ORIGINAL RESEARCH
published: 10 September 2020
doi: 10.3389/fpls.2020.510444

RAP Tag and PMab-2 Antibody: A
Tagging System for Detecting andPurifying Proteins in Plant Cells
Kenji Miura 1,2*, Hideki Yoshida 1,2†, Shohei Nosaki 1,2, Mika K. Kaneko 3
and Yukinari Kato 3,4
1 Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan, 2 Tsukuba-Plant Innovation ResearchCenter, University of Tsukuba, Tsukuba, Japan, 3 Department of Antibody Drug Development, Tohoku University Graduate
School of Medicine, Sendai, Japan, 4 New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan

Edited by:
Goetz Hensel,
Heinrich Heine University Düsseldorf,
Germany
Reviewed by:
Kisung Ko,
Chung-Ang University, South Korea
Kazuhito Fujiyama,
Osaka University, Japan*Correspondence:
Kenji Miura
miura.kenji.ga@u.tsukuba.ac.jp†

Present address:
Hideki Yoshida,
Bioscience and Biotechnology
Center, Nagoya University,
Nagoya, Japan
Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 06 November 2019
Accepted: 26 August 2020
Published: 10 September 2020
Citation:
Miura K, Yoshida H, Nosaki S,
Kaneko MK and Kato Y (2020) RAP
Tag and PMab-2 Antibody: A Tagging
System for Detecting and Purifying
Proteins in Plant Cells.
Front. Plant Sci. 11:510444.
doi: 10.3389/fpls.2020.510444

An afﬁnity tag system requires both high afﬁnity and speciﬁcity. The RAP tag epitope
DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is speciﬁcally recognized by
PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of
PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2
possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a usefultagging system for detecting and purifying proteins from plant cells. The heavy chain of
PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the lightchain of the antibody were introduced into N. benthamiana by agroinﬁltration. The
expression of PMab-2 peaked 4 days after agroinﬁltration, and approximately 0.3 mg/g
fresh weight of the antibody was accumulated. After puriﬁcation, the plant-derived PMab-2
successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost thesame binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed inplant cells were speciﬁcally recognized by PMab-2. These results indicate that PMab-2
can accumulate at high levels in N. benthamiana and is easily puriﬁed and that the RAP
tagging system presents a useful tool for detecting and purifying proteins of interest in
plant cells.
Keywords: transient expression, tagging system, RAP tag, agroinﬁltration, monoclonal antibody, protein
expression, protein puriﬁcation, plant biochemistry

INTRODUCTION
Afﬁnity tag systems are useful for detecting and purifying target proteins and are classiﬁed into
peptide and protein tags. Protein tags, including green ﬂuorescent protein (GFP), b-glucuronidase(GUS), and HaloTag (Lang et al., 2006), are used in plant cells and are generally large in size.
Although these tags are very useful as reporter proteins to detect protein localization or expression
levels (Miura et al., 2013; Ohta et al., 2018), they sometimes affect the characteristics of the target
proteins. Peptide tags, such as the FLAG and HA tags, are also commonly used in plant cells. These
small tags are less likely to affect the structure and function of target proteins (Book et al., 2010).
However, these peptide tags are sometimes not suitable, because of their low speciﬁcity, low afﬁnity,

