Biochemistry and Biophysics Reports 23 (2020) 100780

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

RIEDL tag: A novel pentapeptide tagging system for transmembraneprotein purification
Teizo Asano a, Mika K. Kaneko a, Yukinari Kato a, b, *ab

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Peptide tag
Protein purification
Monoclonal antibody
RIEDL tag
Podoplanin
CD20

Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several
different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an antitag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG
tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional
structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which iscomposed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb),
LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted thepurification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin(wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion(CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were effi­
ciently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot
and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-steppurification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop
structure of multi-pass transmembrane proteins.

1. Introduction[9], Myc [10], and PA [11], are composed of a short polypeptide, whichacts as the tag, and are recognized using anti-tag antibodies that exhibit
high specificity because of the antigen-antibody interaction. Moreover,
epitope tags have low molecular weights, and are therefore not likely toaffect the target protein’s structure. A tag system should be selected byconsidering the advantages of each system. The FLAG tag is one of theuseful epitope tag systems for protein purification and detection [8].
Although the FLAG tag can be added to the N- or C-terminus of a target
protein, it is not applicable for immunoprecipitation assays when it isinserted into a loop structure [1]. When a Myc tag was inserted intoarrestin, anti-Myc antibodies were shown to function as a competitor forarrestin binding to rhodopsin [12]. HA and T7 tags, when inserted into
KdpD, could be recognized by antibodies in an immunoprecipitation
assay [13].
In our previous studies, we established the PA tag [11] and MAP tag
systems [14]. The PA tag was successfully inserted into a loop structure
in integrin and Sema3A, and proved useful for flow cytometry andimmunoprecipitation assays [1]. The MAP tag can also be inserted intoProtein purification and detection are essential techniques for pro­tein research, including for the determination of structure and elucida­tion of function [1]. There exist several methods for protein purification,such as hydrophobic interaction chromatography, ion exchange chro­
matography, and affinity chromatography [2]. However, many steps are
required to isolate a protein from tissues or cells, and it is often difficult
to obtain highly purified proteins. To resolve these problems, several
tagging systems have been developed [3,4]. By using an exogenous tag,a target protein can be purified directly from tissue or cell lysates, which
can express recombinant tagged proteins in one or a few steps.
Affinity tag systems are classified into the “protein tag system” andthe “epitope tag system.” Protein tags, including glutathione S-trans­
ferase (GST) [5], green fluorescent protein (GFP) [6], and maltose
binding protein (MBP) [7], are useful for soluble protein expression;
however, these tags can affect the functions of a target protein, as thesetags have high molecular weights. Epitope tags, such as FLAG [8], HA

Abbreviations: mAb, monoclonal antibody; PBS, phosphate-buffered saline; wPDPN, whale podoplanin.
* Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2020.100780
Received 16 June 2020; Received in revised form 6 July 2020; Accepted 8 July 2020
2405-5808/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Biochemistry and Biophysics Reports 23 (2020) 100780

Fig. 1. Purification of RIEDL-wPDPN using
the RIEDL tag system. (A) Schematic illus­
tration of RIEDL-wPDPN. The epitopes of
LpMab-7 or PMab-237 are indicated. SS;
signal sequence. (B) Flow cytometry using
an anti-RIEDL tag mAb. CHO/RIEDL-wPDPN
was detected by an anti-RIEDL tag mAb(LpMab-7, upper panel) and an anti-wPDPN
mAb (PMab-237, lower panel). (C) Purifica­tion of RIEDL-wPDPN using the RIEDL tag
system. Cell lysates (Lysate) were loaded
onto an LpMab-7-Sepharose column, andunbound proteins passed through (flowthrough). After five washes with PBS con­taining Triton X-100 (Triton wash-1 andTriton wash-5) and five washes with PBS(PBS wash-1 and PBS wash-5), bound pro­teins were eluted with 2 � RIEDL peptide (2� RIEDL 1–5, and -10), followed by acidic
buffer (Glycine-HCl 1–3). Elution fractions
were applied to SDS-PAGE, and the gel was
stained with CBB. (D) Elution fractions fromthe column chromatography were applied to
SDS-PAGE, and the proteins were transferredto PVDF membranes. The membranes were
immunostained with 1 μg/ml of an antiRIEDL tag mAb (LpMab-7; left panel) or ananti-wPDPN mAb (PMab-237; right panel)and incubated with peroxidase-conjugatedsecondary antibody specific for mouse
immunoglobulins.

