Protein Expression and Puriﬁcation 95 (2014) 240–247

Contents lists available at ScienceDirectProtein Expression and Puriﬁcation
journal homepage: www.elsevier.com/locate/yprep

PA tag: A versatile protein tagging system using a super high afﬁnity
antibody against a dodecapeptide derived from human podoplanin
Yuki Fujii a, Mika Kaneko b, Makiko Neyazaki c, Terukazu Nogi c, Yukinari Kato b,⇑, Junichi Takagi a,⇑aLaboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japanc
Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japanba r t i c l e

i n f oArticle history:
Received 6 December 2013
and in revised form 20 January 2014
Available online 28 January 2014
Keywords:
Puriﬁcation method
Immunoafﬁnity chromatography
Monoclonal antibody
Podoplanin
Peptide tag
Immunodetection
Mammalian cell expressiona b s t r a c t
Peptide-based epitope tagging technology is universally used in nearly all kind of research projects thatinvolve biochemical characterization of a target protein, but not many systems are fully compatible with
puriﬁcation purpose. By utilizing an anti-human podoplanin antibody NZ-1, we constructed a novel epitope tag system. NZ-1 possesses exceptionally high afﬁnity toward a dodecapeptide dubbed ‘‘PA tag’’,with a characteristic slow dissociation kinetics. Because of its high afﬁnity, PA-tagged proteins in a dilute
sample can be captured by immobilized NZ-1 resin in a near complete fashion and eluted by a solution offree PA peptide. This enabled efﬁcient one-step puriﬁcation of various proteins including soluble (anectodomain fragment of neuropilin-1) and membrane (epidermal growth factor receptor) proteins
expressed in mammalian cells. Mild regeneration condition of the peptide-bound antibody ensuresrepeated use of the antibody resin, indicating a cost-efﬁcient nature of the system. Together with its outstanding performance in the immunodetection experiments (i.e., Western blotting and ﬂow cytometry),
PA tag/NZ-1 system will offer a great chance to facilitate protein production in many biomedical research
projects.
Ó 2014 Elsevier Inc. All rights reserved.

Introduction
Protein puriﬁcation step represents a serious bottleneck in abasic research as well as industrial applications where high-quality
pure protein sample is required. Efﬁcient puriﬁcation of a recombinant protein is generally achieved by afﬁnity chromatographyusing genetically fused puriﬁcation handle or ‘‘tag’’. Among numerous gene-fusion technologies, peptide tag systems have several
advantages over ‘‘protein tag’’ systems such as GST-fusion. First,
they are less likely to affect structure and function of the target
proteins due to the small size (typically 1–2 kDa). For the same reason, it is often not necessary to remove the tag portion after the
puriﬁcation. Another advantage is that there are wide variety of
peptide tag systems available we can choose from, including His[1], FLAG [2], HA [3], Myc [4], TARGET [5], Softags [6] and manyothers, unlike relatively short list of protein tags. Finally, peptide
fusion is compatible with virtually all expression hosts and canbe expressed either extracellularly or intracellularly. However,
peptide-based tagging often suffers from the low speciﬁcity and/or
afﬁnity. For example, puriﬁcation of oligohistidine-tagged proteins⇑ Corresponding authors. Tel./fax: +81 22 717 8207 (Y. Kato). Tel.: +81 6 6879
8607; fax: +81 6 6879 8609 (J. Takagi).
E-mail addresses: yukinarikato@med.tohoku.ac.jp (Y. Kato), takagi@protein.
osaka-u.ac.jp (J. Takagi).
1046-5928/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pep.2014.01.009

using metal chelate afﬁnity resin usually results in co-puriﬁcationof metal-binding proteins present in the starting material, necessitating further puriﬁcation steps [7]. Epitope tag systems thatutilize peptide tag and anti-peptide monoclonal antibody (mAb)1
generally show high speciﬁcity, but even the most popular systemsuch as FLAG tag/anti-FLAG M2 antibody [8] sometimes suffers from
non-speciﬁc binding to endogenous proteins in certain cell types[9,10]. Another aspect of the tagging system that should be considered is the elution and regeneration condition for the afﬁnity chromatography. Many epitope tag systems are compatible with themild competitive elution of the tagged protein by free antigen peptide. However, high afﬁnity peptide–antibody interaction is difﬁcult
to disrupt and requires high concentration peptide for the elution
and harsh regeneration process after each run, resulting in the limited reusability of the afﬁnity resin. To overcome such difﬁculty,‘‘polyol-responsive’’ antibodies have been developed where boundantigens can be eluted by solutions containing non-denaturing polyols, eliminating the need for harsh elution/regeneration process[6,11]. However, there are limited number of polyol-responsive
anti-tag antibodies available and it is impossible to ‘‘grant’’ such property to a given antibody. Therefore, there is a continuous need for the1
Abbreviations used: GFP, green ﬂuorescent protein; mAb, monoclonal antibody;
SPR, surface plasmon resonance; TBS, Tris-buffered saline; HEK, human embryonickidney.