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or difﬁculty in achieving protein elution. For example, if theexpression level of the His-tagged target protein is low in theplant cells, the antibodies cannot detect the target proteins easily
due to cross-reactions with other metal-binding proteins (Lichty
et al., 2005). To assess different target proteins in plant cells,various tags are required. Immunoafﬁnity chromatography,
which uses anti-peptide monoclonal antibodies (mAbs), is avery powerful tool for purifying scarce recombinant proteins,because of its high speciﬁcity and afﬁnity.
Previously, a mouse mAb (clone PMab-2) was used against
the platelet aggregation-stimulating domain of rat podoplanin(PDPN) (Oki et al., 2015). The PMab-2 antibody has highspeciﬁcity for rat PDPN; however, it does not react withhuman and mouse PDPN. This antibody possesses high afﬁnity
and speciﬁcity for the RAP epitope tag DMVNPGLEDRIE, which
consists of 12 amino acids (Fujii et al., 2017). On expressing
human epidermal growth factor receptor (EGFR) fused with RAP
tag in CHO-K1 and LN229 cells, a single, strong band of EGFRRAP was detected by PMab-2 (Fujii et al., 2017). These results
suggest that the PMab-2 and RAP system can be used in plant
cells. If a high level of antibody production is achieved in plant
cells, the cost of antibody production may be reduced.
Because plants can generate large quantities of proteins at low
cost and present low risk of contamination by animal or humanpathogens, they have been proposed as bioreactors for producing
biosimilar recombinant proteins (Tschofen et al., 2016; Donini andMarusic, 2019). Recombinant proteins are produced in plants using
two different systems, i.e., stable genetic transformation andtransient gene expression (Donini and Marusic, 2019). The
production of recombinant proteins by transgenic plants ishighly time-consuming and often results in a low yield (Tschofen
et al., 2016). In contrast, a high yield of recombinant proteins canbe obtained within 1–2 weeks by a transient gene expression systemwith a “deconstructed” viral vector system (Ibrahim et al., 2019).
The magnICON system, based on the tobacco mosaic virus
replication system, is one of the most renowned deconstructedviral vector systems (Gleba et al., 2005; Marillonnet et al., 2005).
We recently developed one of the most efﬁcient transient protein
expression systems in plant cells, called the Tsukuba system(Yamamoto et al., 2018; Hoshikawa et al., 2019; Suzaki et al.,2019). The pBYR2HS vector exhibits geminiviral replication andincludes a double terminator. On expressing GFP in Nicotiana
benthamiana using this system, approximately 4 mg/g fresh weight(FW) of GFP was able to be produced (Yamamoto et al., 2018).
To determine if a protein complex can also by produced by the
Tsukuba system, PMab-2 was expressed in N. benthamiana in thepresent study. The heavy chain (HC) of PMab-2 was fused with
KDEL, an endoplasmic reticulum (ER) retention sequence, andwas co-expressed with the light chain (LC) of PMab-2, resulting
in the formation of an immune complex. The plant-produced
PMab-2 exhibited similar activity to that produced in CHO cells.
Furthermore, RAP-tagged target proteins were expressed in plant
cells, and then detected speciﬁcally and puriﬁed using the PMab-2
antibody. The results indicated that the RAP tag can be used forboth protein detection in plant cells using western blotting andprotein puriﬁcation from plant soluble extracts.

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MATERIALS AND METHODS
Vector Construction and Preparationof the Agrobacterium Suspension
N. benthamiana codon-optimized HC, fused with SEKEDL at the
C-terminus for retention at the ER, and LC genes of the PMab-2
monoclonal antibody (Fujii et al., 2017) were synthesized using
GeneArt Strings DNA Fragments (Thermo Fisher Scientiﬁc). The
HC and LC genes were ampliﬁed using the primers pBYR2HSMYL-F and pBYR2HS-KDEL-R, and pBYR2HS-MRF-F andpBYR2HS-stopC-R (Supplemental Table S1), respectively. The
PCR products were introduced into SalI-digested pBYR2HS(Yamamoto et al., 2018), using the In-Fusion HD Cloning Kit(Takara Bio). The resulting constructs were designated as either
pBYR2HS-PMab2H or pBYR2HS-PMab2L, respectively.
Agrobacterium tumefaciens GV3101, harboring either pBYR2HSPMab2H or pBYR2HS-PMab2L, were grown separately in L-broth
medium containing 10 mM MES (pH 5.6), 20 µM acetosyringone,
100 mg/L kanamycin, 30 mg/L gentamycin, and 30 mg/L rifampicin,
up to the stationary phase, at 28°C. The Agrobacterium culture wasthen centrifuged at 3,700 ×g for 15 min, the supernatant was
discarded, and the A. tumefaciens pellet was resuspended in theinﬁltration buffer [10 mM MgCl2, 10 mM MES (pH 5.6), and 100µM acetosyringone]. The concentration of A. tumefaciens in thesuspension was adjusted to OD600 = 1. The suspensions of A.
tumefaciens harboring pBYR2HS-PMab2H and pBYR2HSPMab2L were mixed at a ratio of 1:1.