the loop regions of various proteins, and tag antibody interactions weremaintained [15]. Although several tag systems can be inserted into theloop regions of multi-pass transmembrane proteins and are applicablefor some biological assays as described above, much smaller epitope tags
are needed to minimize the effects of the inserted peptides on the pro­teins. In this study, we established a novel tag system, called the “RIEDL
tag system,” composed of LpMab-7 (an anti-human podoplanin mAb)
and a peptide, RIEDL, consisting of only five amino acids, and investi­
gated the utility of this tag system in protein purification.

tag, which is recognized by an anti-RIEDL tag mAb (LpMab-7), wassubcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan) using the In-Fusion HD Cloning Kit (Takara
Bio, Inc., Shiga, Japan). This plasmid was named pCAG-Neo/RIEDLwPDPN. DNA encoding the CD20 gene (IRAL012D02) was provided
by the RIKEN BRC through the National BioResource Project of MEXT,
Japan. The open reading frame of CD20 plus the inserted RIEDL tag
between Pro169 and Ala170 of human CD20 was subcloned into a
pCAG-Ble vector using In-Fusion HD Cloning Kit. This plasmid wasnamed pCAG-Ble/CD20-169RIEDL170.

2. Materials and methods
2.1. Plasmids

2.2. Cell lines

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding
wPDPN (accession No.: XM_007104824.2) plus an N-terminal RIEDL

Chinese hamster ovary (CHO)-K1 cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA). CHO-K1 cells
were transfected with pCAG-Neo/RIEDL-wPDPN using Lipofectamine2

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Biochemistry and Biophysics Reports 23 (2020) 100780

LTX with Plus Reagent (Thermo Fisher Scientific Inc., Waltham, MA,
USA), and stable transfectants were selected by limiting dilution. The
CHO-K1 cells were also transfected with pCAG-Ble/CD20-169RIEDL170
using Neon Transfection System (Thermo Fisher Scientific, Inc.), andstable transfectants were sorted using a cell sorter (SH800; Sony Corp.,
Tokyo, Japan). The CHO-K1 cells and transfectants were cultured in
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific Inc.), 100 units/mL of penicillin, 100 μg/mL streptomycin,
and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.) at 37 � C in ahumidified atmosphere containing 5% CO2. CHO/RIEDL-wPDPN was
cultivated in a medium containing 0.5 mg/ml of G418 (Nacalai Tesque,Inc.). CHO/CD20-169RIEDL170 was cultivated in a medium containing
0.5 mg/mL zeocin (InvivoGen, San Diego, CA).

CA, USA) for 30 min. The membrane was also incubated with anti-CD20
mAb (EP459Y; 1:1000 dilution; Abcam, Cambridge, UK) for 1 h, fol­
lowed by anti-rabbit immunoglobulins (1:1000 dilution; Agilent Tech­
nologies, Inc.) for 30 min. The membranes were visualized with the
ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure
Chemical Corporation) using the Sayaca-Imager. All procedures ofWestern blotting were performed at room temperature.
3. Results
3.1. Establishment of RIEDL tag system for protein purification
We first selected a mAb, LpMab-7, the epitope of which was previ­
ously identified as the pentapeptide Arg79, Ile80, Asp81, Glu82, andLeu83 (RIEDL) [16] from our original mAbs library [17]. We named thismAb-peptide tag combination the “RIEDL tag system.” In our previous
study, LpMab-7 detected the RIEDL sequence in Western blot, immu­
nohistochemistry, and flow cytometry analyses [16]. In this study, we
performed the purification of RIEDL-tagged protein using an
LpMab-7-Sepharose column to investigate whether the RIEDL tag system
can be applied to protein purification.
The RIEDL sequence was added to the N-terminus of wPDPN, andRIEDL-wPDPN was expressed in CHO-K1 cells (Fig. 1A). CHO/RIEDLwPDPN was detected by anti-RIEDL tag mAb (LpMab-7) and antiwPDPN mAb (PMab-237) in flow cytometry (Fig. 1B). The lysate of
CHO/RIEDL-wPDPN was applied to an LpMab-7-Sepharose column.
After five washes with PBS containing Triton X-100, and an additional
five washes with PBS, RIEDL-tagged protein was eluted competitively
with 2 � RIEDL peptide. The total cell lysate, flow-through from the firstcolumn application, several wash fractions (Triton wash-1, Triton wash5, PBS wash-1, and PBS wash-5), elution fractions using 2 � RIEDL(fraction-1, 2, 3, 4, 5, and 10), and elution fractions using glycine-HCl(fraction-1, 2, and 3) were applied to SDS-PAGE, followed by staining
with CBB (Fig. 1C). The 45 kDa RIEDL-wPDPN protein was clearlystained in the 2 � RIEDL peptide elutions, fraction-2 and -3, and weakly
detected in the 2 � RIEDL peptide elutions, fraction-1 and -4. In contrast,
no protein was observed in the additional glycine-HCl elution fractions(Fig. 1C), indicating that RIEDL-wPDPN was completely eluted by 2 �
RIEDL peptide from the LpMab-7-Sepharose column.
Next, we assessed RIEDL-wPDPN protein by Western blot analysis
using LpMab-7 (anti-RIEDL) and PMab-237 (anti-wPDPN mAb). As
shown in Fig. 1D, LpMab-7 (anti-RIEDL) detected a 45 kDa band (gly­cosylated) and a 28 kDa band (non-glycosylated) in the 2 � RIEDL
elution fractions. In contrast, PMab-237 (anti-wPDPN mAb) detected
only the 45 kDa band in the 2 � RIEDL peptide elution fractions. No
protein was observed in acidic buffer elution fractions, also indicatingthat RIEDL-wPDPN was completely eluted by 2 � RIEDL peptide fromthe LpMab-7-Sepharose column.