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Y. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

development of new afﬁnity tag system with high afﬁnity, high speciﬁcity, and high reusability.
We have established a unique rat monoclonal antibody NZ-1 directed against 14-residue peptide segment in human podoplanin
PLAG domain [12]. Podoplanin is a type I transmembrane protein
highly expressed on malignant cancer cells and is implicated in tumor-induced platelet aggregation by binding to CLEC-2 on platelet
surface [13]. NZ-1 inhibits binding of podoplanin to CLEC-2 and is apotential lead for the antibody-based therapy against glioblastoma[14]. During the above investigation, we realized that NZ-1 possesses desirable character as an anti-tag antibody. Here, we reporta development of a novel afﬁnity tag system consisting of NZ-1 andits epitope peptide dubbed ‘‘PA tag’’. PA tag system proves to outperform many existing afﬁnity tag systems owing to its high afﬁnity, high selectivity, and extended reusability.
Materials and methods
Protein expression
Expression constructs for the T4 lysozyme (T4L) attachedwith various tags shown in the Supplementary Fig. 1 (PA10-T4L,
PA12-T4L, PA14-T4L, T4L-PA10, T4L-PA12, T4L-PA14, T4L-FLAG,
T4L-HA and T4L-Myc) were prepared by extension PCR and cloned
into NcoI-BamHI site of pET16b vector (Novagen). All constructs
contained either C-terminal His8 or N-terminal His10 tags tofacilitate protein puriﬁcation. Single-residue alanine substitution
mutants (G1A, V2A, M4A, P5A, G6A, E8A, D9A, D10A, V11A and
V12A) of T4L-PA12 were produced using QuikChange strategy(Agilent). Construct for GFPUV-PA12 was prepared by replacing
T4L segment of T4L-PA12 with the coding sequence of GFPUV (Clontech). Full-length DNA for human isocitrate dehydrogenase 1(IDH1) was attached with tandem FLAG and PA12 tags at the C-terminus and cloned into pcDNA3.1 vector (Life Technologies Corp.)
as described previously [15]. For the construction of mNrp1ecPA12, DNA encoding the entire ectodomain of mouse neuropilin1 (a gift from A. Kumanogoh) and the linker + PA12 segment from
T4L-PA12 were fused by extension PCR and cloned into pcDNA3.1
vector. DNA sequence coding for C-terminally truncated constructof human epidermal growth factor receptor (EGFR), which included amino acids 25–1022, was fused with the PA12 tag at the
N-terminus and cloned into the pcDNA3.1 vector. Three cysteine
residues present in the cytosolic kinase domain (Cys-775, -781
and -797) were replaced with serine using the QuikChange strategy. For the construction of full-length rat neuropilin-2 taggedwith PA12 tag, rNrp2-mCherry in pSecTag2A vector (a gift from
Y. Goshima) was used to insert PA12 + linker segment (taken fromthe PA12-T4L) after the signal sequence. All constructs were veriﬁed by DNA sequencing.
The tagged T4L proteins were expressed in transformed Escherichia coli BL21 (DE3) cultured for 3 h at 37 °C in the presence of
1 mM IPTG. The T4L proteins were puriﬁed from the soluble fraction of the bacterial lysate by Ni–NTA chromatography and dialyzed against phosphate-buffered saline (PBS), pH 6.0. Expressionof GFPUV-PA12 was similarly conducted except that the transformed bacteria were cultured for 24 h at 16 °C in the presenceof 1 mM IPTG.