Plant Growth Conditions andAgroinﬁltration
N. benthamiana plants were grown for 5–6 weeks, at 25°C andunder a 16-h light/8-h dark photoperiod. Five hundred millilitersof the mixed A. tumefaciens suspension was poured into abeaker. Plant leaves were soaked in the suspension, andvacuum inﬁltration was performed (Leuzinger et al., 2013).
After inﬁltration, N. benthamiana were incubated at 20°C in a
growth chamber, under a 16-h light/8-h dark photoperiod.

Puriﬁcation of Recombinant PMab-2
Antibodies From Agroinﬁltrated Leaves
PMab-2 was puriﬁed following a previously described method(Ocampo et al., 2016) with some modiﬁcations. Brieﬂy, 10 g ofagroinﬁltrated N. benthamiana leaves were ground in liquid
nitrogen using a mortar and pestle. The powdered tissue was
mixed with 40 ml of extraction buffer (0.5 NaCl, 45 mM TrisHCl, 1 mM EDTA, 40 mM ascorbic acid, 1 mM PMSF; pH 7.5)
and then agitated on ice for 1 h. The suspension was ﬁlteredthrough Miracloth (Meck Millipore), and centrifuged twice at
44,000 ×g for 30 min at 4°C. The supernatant was ﬁlteredthrough an Omnipore Membrane Filter (pore size 0.2 µm;
Merck Millipore). PMab-2 was then puriﬁed using the AKTA
start system equipped with a HiTrap Protein G HP column (GEHealthcare). Once the antibodies had bound to Protein G, thecolumn was washed with the aforementioned extraction buffer
without ascorbic acid. The antibodies were eluted with 0.1 M
glycine-HCl (pH 2.7) and neutralized with 60 µl of 1 M Tris-HCl

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FW/ml). To load a crude extract equivalent to 0.1 mg FW, 5 ml of
the sample solution was applied onto an SDS-PAGE gel, which
was then stained with Coomassie Brilliant Blue (CBB). The
protein was transferred onto a PVDF membrane (Amersham
Hybond P PVDF, GE Healthcare). The blot was probed withplant-derived PMab-2 and detected using Luminata Forte
Western HRP substrate (Millipore).
To compare the protein expression levels, all proteins wereapplied onto the same gel. The band intensity was measured using
ImageJ software, and the protein concentration was calculated as
described previously (Yamada et al., 2020). As shown in Figure 3,
the indicated weight of the puriﬁed PMab2 antibody was loaded
onto the gel. The standard line was calculated based on the band
intensities and protein concentration. Protein weight wascalculated using the standard line and then divided by 0.1 mg FW.

(pH 9.0) per ml fraction. The elutant was concentrated using
Vivaspin (Sartorius) with 1 × PBS, at approximately 20 times.

Construction of RAP Tag Fused With
Phytochrome Interacting Factor 4 (PIF4),
the C-Terminal Region of BIG, and Bet v 1
To introduce the RAP tag into the pBYR2HS vector, PCR products
containing the His-tag (HHHHHH), RAP tag (DMVNPGLEDRIE),
and recognition site for HRV 3C protease (LEVLFQGP), were
produced using the primers pBYR2HS-Hisx6-F, His-RAPHRV3C, and pBYR2HS-HRV3C-R, or pBYR2HS-HRV3C-F,
HRV3C-RAP-His, and pBYR2HS-stopHis-R, respectively(Supplemental Table S1). These two PCR products were thenintroduced into SalI-digested pBYR2HS, using the In-Fusion HD
Cloning Kit. The resulting constructs were designated as either
pBYR2HS-NHisRAP or pBYR2HS-CRAPHis, respectively. The SalI
recognition site in these vectors functioned as a cloning site.
Total RNA was extracted from Arabidopsis thaliana aspreviously described (Ohta et al., 2018). Brieﬂy, total cDNA
was synthesized using an oligo(dT) primer and SuperScript III
Reverse Transcriptase (Thermo Fisher Scientiﬁc), according to
the manufacturer’s instructions. PIF4 was ampliﬁed using theprimers pBYR2HS-dNPIF4-F and pBYR2HS-dNPIF4RH-R(Supplemental Table S1). The PCR products were introduced
into SalI-digested pBYR2HS-CRAPHis, using the In-Fusion HD
Cloning Kit, and the resulting construct was designated as
pBYR2HS-dNPIF4RH.
Speciﬁc cDNA for the BIG gene was synthesized from the total
RNA of A. thaliana, using the BIG-15297R primer (Supplemental
Table S1) and SuperScript III Reverse Transcriptase. This BIG
cDNA was then used as a template to amplify the C-terminal
region of Arabidopsis BIG gene, using the primers pBYR2HS-HRBIG12466-F and pRI201-AtBIG-R (Supplemental Table S1). The
PCR products were then introduced into SalI-digested pBYR2HSNHisRAP, using the In-Fusion HD Cloning Kit, and the resulting
construct was designated as pBYR2HS-HRBIG12466.
Nicotiana tabacum codon-optimized Bet v 1A gene (Yamada
et al., 2020) was ampliﬁed using the primers HRV3C-Betv1Nt-F
and pRITetI-Betv1Nt-R (Supplemental Table S1). The PCR
products were introduced into SalI-digested pBYR2HSNHisRAP, using the In-Fusion HD Cloning Kit, and theresulting construct was designated as pBYR2HS-HRBetv1Nt.