2.3. Flow cytometry
Cells were harvested by brief exposure to 0.25% trypsin/1 mM eth­ylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin in phosphate-buffered saline(PBS), cells were treated with primary mAbs (1 μg/ml) for 30 min at 4 � C
and subsequently with Alexa Fluor 488-conjugated anti-mouse IgG(1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluores­
cence data were collected using an EC800 Cell Analyzer (Sony Corp.).
2.4. Expression and purification of RIEDL-tagged proteins
LpMab-7-Sepharose column (2 mg IgG/ml gel) was prepared using
CNBr-activated Sepharose 4 Fast Flow (GE Healthcare, Bio-Sciences,
Pittsburgh, PA) according to the protocol provided by the manufac­
turer. The peptide used in the elution was synthesized and purified byreverse-phase HPLC (Sigma-Aldrich Corp., St. Louis, MO). CHO/RIEDLwPDPN and CHO/CD20-169RIEDL170 were collected by centrifugation at
190�g for 5 min, and lysed with PBS containing 1% Triton X-100 and
0.05 mg/ml aprotinin for 15 min on ice. Supernatant was collected bycentrifugation at 22,140�g for 15 min, applied to the LpMab-7Sepharose column (1 ml bed volume), and washed with PBS contain­
ing 1% of Triton X-100, followed by washing with PBS containing 0.05%
of Tween-20 (PBST). The bound protein was eluted with 0.1 mg/ml
decapeptide (RIEDLRIEDL sequence, named 2 � RIEDL peptide), fol­
lowed by eluting with glycine-HCl buffer (pH 2.7) (Polysciences, Inc.,
Warrington, PA). In another experiment, the bound protein was eluted
with glycine-HCl buffer (pH 2.7), followed by 0.1 mg/ml of 2 � RIEDL
peptide.
2.5. SDS-PAGE and gel staining
Elution fractions and cell lysates were boiled in sodium dodecyl
sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). The samples were
electrophoresed on 5%–20% polyacrylamide gels under reducing con­dition (Nacalai Tesque, Inc.). The gels were stained using Coomassie
Brilliant Blue (CBB, Bio-Rad Laboratories, Inc., Berkeley, CA) for 30 min.
The gels were also stained using Oriole Fluorescent Gel Stain (Bio-Rad
Laboratories, Inc.) for 2 h and were visualized with a Sayaca-Imager(DRC Co., Ltd., Tokyo, Japan).