monoclonal antibody 4B2 and the mouse anti-Myc monoclonal
antibody 9E10 were obtained from Wako Pure Chemical Industries
Ltd. The FITC-conjugated goat anti-rat IgG polyclonal antibody was
obtained from Beckman Coulter Inc.
Bio-layer interferometry (BLI)
Binding kinetics of various antibodies toward T4L protein
tagged with their respective antigen peptide was analyzed by BLI
using Octet RED system (Pall ForteBio). Binding assays were performed in 96-well microtiter plates at 30 °C with orbital sensor
agitation at 1000 rpm. Amine Reactive (AR2G) sensors were immobilized with each antibody dissolved at 10–20 lg/ml in 10 mM sodium acetate buffer, pH 6.0, followed by quenching with 1 Methanolamine, pH 8.5. Tagged proteins were serially diluted in arunning buffer (PBS containing 0.005% Tween 20 and 1% BSA)
and placed in different wells at a volume of 200 ll. Binding wasmonitored by dipping the sensors into the wells for 60 s, followedby dissociation in the running buffer for 120 s. Binding curves wereanalyzed by BIA evaluation software (GE Healthcare) with a curveﬁtting using a 1:1 binding model.
Binding kinetics analysis using surface plasmon resonance (SPR)
NZ-1 was immobilized on the CM5 sensor chip using amino
coupling chemistry according to the method provided by the manufacturer. The PA tagged T4L proteins were diluted in PBST (PBS
(pH 6.0) containing 0.005% Tween 20) and injected at a ﬂow rateof 20 ll/min. For scouting the condition for the mild dissociationof antigen, the NZ-1 surface was ﬁrst injected with a 100 nM solution of T4L-PA12 to saturate binding sites, incubated with PBST for
200 s to allow free dissociation, followed by an injection of various
buffers for 30 s (Supplementary Fig. 2). Dissociation efﬁciencies
were calculated by the ratio of the binding signal (in RU) before
and after the injection of each solution (Table 1). All SPR experiments were performed using BIACORE 2000 instrument (GEHealthcare).
Western blot analysis
Human osteosarcoma U-2 OS cells (obtained from the American
Type Culture Collection (ATCC), Manassas, VA) were transiently

Table 1
Effect of various buffers on the interaction between PA tag and NZ-1.
Salt, additive

pH (buffer)

Dissociation
efﬁciency (%)

0.15 M NaCl
No

7.5 (Tris)
2.0 (Gly–HCl)091

0.15 M NaCl + 40%
propylene glycol

7.5 (Tris)

8

2 M NaCl

7.0 (PIPES)
6.0 (MES)
5.0 (Acetate)
4.0 (Acetate)

28
30
40
87

2 M NaBr

7.0 (PIPES)
6.0 (MES)
5.0 (Acetate)
4.0 (Acetate)

52
59
77
97*

2 M MgCl2

7.0 (PIPES)
6.0 (MES)
5.0 (Acetate)
4.0 (Acetate)

93
94
96*
98*

3 M MgCl2

6.0 (MES)

98*

Antibodies used
The rat anti-PA monoclonal antibody NZ-1 (IgG2a, k) was described previously [12]. The mouse anti-FLAG monoclonal antibody M2, the HRP-conjugated rabbit anti-rat IgG polyclonal
antibody, and the HRP-conjugated goat anti-mouse IgG polyclonal
antibody were obtained from Sigma–Aldrich. The mouse anti-HA*

Conditions suitable for antigen elution.

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Y. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

transfected with IDH1-FLAG-PA12 and the cell lysate was prepared
as described previously [15]. The lysate was separated on 10% SDS–
PAGE and transferred to a PVDF membrane, followed by blocking
with 4% skim milk in PBS (pH 7.4) with 0.05% Tween 20 for
15 min. For the primary antibody step, the membranes were incubated with varying concentrations of NZ-1 (0.01, 0.1, 1, or 3.5 lg/ml) or 3.5 lg/ml anti-FLAG M2 diluted in Tris-buffered saline, pH
8.0, containing 0.05% Tween 20 (TBST), for 1–60 min. For the secondary antibody step, membranes were incubated for 30 min witheither HRP-conjugated rabbit anti-rat polyclonal antibody or HRPconjugated goat anti-mouse polyclonal antibody (both from Sigma–Aldrich) diluted at 1:6000 in TBST. Four consecutive washes(TBST, 5 min) were conducted between each step. The membranes
were developed with ECL™ Prime reagent (GE Healthcare) andanalyzed using ImageQuant LAS 4000mini (GE Healthcare).
Flow cytometry
CHO-K1 cells (obtained from ATCC) were transiently transfectedwith PA12-rNrp2-mCherry construct. After 48 h, the cells were detached, washed, and resuspended in DMEM with 5% FCS (DMEM/
FCS). The cells were then incubated with DMEM/FCS containing
NZ-1 at various concentrations (0.01, 0.1, 1, or 10 lg/ml) on ice
for 30 min, washed twice with DMEM/FCS, and resuspended in
DMEM/FCS containing FITC-conjugated goat anti-rat IgG antisera(Beckman Coulter Inc.) diluted at 1:100. After 30 min incubation onice, the cells were washed once and suspended in PBS (pH 7.4), andanalyzed on a ﬂow cytometer (Guava EasyCyte, Millipore).