Puriﬁcation of RAP-Tagged and FLAGTagged Target Proteins
PIF4-RAP was expressed in N. benthamiana by inﬁltration withpBYR2HS-dNPIF4RH. Soluble extracts were prepared from N.
benthamiana leaves. Plant-derived PMab-2, puriﬁed from the N.
benthamiana leaves, was incubated with 50 ml of Dynabeads
Protein G (Thermo Fisher Scientiﬁc). The Dynabeads Protein GPMab-2 complex was then rinsed off with PBS, and the soluble
extracts containing the PIF4-RAP protein were added to thecomplex and incubated. After washing the complex with PBS, theRAP-tagged proteins were extracted using 50 ml of elution buffer(50 mM glycine; pH 2.7) and neutralized with 3 ml of 1 M TrisHCl (pH 9.0).
His-Flag-Bet v 1 or His-RAP-Bet v 1 was expressed in N.
benthamiana by inﬁltration with pBYR2HS-HFBetv1Nt (Yamada
et al., 2020) or pBYR2HS-HRBetv1Nt, respectively. Afterincubation for 4 days, plant leaves (5 g) were ground, the lysis
buffer was added to the powdered leaves to obtain a concentrationof 0.5 g FW/ml, and the soluble extract was prepared.
Dynabeads Protein G (50 ml; Thermo Fisher Scientiﬁc) was
added to a magnetic stand to remove the supernatant. Then, 1 mgof the anti-FLAG antibody (anti-DYKDDDDK antibody,
Fujiﬁlm Wako Pure Chemical Corp.) or plant-derived PMab-2
was added. The Dynabeads Protein G-PMab-2 complex was thenrinsed off with PBS, and the soluble extracts (5 ml) were added tothe complex and incubated for 1 h. After washing the complex
with PBS, the RAP- or FLAG-tagged proteins were extractedusing an SDS buffer (60 mM Tris-HCl, 2% SDS, 10% glycerol, 5%2-mercaptoethanol, and 0.025% bromophenol blue; pH 6.8). The
extract was separated by SDS-PAGE, and western blot analysis
was performed using anti-poly-His tag antibody (Abcam).

Protein Extraction and Immunoblot Analysis
The soluble protein was prepared following a previously
described protocol (Miura et al., 2012; Yamamoto et al., 2018).
Brieﬂy, plant leaves (100–200 mg) were ground, and lysis buffer[50 mM Tris-HCl, 120 mM NaCl, 0.2 mM sodium
orthovanadate, 100 mM NaF, 10% glycerol, 0.2% Triton X-100,
5 mM DTT, and 1× protein inhibitor cocktail (Nacalai Tesque,Inc., Kyoto, Japan); pH 8.0] was added to the powdered leaves toobtain a concentration of 0.2 mg FW/ml. The mixture wasincubated for 1 h. The liquid solution obtained after removing
debris by centrifugation served as the soluble protein extract. The
solution was the diluted 10 folds using the lysis buffer (0.02 mg

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Flow Cytometry
Cells were harvested by brief exposure to 0.25% trypsin/1 mM
EDTA (Nacalai Tesque, Inc.). After rinsing with 0.1% BSA/PBS,
the cells were treated with primary mAbs for 30 min at 4°C, andthen with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000;
Cell Signaling Technology, Inc., Danvers, MA). Fluorescencedata were collected using EC800 Cell Analyzer (Sony Corp.).