3.2. Comparison of elution efficiency between 2x RIEDL peptide andacidic buffer
RIEDL-wPDPN was efficiently eluted from the LpMab-7-Sepharosecolumn with 2 � RIEDL peptide, but not eluted by additional glycineHCl buffer (Fig. 1C), indicating that RIEDL-wPDPN was completely
eluted by 2 � RIEDL peptide from the LpMab-7-Sepharose column. To
compare the elution efficacy, we changed the elution sequence, where
we first eluted using glycine-HCl buffer, followed by a 2 � RIEDL pep­tide elution. As detected by LpMab-7 in Western blot, RIEDL-wPDPN
was clearly eluted using glycine-HCl, but further eluted by an addi­
tional 2 � RIEDL peptide elution (Suppl. Fig. 1A). These results wereconfirmed by Western blot using PMab-237 (Suppl. Fig. 1B). Because thefirst 2 � RIEDL peptide completely eluted RIEDL-wPDPN from the
LpMab-7-Sepharose column, and additional glycine-HCl buffer elution
was not necessary (Fig. 1C), an elution procedure using only the 2 �
RIEDL peptide was determined to be more efficient and more useful than

2.6. Western blotting
Elution fractions and cell lysates were boiled in SDS sample buffer(Nacalai Tesque, Inc.). The samples were electrophoresed on 5%–20%
polyacrylamide gels (Nacalai Tesque, Inc.), and were transferred onto
polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt,
Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for
1 h, the membranes were incubated with LpMab-7 (1 μg/ml) or PMab237 (1 μg/ml) for 1 h, followed by HRP-conjugated anti-mouse immu­
noglobulins (1:1000 dilution; Agilent Technologies, Inc., Santa Clara,
3

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Biochemistry and Biophysics Reports 23 (2020) 100780

Fig. 2. Purification of CD20-169RIEDL170
using the RIEDL tag system. (A) Schematic
illustration of CD20-169RIEDL170. The epi­
topes of LpMab-7 and anti-CD20 mAb (clone
EP459Y) are indicated. (B) Flow cytometry
using an anti-RIEDL tag mAb. CHO/
CD20-169RIEDL170 was detected by antiRIEDL tag mAb (LpMab-7, upper panel)and anti-CD20 mAb (C20Mab-11, lower
panel). (C) Cell lysates (Lysate) were loaded
onto an LpMab-7-Sepharose column, andunbound proteins passed through (flowthrough). After five washes with PBS con­taining Triton X-100 (Triton wash-1 andTriton wash-5) and five washes with PBS(PBS wash-1 and PBS wash-5), bound pro­teins were eluted with 2 � RIEDL peptide (2� RIEDL 1–5, and -10) followed by acidic
buffer (glycine-HCl 1–3). Elution fractions
were applied to SDS-PAGE, and the gel was
stained with Oriole Fluorescent Gel Stain.
(D) Elution fractions from the column chro­
matography were applied to SDS-PAGE, andthe proteins were transferred to PVDF
membranes. The membranes were immuno­
stained with 1 μg/ml of an anti-RIEDL tag
mAb (LpMab-7; left panel) or an anti-CD20
mAb (clone EP459Y; right panel) and incu­
bated with peroxidase-conjugated secondary
antibody
specificformouse
immunoglobulins.

a glycine-HCl buffer elution.

Gel Stain. The 35 kDa protein was detected in the 2 � RIEDL peptide
elution fractions (fraction-2, 3, 4, 5, and 10) and in the glycine-HCl
elution fractions (fraction-1, 2, and 3), indicating that
CD20-169RIEDL170 was not completely eluted by 2 � RIEDL peptide fromthe LpMab-7-Sepharose column, and instead could be further eluted
using an additional glycine-HCl elution. (Fig. 2C).
Next, we assessed CD20-169RIEDL170 protein by Western blot anal­
ysis using LpMab-7 (anti-RIEDL) and clone EP459Y (anti-CD20 mAb). As
shown in Fig. 2D (left panel), LpMab-7 clearly detected a 35 kDa-band in
the 2 � RIEDL peptide elution fractions (fraction-2, 3, 4, 5, and 10), andin the glycine-HCl elution fractions (fraction-2 and 3) at a higher in­tensity, indicating that the glycine-HCl elution is more effective than the
2 � RIEDL peptide elution. Anti-CD20 mAb also detected the same 35
kDa-band (Fig. 2D, right panel).
These results demonstrated that the RIEDL tag, which was inserted
into a loop structure of CD20, was sensitively detected by LpMab-7 inflow cytometry, and was useful for the purification of CD20 using