0.1 mg/ml epitope peptide in a step-wise manner (100 ll  10)
as described above.
Results and discussion
Kinetic analysis of NZ-1/PA tag interaction
Previous investigations have indicated that NZ-1 possesses very
high afﬁnity toward podoplanin. Because successful anti-tag antibody system requires the high afﬁnity interaction, we investigatedbinding kinetics between NZ-1 and its antigenic peptide and compared it with three other tag/mAb pairs that are commerciallyavailable as puriﬁcation systems. Antibodies used were M2 (antiFLAG, available from Sigma–Aldrich) [17], 4B2 (anti-HA, available
from WAKO Pure Chemical Industries Ltd.), and 9E10 (anti-Myc,
available from WAKO Pure Chemical Industries Ltd.) [18]. In orderto make fair comparison, all peptide tags were fused C-terminallyto the same model protein T4 lysozyme (T4L) using identicallength of linker (Supplementary Fig. 1). Although all anti-tag mAbs
exhibited reasonably good binding behavior toward each matchedantigenic peptide in the biolayer interferography experiments
using Octet devise (Fig. 1A), NZ-1/PA tag interaction was exceptional in that it did not show dissociation over the assay time offew minutes. In fact, curve ﬁtting of each data set showed exceptionally slow dissociation rate constant for NZ-1/PA interaction,
resulting in much higher overall afﬁnity than other tag systems(Fig. 1B). The very slow dissociation is a property highly desirable
for isolation purpose, because it assures that tagged protein arecompletely captured and does not leak during the washing steps.

Puriﬁcation of PA-tagged proteins
Minimal sequence requirement for the PA tag
Coupling of NZ-1 to CNBr-activated Sepharose 4 Fast Flow (GEHealthcare) was performed according to the protocol provided by
the manufacturer and routinely yielded coupling levels of 2 mg
IgG/ml gel. For the puriﬁcation of soluble mNrp1ec-PA12 secreted
from mammalian cells, HEK293T (obtained from ATCC) cells plated
in a 15-cm culture dish were transiently transfected with the plasmid and 25 ml of culture supernatant was harvested 72 h after
the transfection. Cleared supernatant was mixed with NZ-1-Sepharose (250 ll bed volume) and incubated for 2 h at 4 °C undergentle agitation. The beads were then transferred to a small
column, washed with 5 ml of TBS, and the bound protein was
eluted with TBS containing 0.1 mg/ml epitope peptide (EGGVAMPGAEDDVV). The peptide used in the elution was synthesized
by standard solid-phase synthesis and puriﬁed by reverse-phase
HPLC (Scrum Inc.). The cost of the peptide solution for the elution
would be in the order of $100/100 ml, when using a typical peptide synthesis service. The elution was conducted at room temperature in a step-wise manner (250 ll  10), where dissociation timeof 5 min was given for each elution step. For the puriﬁcation of
EGFR from cell membrane, HEK293S GnT1-cells [16] plated in
two 15-cm culture dishes were transiently transfected with theplasmid and harvested 48 h after the transfection. Cells were detached from the plates using PBS containing 0.5 mM EDTA. The pelleted cells were resuspended in 5 ml of a solution containing
20 mM Tris–Cl (pH 8.0), 400 mM NaCl, 10% glycerol and 1 mM
EDTA and solubilized by adding 5 ml of the same solution containing 0.4% (w/v) Triton X-100. The solubilized cell lysates wereincubated at 4 °C for 1 h with gentle mixing and further ultracentrifuged 30 min at 50,000g at 4 °C. The cleared lysates weremixed with NZ-1-Sepharose (100 ll bed volume) and incubatedat 4 °C for 2 h under gentle agitation. The beads were washedin a small column with 2 ml of a solution containing 20 mM
Tris–Cl (pH 8.0), 400 mM NaCl, 10% glycerol, 1 mM EDTA and
0.1% Triton X-100 and eluted with the same solution containing

NZ-1 was raised against 14-residue peptide (EGGVAMPGAE
DDVV; PA14) corresponding to residues 38–51 of human podoplanin [12]. Previous study showed that the central 10-residue portion (GVAMPGAEDD; PA10) was indispensable for the recognition[19]. In order to determine the minimal peptide region that warrants observed high afﬁnity, we performed surface plasmon resonance (SPR) binding experiments between NZ-1 and various
versions of PA tagged protein. As shown in Fig. 2A and B, either
PA14 or PA12 (GVAMPGAEDDVV) tags attached C-terminally to
T4L bound to NZ-1 with high afﬁnity in an indistinguishable manner, indicating that the N-terminal two residues (Glu-Gly) weredispensable. In contrast, PA10-tagged version showed greatly reduced afﬁnity (Fig. 2C), suggesting that the last two valines were
important to ensure the high afﬁnity. We next tested whether PA
tag was compatible with N-terminal fusion. As shown in Fig. 2D
and E, both PA14 and PA12 mediated high afﬁnity binding to NZ1 when appended at N-terminus of T4L, although the dissociation
was slightly faster than the C-terminal versions. Interestingly,
afﬁnity of PA10-T4L was decreased by only 1.5-fold from PA12
and PA14. It seems likely that the presence of the last two residues
is important for the high afﬁnity but they can be substituted byother amino acids (such as Arg–Glu in PA10-T4L). Hereafter, we
will use PA12 sequence for all applications.
To determine which amino acid residues within the PA12 are
critical for the recognition by NZ-1, ten non-alanine residues weremutated to alanine and each mutant (in the format of T4L-PA12)
was evaluated in SPR assay. As clearly shown in the overlaid sensorgrams in Fig. 3, mutations at M4 and D10 had the most severe
effect, indicating the major contribution of these residues in thebinding. Effects of four other mutations (P5A, G6A, E8A, and D9A)
were less pronounced, and the rest of the mutants exhibited virtually identical binding behavior to the original PA tag. Therefore,
NZ-1 seems to achieve the very high afﬁnity toward PA tag using