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weights of 50 and 25 kDa, respectively (Figure 2). The fulltetrameric assembly of PMab-2 (2 HCs and 2 LCs) was
conﬁrmed by immunoblot analysis using native-PAGE(Figures 3A, B). Accumulation of PMab-2 was the highest 4
days post inﬁltration, exhibiting a value of approximately 0.3 mg/
gFW (Figure 3C).
N. benthamiana expressing PMab-2 HC and LC were puriﬁedusing a Protein G afﬁnity column, as previously described (Husk
et al., 2014). SDS-PAGE and CBB staining indicated that PMab-2
produced in N. benthamiana can be puriﬁed by a single afﬁnity
chromatography step, with a high level of purity and intact HC
and LC (Figure 3D), indicating that the current puriﬁcation
method of PMab-2 worked well.

RESULTS
Transient Expression of RecombinantPMab-2 in N. benthamiana Leaves
Codon-optimized PMab-2 HC and LC were introduced intopBYR2HS to obtain pBYR2HS-PMab2H and pBYR2HS-PMab2L,
respectively (Figure 1). A previous study demonstrated that theexpression level of IgG in N. benthamiana leaves was higher when
the HC of the antibody was expressed in the ER (Phoolcharoen
et al., 2011). Thus, PMab-2 HC was fused with KDEL, an ER
retention sequence. Agrobacterium harboring either pBYR2HSPMab2H or pBYR2HS-PMab2L were incubated separately
overnight. The two Agrobacterium solutions were then mixed andinﬁltrated into N. benthamiana.
Soluble protein extracts were prepared from N. benthamianaleaves 3 to 7 days post inﬁltration. PMab-2 HC and LC weredetected using anti-mouse IgG (H) antibody (Figure 2A) andanti-mouse IgG (L) antibody (Figure 2B), respectively. Western
blot analyses revealed that PMab-2 HC and LC were expressed in
N. benthamiana leaves, exhibiting the expected molecularComparison of RAP Tag Recognition by
Plant- and CHO Cell-Derived PMab-2
The RAP epitope tag sequence, DMVNPGLEDRIE, is includedin PDPN (Fujii et al., 2017). PDPN was expressed in CHO-K1
cells, and recognition of the expressed PDPN by plant-derived
PMab-2 was examined.

FIGURE 1 | Schematic representation of the T-DNA regions of the plasmids, including pBYR2HS-PMab2H and pBYR2HS-PMab2L, used for protein expression.
35S-p × 2, CaMV 35S promoter with double-enhanced element; AtADH5′, 5′-untranslated region (UTR) of Arabidopsis thaliana alcohol dehydrogenase gene; KDEL,
Lys-Asp-Glu-Leu sequence for endoplasmic reticulum retention; HSPter, heat shock protein gene terminator; Ext3′, tobacco extensin gene 3′ element; LIR, longintergenic region of the bean yellow dwarf virus (BeYDV) genome; SIR, short intergenic region of the BeYDV genome; C1/C2, BeYDV ORFs C1 and C2 encoding
replication initiation protein (Rep) and RepA, respectively; LB and RB, left and right borders of the T-DNA region, respectively; Nos-p and Nos-t, NOS promoter andterminator, respectively; p19, a gene-silencing suppressor gene from tomato bushy stunt virus; HRV3C, HRV 3C protease recognition site.

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A

B

FIGURE 2 | Expression of PMab-2 in N. benthamiana leaves. N. benthamiana leaves were inﬁltrated with an Agrobacterium mixture containing pBYR2HS-PMab2H andpBYR2HS-PMab2L and harvested at the indicated days post inﬁltration. Total protein extracts of N. benthamiana leaves were separated by 12% SDS-PAGE andtransferred onto PVDF membranes. The membranes were incubated with anti-mouse IgG (H) to detect the HC (A) or anti-mouse IgG (L) to detect the LC (B). Four
samples each for HC and LC were subjected to SDS-PAGE. The large subunit of rubisco was detected by CBB staining as a loading control.

