from 3 ng/mL to 10 mg/mL and was incubated overnight. After washing, the plates wereincubated with 0.5 mg/mL biotinylated NZ-1 (anti-PA tag), followed by 1:5000 diluted peroxidase-conjugated streptavidin(GE Healthcare Bio-Sciences, Pittsburgh, PA). The enzymatic reaction was performed using 1-Step Ultra TMB-ELISA(Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-RadLaboratories, Inc., Berkeley, CA). Data are means of four replicates – SEM. (D) Purification of soluble ectodomain fragmentof EGFR (EGFRec). LN229/EGFRec was cultured and 1 L of culture supernatant was harvested. The filtered supernatant waspassed through PMab-2-Sepharose (4 mL bed volume), and the same process was repeated three times. The beads were thenwashed with 80 mL of Tris-buffered saline (TBS; pH 7.5) and eluted with 0.1 mg/mL epitope peptide in a step-wise manner(4 mL · 10). Ten microliters of the 5th of 5 washes in TBS (wash) and 10 peptide-eluted fractions (lanes 1–10) during thecolumn chromatography were subjected to 5%–20% SDS-PAGE under reducing conditions and were stained with Coomassie brilliant blue. Arrow: EGFRec. ec, ectodomain; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked
immunosorbent assay; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
SS, signal sequence.
69

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FUJII ET AL.

51 and AY13) recognized CHO-K1/EGFR (Fig. 1B, middle
and right). We further investigated whether the RAP tag
system is useful in sandwich enzyme-linked immunosorbent
assay (ELISA). PMab-2 was immobilized, and the solubleectodomain fragment of EGFR (EGFRec) with RAP tag wasadded at a concentration from 3 ng/mL to 10 mg/mL and detected by biotinylated NZ-1. As shown in Figure 1C, EGFRec
was detected in a dose-dependent manner. These results indicate that the RAP tag system is useful for Western blotting,flow cytometry, and sandwich ELISA.
Next, we purified three different RAP-tagged proteinsusing the RAP tag system. The EGFRec and the soluble ectodomain fragment of human epidermal growth factor receptor 2 (HER2ec) were expressed in LN229 and werepurified from the culture supernatant (Fig. 1D and Supplementary Fig. S1A). The fragment of a-thalassemia/mentalretardation-syndrome-X-linked (ATRXepi; *25 kDa) wasexpressed in Escherichia coli and purified from the soluble
fraction of the bacterial lysate (Supplementary Fig. S1B). All
RAP-tagged proteins were captured onto PMab-2-Sepharoseand eluted from the resin by a solution containing 0.1 mg/mL
free epitope peptide (GDDMVNPGLEDRIE). These proteins
were efficiently eluted using the RAP tag peptide becauseof the high dissociation constant for PMab-2–RAP tag interaction. EGFRec and HER2ec were eluted in elution fractions 2–4 (EGFRec) or 2–3 (HER2ec) at a high concentration (Fig. 1Dand Supplementary Fig. S1A). EGFRec and HER2ec are highly
glycosylated(34,35); therefore, they were electrophoresed at a‘‘heavier’’ position compared with that suggested by the theoretical molecular weight. ATRXepi was eluted in elution
fractions 2–10 (Supplementary Fig. S1B). These purified
proteins showed high purity and were ready for use in downstream experiments without further purification, indicatingthat the RAP tag system is a powerful protein purification tool.
In conclusion, we successfully developed a novel affinity
tagging system, ‘‘RAP tag,’’ by employing a unique mAb
PMab-2 against rat podoplanin. The RAP tag system could be
advantageous for protein purification and detection in thefield of protein science.
Acknowledgments

This work was supported mainly by the Basic Science andPlatform Technology Program for Innovative Biological
Medicine from Japan Agency for Medical Research andDevelopment, AMED (Y.K.). This work was also supportedin part by the Platform for Drug Discovery, Informatics, andStructural Life Science (PDIS) from AMED (Y.K.), by Project for utilizing glycans in the development of innovative
drug discovery technologies from AMED (Y.K.), by Translational Research Network Program from AMED (Y.K.), bythe Regional Innovation Strategy Support Program from theMinistry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Y.K.), by JSPS KAKENHI Grant
Numbers 26440019 (M.K.K.) and 16K10748 (Y.K.). This
work was performed, in part, under the Cooperative Research
Program of Institute for Protein Research, Osaka University,CR-16-05. The authors thank Enago (www.enago.jp) for the
English language review.
Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: December 21, 2016
Accepted: February 21, 2017