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Y. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

A
PA
NZ-1

FLAG
M2

HA
4B2

Myc
9E10

B
Tag(Antibody)ka (1/M•s)k d (1/s)

KD (M)

χ2

PA(NZ-1)

1.4 x 104

5.8 x 10-6

4.0 x 10-10

3.4 x 10-5

FLAG (M2)

6.1 x 104

1.7 x 10-3

2.8 x 10-8

1.2 x 10-5

HA (4B2)

6.7 x 105

1.1 x 10-3

1.6 x 10-9

1.4 x 10-5

Myc (9E10)

1.1 x 106

2.5 x 10-3

2.2 x 10-9

1.3 x 10-5

Fig. 1. Bio-layer interferometry kinetic analysis of various anti-epitope tag antibodies. (A) Representative BLI binding data for C-terminally tagged T4L proteins (T4L-PA12,
T4L-FLAG, T4L-HA, T4L-Myc). Each anti-tag mAb was immobilized on sensor tips. Association (from 60 s to 120 s) and dissociation (from 120 s to 240 s) phases are shown forserial dilution series of the tagged T4L (red, 30 nM; blue, 10 nM; purple, 3 nM, green, 1 nM). Black lines represent a global ﬁt of the data to a 1:1 binding model. (B) Kinetic
binding parameters for each interaction obtained by the global ﬁt shown in (A). Successful curve ﬁtting to the model was indicated by the very low v2 values. (Forinterpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

B

(M)

300
Response (RU)

Response (RU)

-12

KD = 1.8 x 10

(T4L-PA14)

200
100
0

C
(M)

300

200
100
0

0

100

200

0

100

E
-9

300

(M)
Response (RU)

KD = 8.7 x 10

200
100
0
100
Time (s)

100

0

100

200

200

Time (s)-9KD = 9.1 x 10

F

(M)

300

(PA12-T4L)

200
100
0

0

(M)

200

200

Response (RU)

(PA14-T4L)-8

KD = 7.4 x 10

Time (s)D300(T4L-PA10)

0Time (s)Response (RU)

-12

KD = 4.3 x 10

(T4L-PA12)

Response (RU)

A
300

-8

(PA10-T4L)

KD = 1.5 x 10

(M)

200
100
0

0

100
Time (s)

200

0

100

200

Time (s)Fig. 2. Surface plasmon resonance kinetic analysis of NZ-1 binding toward various PA tags. Serially diluted PA-tagged T4L proteins (3.125, 6.25, 12.5 and 25 nM) were injected
over the CM5 sensor chip immobilized with NZ-1 for 60 s, followed by dissociation in PBST for 120 s at a ﬂow rate of 20 ll/min. PA14 (A and D), PA12 (B and E), or PA10 (C andF) peptides were fused either C-terminally (A–C) or N-terminally (D–F) to T4L protein. Each set of binding curves was globally ﬁtted to 1:1 binding model to derivedissociation equilibrium constant (KD) values shown in the boxes.

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Y. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

E8A•D9A
P5A

100

Cell number

Response (RU)200

% positive cells

WT•G1A•
V2A•V11A•
V12A

300

G6A
M4A•D10A

0
0

50

100
150
Time (s)200

positive region

Fig. 3. Effect of alanine substitutions of PA tag sequence on the binding to NZ-1
evaluated using SPR. Puriﬁed alanine-substituted versions of T4L-PA12 diluted at
20 nM were injected over the surface immobilized with NZ-1 as described in thelegend to Fig. 2 and sensorgrams after subtraction of the buffer control wereoverlaid.

a combination of many interactions to the central core segment(M4-D10), with the contribution of M4 and D10 being dominant.