were prepared from N. benthamiana leaves 4 days after incubationand separated by SDS-PAGE. PIF4-RAP and RAP-BIG-C proteins
were detected using PMab-2 puriﬁed from N. benthamiana(Figures 5A, B).
Proteins were then puriﬁed using the RAP tag. The plantderived PMab-2 antibody interacted with Dynabeads Protein G.
Crude extracts from N. benthamiana inﬁltrated with pBYR2HSdNPIF4RH were incubated with the PMab-2-bonded Dynabeads
Protein G. The RAP-tagged target protein and PMab-2 complex
were puriﬁed using Dynabeads Protein G. The immunoprecipitate
was separated by SDS-PAGE and analyzed by immunoblottingwith PMab-2 antibody. As shown in Figure 5C, the PIF4-RAP
protein could be puriﬁed from crude plant extracts using the RAP
tag and PMab-2.
To assess if the RAP tagging system could be applied for
detecting and purifying proteins, we compared its efﬁciency withthat of the FLAG tagging system. We used Bet v 1 because HisFLAG-Bet v 1 (HF-Betv1) is known to be well-expressed in N.
benthamiana (Yamada et al., 2020). His-RAP-Bet v 1 (HRBetv1) was also expressed in N. benthamiana, and both HFBetv1 and HR-Betv1 exhibited similar expression levels in N.
benthamiana (Figure 6B). Then, the same amount of soluble
proteins was used for immunoprecipitation with anti-FLAG
antibody or plant-derived PMab-2, and western blot analysis
was performed using anti-poly-His antibody (Figure 6A). Basedon the resulting blot, the puriﬁcation efﬁciency was found to be
similar for both RAP and FLAG tagging systems.

Fluorescence-activated cell sorting (FACS) analysis indicatedthat plant-derived PMab-2 reacted with rat PDPN expressed in
CHO-K1 cells in a manner similar to CHO-derived PMab-2(Figure 4A). No signiﬁcant differences were observed between
CHO-K1 cells treated with plant-derived and CHO-produced
PMab-2 (Figure 4B).

The RAP Tag Is a Useful Peptide Tag for
Plant Cells
PMab-2 shows high afﬁnity and speciﬁcity for detecting RAPtagged target proteins in mammalian cells (Fujii et al., 2017). To
conﬁrm the applicability of this afﬁnity tag system in plant cells,
RAP-tagged proteins were expressed and detected in plant cells
using plant-derived PMab-2. Arabidopsis PIF4 is a basic helixloop-helix-type transcription factor that interacts with the red
light receptor phytochromes (Huq and Quail, 2002). The
Arabidopsis BIG protein is a large, calossin-like protein
required for polar auxin transport (Gil et al., 2001). The
coding sequence of the BIG gene is approximately 15 kb long,hence the name “BIG”. In this study, the C-terminal region of
BIG (2,769 bp) was used. It is difﬁcult to produce PIF4 in an E.
coli expression system. The BIG protein has not been analyzed
extensively because of its large size. Thus, we determined if these
proteins could be produced using our system.
The two genes were introduced into pBYR2HS. Agrobacterium
harboring either pBYR2HS-dNPIF4RH or pBYR2HS-HRBIG12466
were then inﬁltrated into N. benthamiana. Soluble protein extracts

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A

B

D

C

FIGURE 3 | Expression and puriﬁcation of PMab-2. N. benthamiana leaves were inﬁltrated with an Agrobacterium mixture containing pBYR2HS-PMab2H andpBYR2HS-PMab2L and harvested at the indicated days post inﬁltration. Total protein extracts of N. benthamiana leaves were separated by native-PAGE andtransferred onto PVDF membranes. The membranes were incubated with anti-mouse IgG (H) to detect the HC (A) or anti-mouse IgG (L) to detect the LC (B).
Because these proteins formed a complex, bands of similar size were detected. Four samples each for HC and LC were loaded onto SDS-PAGE. (C) Protein
concentration was measured based on the western blot band intensity using ImageJ software. (D) Puriﬁcation of PMab-2 from protein extracts of N. benthamianaleaves inﬁltrated with pBYR2HS-PMab2H and pBYR2HS-PMab2L using the Protein G column. Puriﬁed PMab-2 were separated by SDS-PAGE and the gel was
stained with CBB. Two representatives of four independent experiments are shown.