3.3. Insertion of the RIEDL tag into a loop region of CD20
We investigated whether anti-RIEDL tag mAb (LpMab-7) could stilldetect the RIEDL tag when the tag was inserted into a loop region of
CD20 (Fig. 2A). We inserted the RIEDL sequence between Pro169 and
Ala170, and called this construct CD20-169RIEDL170. In flow cytometry,
LpMab-7 detected CD20-169RIEDL170, which was overexpressed in CHOK1 cells (Fig. 2B). As a positive control, an anti-CD20 mAb (C20Mab-11)
also detected CD20-169RIEDL170.
CD20-169RIEDL170 was purified using the same purification strategy
with RIEDL-wPDPN. Cell lysate, flow-through of the first application,
several wash fractions (Triton wash-1, Triton wash-5, PBS wash-1, andPBS wash-5), elution fractions using 2 � RIEDL (fraction-1, 2, 3, 4, 5,
and 10), and elution fractions using glycine-HCl (fraction-1, 2, and 3)
were applied to SDS-PAGE, followed by staining with Oriole Fluorescent4

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Biochemistry and Biophysics Reports 23 (2020) 100780

LpMab-7.

Declaration of competing interest

4. Discussion

The authors declare no conflicts of interest involving this article.

Tag systems are an essential tool for biological experiments. In thisstudy, we sought to construct a novel tag system by searching for a highaffinity mAb with a corresponding small-sized epitope from our original
mAbs library [17]. We selected LpMab-7, the epitope of which was
identified as the pentapeptide Arg79, Ile80, Asp81, Glu82, and Leu83(RIEDL) [16]. We named the combination of RIEDL tag and LpMab-7
mAb the “RIEDL tag system.” To investigate the utility of this tag sys­
tem in protein purification, we purified two RIEDL-tagged proteins,
including RIEDL-wPDPN and CD20-169RIEDL170, using affinity column
chromatography with LpMab-7-Sepharose. The RIEDL-tagged proteins
were purified by competitive elution with 2 � RIEDL peptide, and theresulting purified proteins exhibited high purity (Figs. 1C and 2C),
indicating that RIEDL-tagged proteins can be eluted without denatur­
ation and purified in one step by affinity chromatography.
RIEDL-wPDPN was efficiently and completely eluted using 2 �
RIEDL peptide elution from the LpMab-7-Sepharose column, as we
observed no further elution with acidic buffer (Fig. 1D). Furthermore,
RIEDL-wPDPN could be eluted using 2 � RIEDL peptide even after anacidic buffer elution step (Suppl. Fig. 1), demonstrating that the 2 �
RIEDL peptide could elute RIEDL-wPDPN more efficiently than acidic
buffer. In contrast, CD20-169RIEDL170 could not be completely eluted
from the LpMab-7-Sepharose column using this method, and required an
additional elution step using acidic buffer (Fig. 2D). This result indicatesthat LpMab-7 might bind more strongly to the RIEDL tag, which wasinserted into a loop structure in CD20. Although the 2 � RIEDL peptide
did not completely elute CD20-169RIEDL170 in one step, the RIEDL tag
system was shown to be useful not only for detecting CD20 in flow
cytometry, but also for purifying CD20 when the RIEDL sequence wasinserted into a loop structure of CD20.
The lengths of well-known tags, including FLAG [8], Myc [10], andPA [1], are 8 amino acids, 10 amino acids, and 12 amino acids,
respectively. Small peptide tags do not interfere with the structure andfunction of target proteins; therefore, these tag systems are very usefulfor detecting functional proteins. However, it was shown that FLAG and
Myc tags inserted into loop structures in integrin could not be detectedby anti-tag mAbs [1], and as a result, tags are usually added to the N- or
C-termini of target proteins. In contrast, the PA tag inserted into loop
structure could be detected by an anti-PA mAb, even though this tag is adodecapeptide [1]. The most critical characteristic of our new RIEDL tag
system is its use of a very short peptide tag sequence, the pentapeptide“RIEDL.” Therefore, the RIEDL tag system might be useful not only for
detection and purification of transmembrane proteins, but also forfurther functional biological analyses without interfering with proteinfunction. If needed, we further insert protease recognition sequences
between RIEDL tag and proteins. We did not confirm whether the
structure or function of RIEDL tagged proteins were affected by insertingRIEDL tag. In the future study, we should perform additional experi­ments for investigating the structure or function of RIEDL-tagged pro­teins compared to RIEDL tag-removed proteins.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2020.100780.
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Funding
This research was supported in part by AMED [grant numbers:
JP20am0401013, JP20am0101078, JP20ae0101028] (Y⋅K.).

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