Utility of NZ-1 as a detection antibody
The unusually high afﬁnity of NZ-1 toward PA tag made us
speculate that it may be used as a mean to detect proteins thatare tagged with PA peptide. To test this idea, a cytoplasmic enzyme
isocitrate dehydrogenase 1 (IDH1) was doubly tagged with FLAGand PA (Supplementary Fig. 1), and expressed in human osteosarcoma cell line U-2 OS that expresses low endogenous podoplanin[20,21]. As shown in Fig. 4A, Western blotting of the resultant celllysate with NZ-1 produced a strong band corresponding to the expressed IDH1 (45 kDa). NZ-1 immunoblot of the untransfected
cells did not show any reactive bands (data not shown), indicating
very high speciﬁcity of the detection. In contrast, numerous nonspeciﬁc bands other than tagged IDH1 were seen in the anti-FLAG
M2 antibody immunoblot (Fig. 4A, middle lane). In addition to thehigh speciﬁcity, detection by NZ-1 was proven highly sensitive, because the signal intensity of the reactive band was saturated after
incubating with NZ-1 for as short as 5 min (Fig. 4B), and at concentration as low as 0.1 lg/ml (Fig. 4C).
Next we applied NZ-1/PA tag combination to the ﬂow cytometric analysis of cell surface receptors. An expression construct for a
type I transmembrane protein neuropilin (Nrp)-2 was engineered

A

Fluorescence intensity
Fig. 5. Flow cytometric analysis of PA-tagged membrane protein. CHO-K1 cells(gray area) or CHO-K1 cells transiently transfected with PA12-rNrp2-mCherry (solidline) were incubated with 10 lg/ml NZ-1 and stained with FITC-labeled secondary
antibody speciﬁc for rat IgG. Less than 1% of parental CHO cell population was
positive with NZ-1 (region indicated by the bracket). Staining efﬁciency was
expressed as the % positive cells and plotted against the concentration of primary
antibody (inset).

to carry PA tag at its N-terminus (extracellular domain) andmCherry at its C-terminus (cytoplasmic domain), and was transiently transfected into CHO-K1 cells. Inspection of cells by ﬂuorescence microscopy after transfection indicated that 20% cells werepositive for the Nrp2-mCherry. Similar level of surface expression
was conﬁrmed by the ﬂow cytometric analysis of the same cellswith NZ-1 (Fig. 5, solid line histogram). Furthermore, efﬁcient
staining was achieved even when the concentration of the primary
antibody was reduced to 0.1 lg/ml NZ-1 (Fig. 5, inset), again
underscoring the great potential of PA tag as a detection system.
One potential shortcoming of the NZ-1/PA tag system is the incompatibility with human cell lines expressing endogenous podoplanin. However, the NZ-1-reactive sequence is unique to human
podoplanin and both M4 and D10 are absent in mouse, rat, hamster, or dog podoplanin (Supplementary Fig. 3), indicating that celllines from these animals are compatible with this system. In fact,

B

C

kDa

kDa

kDa

250
150
100
75

250
150

250
150

100

100

75

75

50

50

50

37

37

37

25

25

25

NZ-1

M2

(PA)

(FLAG)

CBB

1

5

30

incubation time (min)

60

0.01

0.1

1

3.5

NZ-1 concentration (µg/ml)

Fig. 4. Western blot analysis of PA tagged IDH1 with NZ-1. (A) Total cell lysates from human osteosarcoma cells expressing the IDH1-FLAG-PA12 tag were electrophoresed
under reducing conditions using 10% SDS–PAGE gel and transferred to a membrane. Three strips of the membrane containing the same amount of lysate were immunoblottedwith NZ-1 (left), M2 (center), or directly stained with Coomassie Brilliant Blue (right). The same membrane strips were used to assess the time course (B) or dose-dependency(C) of the NZ-1 incubation. In (B), membranes were incubated with 1 lg/ml NZ-1 for the indicated period. In (C), membranes were incubated with varying concentrations of
NZ-1 for 30 min. All steps after the NZ-1 incubation, including secondary antibody reaction and chemiluminescence visualization, were conducted under the exactly same
condition for all membranes. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

245

A
kDa

B
wash
elution1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250
150
100
75

kDa

c. supflowthrough

x1
/
x1 2
x1/4
/8
x1
/2
x1
x1/4
/8

c. supflowthroughY. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

250
150
100

50

37

37

25

25

29
21
18

50

2.
7
0.
0. 9
04

75

signal (arbitrary unit)

cell lysate
flowthroughwashCelution1 2 3 4 5 6 7 8 9 10

kDa
200
160
100
70
50
40
30

Fig. 6. One-step puriﬁcation of PA12-tagged proteins. (A) Puriﬁcation of soluble ectodomain fragment of mouse neuropilin-1. Five microliters of the culture supernatant fromthe transfected cells before (c. sup) and after (ﬂowthrough) NZ-1-Sepharose capture, as well as 10 ml of the ﬁve wash (lanes 1–5) and ten peptide-elution fractions(lanes 6–15) during the column chromatography were subjected to 10% SDS–PAGE under reducing condition and stained with CBB. (B) The culture supernatant and theﬂowthrough fractions from the chromatography shown in (A) were diluted with TBS as indicated and subjected to Western blotting using NZ-1. Intensity of the 120-kDa band
was quantiﬁed by densitometric analysis and shown below each lane. (C) One step puriﬁcation of EGFR from the HEK cells. Ten microliters of the detergent-solubilized celllysates from transfectants before (cell lysate) and after (ﬂowthrough) NZ-1-Sepharose capture, as well as the 10 ml of the wash and ten peptide-elution fractions during thecolumn chromatography were subjected to 10% SDS–PAGE under reducing condition and stained with CBB.