ampliﬁcation of two different replicons can be achieved using ageminiviral replication vector (Phoolcharoen et al., 2011; Diamos
et al., 2016; Rattanapisit et al., 2017). The most renowned andwidely used transient expression system is the magnICON
system (Marillonnet et al., 2005). In this system, viral vectors
derived from the tobacco mosaic virus and potato virus X areused to produce recombinant proteins with two heterosubunits(Giritch et al., 2006). Secretory IgA, IgM, and heteromultimericvirus-like particles can be efﬁciently produced using these twoviral vectors (Chen and Lai, 2013). Production of more than twoheterosubunits is difﬁcult because of the competing nature ofmany RNA viruses. Previously, four heterosubunits weresuccessfully assembled using a non-replicating system, basedon the ability of the cowpea mosaic virus to produce
bluetongue virus-like particles (Thuenemann et al., 2013),
albeit with a low recombinant protein yield.
In this study, the yield of PMab-2 in N. benthamiana wasapproximately 0.3 mg/gFW. Increasing the antibody yield would

DISCUSSION
An afﬁnity tag system should possess both high afﬁnity andspeciﬁcity. PMab-2, a rat anti-mouse PDPN mAb, speciﬁcallyrecognizes the RAP tag epitope DMVNPGLEDRIE. In this study,
high levels of PMab-2 production, reaching approximately 0.3
mg/gFW, were achieved in N. benthamiana using a newly
developed expression system. The results conﬁrmed that plantproduced PMab-2 retained its antigen-binding activity andspeciﬁcity, similar to its mammalian CHO cell-derived
counterpart (Figure 4). Furthermore, the efﬁciency of the RAP
tag as a tool for protein detection in plant cells was demonstrated.
RAP-tagged proteins expressed in N. benthamiana could be
detected using plant-derived PMab-2 (Figure 5). In addition,
the RAP tagging system was found to be as useful for protein
puriﬁcation as the FLAG tagging system (Figure 6).
Efﬁcient complex formation is required to produce antibodies
in plant cells. It has previously been shown that non-competing

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B

FIGURE 4 | Fluorescence-activated cell sorting (FACS) analysis of rat podoplanin (PDPN) expressed in CHO-K1 cells. PMab-2 derived from N. benthamiana leaves
or CHO cells was used. CHO-K1/rat PDPN cells (A) and CHO-K1 cells (B) were treated with the indicated concentration of PMab-2 puriﬁed from plant leaves or
CHO cells, for 30 min at 4°C, followed by 1:1,000 dilution with Oregon Green 488 goat anti-mouse IgG. Fluorescence data were collected using Cell Analyzer
EC800. Because it has previously been shown that CHO-derived PMab-2 can recognize the RAP tag in PDPN (Fujii et al., 2017), detection of PDPN from CHO-K1/
rat PDPN cells by CHO-derived PMab-2 was established as the positive control non-detection of any protein from CHO-K1 cells by CHO-derived PMab-2 wasestablished as the negative control.

consisting of a series of 6–12 histidines. This tag is used for
puriﬁcation by immobilized metal afﬁnity chromatography(IMAC). IMAC is a cheap afﬁnity chromatography techniquewhen compared with afﬁnity chromatography using an antibody.
Although this is a clear advantage, IMAC captures other metalbinding proteins in addition to the His-tagged target proteins(Lichty et al., 2005). The His-tagging system with IMAC iseffective for purifying large amounts of recombinant proteins.
Conversely, other small tagging systems are useful for identifying
protein functions. Several peptide tags, such as the FLAG (Xiaoet al., 2019; Cui et al., 2020; Miura et al., 2020; Uemura et al.,2020), HA (Shinozawa et al., 2019; Kuhnert et al., 2020), and cMYC (Earley et al., 2006; Baudisch et al., 2018; Roshan et al.,2018) tags are used in plants because of their speciﬁcity. Thesetags may affect the solubility or insolubility of the expressed
proteins and their activities, depending on the characteristics ofthe expressed proteins (Ki and Pack, 2020). Thus, availability ofdifferent protein tagging systems is desirable.