NZ-1 does not exhibit any binding toward CHO cells in ﬂow cytometry (Fig. 5, shaded histogram) or in Western blotting (data notshown). Furthermore, presence of endogenous podoplanin does
not cause problems when the system is used for a puriﬁcation purpose as will be described in the next section.
Application to the protein puriﬁcation system
We next explored the possibility to apply NZ-1/PA tag interaction in a protein puriﬁcation system. First we focused on the puriﬁcation of recombinant proteins secreted into culture media fromtransfected mammalian cells, because such project generally suffers from the lack of robust puriﬁcation method due to the low
abundance of the protein in the starting material (i.e., culture
supernatants). As shown in Fig. 6A, soluble ectodomain fragmentof mouse Nrp1 tagged C-terminally with PA12 could be efﬁciently
captured onto NZ-1-Sepharose from the dilute culture supernatants of the transfected HEK293T cells, and eluted from the resinby a solution containing 0.1 mg/ml free PA14 peptide. The abilityof free peptide to competitively elute bound antigen was rather
unexpected, because the interaction seemed to be almost irreversible in the kinetic binding assays shown in Figs. 1 and 2. In fact, wefound it essential to perform the elution in a step-wise manner
rather than under continuous ﬂow, and allow 5 min of dissociation time for each fraction. The near-perfect capture of tagged protein during the afﬁnity chromatography was conﬁrmed by thepresence of less than 5% antigen in the ﬂowthrough fraction(Fig. 6B). Importantly, protein eluted from the resin was extremely
pure, and ready for use in the downstream experiments without
further puriﬁcation. We have so far applied PA tag/NZ-1 system
to purify more than 15 secreted proteins having widely different
molecular weight (from 15 to >300 kDa), where >95% purity
was regularly attainable in one step by NZ-1-Sepharose
chromatography.
We also tested if PA tag system is compatible with the puriﬁcation of membrane proteins solubilized with detergents. HEK293S
GnT1- cells were transfected with N-terminally PA-tagged human
EGFR, and the harvested transfectants were solubilized with 0.2%
Triton X-100. As clearly shown in Fig. 6C, PA-tagged EGFR was captured by NZ-1-Sepharose and eluted efﬁciently with 0.1 mg/ml free
PA14 peptide solution. A great separation power of the PA tag/NZ-1
system is demonstrated by the successful puriﬁcation of the 145kDa EGFR protein from the low abundance cell lysate, with virtually no contaminations. To our surprise, podoplanin (37 kDa)
was not co-puriﬁed with the PA-tagged proteins even though thecell line expresses endogenous human podoplanin. It is possiblethat the effective afﬁnity of NZ-1 for the epitope region (PLAG domain) of native podoplanin is relatively low, probably due to thesteric hindrance of the peptide sequence by the nearby glycan
chains [22], resulting in the inefﬁcient capture during the afﬁnity
chromatography. In any case, this result indicates that NZ-1/PA
tag is fully compatible with membrane protein puriﬁcation in thepresence of detergents, even from the cell lysates containing
endogenous human podoplanin.

246

Y. Fujii et al. / Protein Expression and Puriﬁcation 95 (2014) 240–247

Fluorescence (arbitrary unit)

treatment with 3 M MgCl2 at pH 6.0 without causing any damageon the immobilized antibody. It is important to note that direct
elution with 3 M MgCl2 rather than the expensive 0.1 mg/ml peptide solution can be employed during the puriﬁcation step, when
the target protein is known to survive brief exposure to thiscondition.
Conclusion

Elution cycle
Fig. 7. Effect of the repeated column regeneration treatments on the yield of PAtagged GFPUV puriﬁcation by NZ-1-Sepharose. One puriﬁcation cycle constituted of(i) application of bacterial lysate containing GFPUV-PA12 to a small column of NZ-1
Sepharose, (ii) washing with 4 column volumes of TBS, and (iii) elution/regeneration by adding 5 column volumes of 3 M MgCl2 in 10 mM MES, pH 6.0. The graphshows the amount of GFPUV-PA12 contained in the pooled eluate fraction from each
puriﬁcation cycle quantiﬁed with a ﬂuorescence measurement.