make it easier to obtain and purify antibodies from N. benthamiana.
Changing the target location of recombinant proteins also affects
their accumulation in N. benthamiana. Accumulation of HA washigher when the protein targeted the ER instead of the apoplast. The
effect of leaf tissue necrosis caused by ER-targeted HA was
suppressed at lower temperatures (Matsuda et al., 2017). On
expressing the monoclonal antibody 14D9, which catalyzes
enantioselective protonation (Zheng et al., 2004), fused with thevacuolar targeting signal (KISIA or NIFRGF) or ER retention signal(SEKDEK) in N. benthamiana, the accumulation of ER-mAb andvacuolar mAb was 10–15 folds higher than that of the secreted mAb(Ocampo et al., 2016). In the present study, PMab-2 was fused with
KDEL. Another strategy is required to increase the antibody yield.
Small peptide tags are more useful than large peptide tags
because the latter may perturb protein trafﬁcking and function
and protein-protein interactions (Lotze et al., 2016), and theformer were developed to avoid potential interference withprotein function. The His-tag is a well-known small tag

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RAP-Tag and PMab-2 Antibody in Plant Cells

A

B

C

FIGURE 5 | Applicability of the RAP tag in plant cells. PIF4-RAP (A) or RAP-BIG-C (B) were expressed in N. benthamiana. Total soluble extracts were separated
by 12% SDS-PAGE and transferred onto PVDF membranes. PIF4-RAP and RAP-BIG-C were detected using PMab-2 puriﬁed from N. benthamiana leaves.
(C) Puriﬁcation of RAP-tagged PIF4 proteins from soluble extracts of N. benthamiana leaves. PIF4-RAP was puriﬁed using Dynabeads Protein G with plant-derived
PMab-2.

PMab-2 exhibited the same binding activity as the CHOderived PMab-2. Furthermore, the RAP-tagged proteins were
speciﬁcally recognized by PMab-2 in plant cells, allowing
puriﬁcation by afﬁnity chromatography. These results indicate
the potential of the RAP tag for use in plant cells anddemonstrate that PMab-2 can be easily obtained using ourtransient expression system in N. benthamiana.

A

B

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding author.

AUTHOR CONTRIBUTIONSFIGURE 6 | Comparison of the puriﬁcation of RAP-tagged and FLAG-tagged
Bet v 1. His-FLAG-Bet v 1 (HF-Betv1) and His-RAP-Bet v 1 (HR-Betv1) wereexpressed in N. benthamiana agroinﬁltrated with pBYR2HS-HFBetv1Nt andpBYR2HS-HRBetv1Nt, respectively. (A) HF-Betv1 and HR-Betv1 were
immunoprecipitated with anti-FLAG antibody (IP: FLAG) and PMab-2 (IP: RAP),
respectively. N. benthamiana leaves (NT) were used as a negative control for
immunoprecipitation. Immunoprecipitates were separated by SDS-PAGE, andwestern blot analysis was performed using anti-poly-His antibody to detect both
HF-Betv1 and HR-Betv1. (B) Expression levels of HF-Betv1 and HR-Betv1 in N.
benthamiana were conﬁrmed by western blot analysis using anti-poly-His
antibody. Agroinﬁltrated leaves (100 mg) were harvested and mixed with thelysis buffer (500 ml). Soluble proteins were obtained by centrifugation. The same
volume of the soluble protein mixture was separated by SDS-PAGE. Western
blot analysis was performed using anti-poly-His antibody.

KM and YK conceived and designed the experiments. KM, HY, SN,
and MK conducted the experiments and collected the data. KM
and YK drafted and edited the manuscript.

FUNDING
This work was supported by a Cooperative Research Grant #2020of the Plant Transgenic Design Initiative (PTraD) by Gene
Research Center, Tsukuba-Plant Innovation Research Center(T-PIRC), University of Tsukuba, JSPS Grant-in-Aid for
Scientiﬁc Research on Innovative Areas (19H04637) to KM
and AMED Grant Numbers JP19am0401013, JP19am0101078,
and JP19ae0101028 to YK.

In conclusion, the monoclonal antibody PMab-2 wasexpressed in N. benthamiana, and the RAP-tagged protein
could be detected and puriﬁed using PMab-2. Plant-derived

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RAP-Tag and PMab-2 Antibody in Plant Cells

ACKNOWLEDGMENTS

SUPPLEMENTARY MATERIAL

We thank Ms. Yuri Nemoto and Ms. Yuriko Nagai at the
University of Tsukuba for technical support and Editage(www.editage.com) for English language editing.

The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2020.
510444/full#supplementary-material

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Conﬂict of Interest: The authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that could be construed as apotential conﬂict of interest.
Copyright © 2020 Miura, Yoshida, Nosaki, Kaneko and Kato. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, provided
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