Reusability of the NZ-1 afﬁnity resin
In general, antibody-based puriﬁcation systems tend to be
costly compared to other methods that use afﬁnity resins withimmobilized chemical compounds [7]. Although the major reason
for the high cost is the requirement of the use of synthetic peptide
for the elution process, it is often noted that the limited reusabilityof the antibody resin is another cost-elevating factor. This is especially critical in an immunoafﬁnity system where the antibody
binds its antigen tightly, because potentially damaging condition(e.g., high or low pH, denaturing agents, etc.) is required for theregeneration of the column to remove the peptide that was boundduring the elution process. We therefore sought to ﬁnd conditions
that can completely disrupt NZ-1:PA tag interaction while maintaining the functional integrity of the antibody. To this end, NZ-1
was covalently immobilized on a Biacore sensorchip and allowed
to saturate with T4L-PA12, followed by an injection of various buffers (Supplementary Fig. 2). Table 1 shows the effect of brief (30 s)
exposure to a variety of conditions on the dissociation of the boundantigen. Unlike some ‘‘polyol-responsive’’ antibodies [6,11], inclusion of water-miscible organic solvent such as propylene glycol
did not have dissociating effects. Strongly acidic condition (pH2.0) was effective in disrupting the interaction, but it severely
damaged the NZ-1 itself, because the surface lost binding ability(data not shown). Lowering the pH of the buffer was generally
effective, especially when high concentration of salt was included.
After testing several combinations, we found that a slightly acidic(i.e., pH 6.0) buffer containing 3 M MgCl2 could completely remove
bound antigen from the antibody. It was conﬁrmed that brief injection of this buffer onto the NZ-1 surface did not affect the binding
capacity.
We adopted the above buffer as a column-regeneration condition after each cycle of protein puriﬁcation using NZ-1-Sepharose.
In order to assess potential damaging effect of the regeneration
condition, GFPUV-PA12 was expressed in bacterial cytosol and an
aliquot of cell lysate was applied to a small column of NZ-1-Sepharose, followed by direct elution with 3 M MgCl2, pH 6.0. Measurement of the total ﬂuorescent intensity of the cell lysate
before and after passing through the column conﬁrmed more than
95% capture of GFPUV-PA12, as well as 100% recovery of the captured protein in the eluted fraction (data not shown). Most importantly, we observed sustained ﬂuorescence recovery when thisabsorption-elution/regeneration cycles were repeated up to
60 times, indicating that the NZ-1 column could withstand60 times of regeneration cycles (Fig. 7). It is therefore clearthat the NZ-1-Sepharose can be efﬁciently regenerated by theWe have successfully developed a novel afﬁnity tagging system‘‘PA tag’’ by applying a unique mAb NZ-1 against human podoplanin. Owing to its unusually high afﬁnity toward linear dodecapeptide antigen, NZ-1 can be used to detect, capture, and purify tagged
proteins in various assay formats. The biggest advantage of thissystem is the ability to purify low abundant proteins at high yield
in one step, along with its extended reusability. Major limitation
does exist, however, in that it is not fully compatible with the human tissues/cells that express endogenous podoplanin. Among
commonly used cell lines, we have conﬁrmed that HEK cells and
COS cells are positive for NZ-1 staining. Many but not all cancer celllines express podoplanin [12,21]. Nevertheless, these cells can beused as expression hosts if the purpose is to purify PA-tagged recombinant proteins, because the contamination of endogenous
podoplanin is negligible (see Fig. 6C). On the other hand, PA tag
system is universally useful in a wide variety of applications dealing with non-human tissues/cells. Because the two most criticalresidues in the PA tag for the high afﬁnity recognition by NZ-1
(i.e., M4 and D10) are substituted to very different amino acids
in mouse, rat, hamster, and dog podoplanins (Supplementary
Fig. 3), cells/tissues from these animals should be fully compatible
with the PA tag/NZ-1 system, as exempliﬁed by experiments with
CHO-K1 cells (Fig. 5). Furthermore, utility of the PA tag system may
not be limited to the applications described in the current study.
For example, PA-tagging and NZ-1 capture may be used in a proteomic interactome analyses, because such study requires highafﬁnity (or more accurately, slow dissociation) between the tagged
bait and the capture device to withstand extensive washing toeliminate nonspeciﬁc binders. Overall, it is anticipated that the
PA tag system will be a unique and powerful addition to our listof experimental tools in the ﬁeld of protein expression andpuriﬁcation.
Acknowledgments
We would like to thank Keiko Tamura-Kawakami, Emiko Mihara, and Rika Oi for their excellent technical support, and Mie Sakai
for preparation of the manuscript. This work was supported by the
Platform for Drug Discovery, Informatics, and Structural Life Science grant from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (MEXT).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.pep.2014.01.009.
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