Received: 26 June 2021

Revised: 5 August 2021

Accepted: 6 August 2021

Accepted article online: 19 August 2021

DOI: 10.1002/pmic.202100070

RESEARCH ARTICLE

Single polypeptide detection using a translocon EXP2 nanopore
Mitsuki Miyagi
Ryuji Kawano

Sotaro Takiguchi

Department of Biotechnology and Life Science,
Tokyo University of Agriculture andTechnology, Koganei-shi, Tokyo, Japan
Correspondence
Ryuji Kawano, Department of Biotechnology
and Life Science, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo
184–8588, Japan.
Email: rjkawano@cc.tuat.ac.jp

Kazuaki Hakamada

Masafumi Yohda

Abstract
DNA sequencing using nanopores has already been achieved and commercialized; thenext step in advancing nanopore technology is towards protein sequencing. Although
trials have been reported for discriminating the 20 amino acids using biological
nanopores and short peptide carriers, it remains challenging. The size compatibility
between nanopores and peptides is one of the issues to be addressed. Therefore,
exploring biological nanopores that are suitable for peptide sensing is key in achieving

Mitsuki Miyagi and Sotaro Takiguchi contributed equally to this work.

amino acid sequence determination. Here, we focus on EXP2, the transmembrane pro-

Funding information
KAKENHI from the Ministry of Education,
Culture, Sports, Science and Technology,
Grant/Award Numbers: 19H00901, 21K19786

the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled

tein of a translocon from malaria parasites, and describe its pore-forming properties infrom 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL)at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution
to distinguish the difference in molecular weight between two individual PLL, long PLL(Mw: 30,000–70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.
KEYWORDS

lipid bilayer, microfluidics, nanopore, peptide sensing, translocon

1

INTRODUCTION

sensing, have also been used for peptide sensing [12–19]. Regarding
peptide detection using the αHL pore, it has been shown that equipping

Nanopore technology is an emerging tool with great potential for

αHL nanopores with Au25 (SG)18 clusters can improve the mass reso-

single-molecule sensing. When a molecule enters a nanopore under

lution up to two-fold by increasing the on/off rate of peptides to thean applied voltage, it can be recognized as an ionic current blockage,

nanopore [20]. Fragaceatoxin C (FraC) nanopores [21] have recently

allowing for the detection of individual molecules with high sensitivity

enabled the discrimination of a 1.3 kDa difference in peptides by using[1–7]. As a commercialized application of this technology, nanopore-a model biomarker [22]. Huang et al. successfully improved this res-based DNA sequencing has already been achieved by identifying theolution down to 44 Da by using variant FraC that had a smaller-sized

four individual nucleobases [8, 9]. The current challenge is the applica-

constriction [23]. Aerolysin (AeL) nanopores [24, 25] have also beention of nanopore technology to peptide and protein sequencing.

employed for the structural discrimination of peptides. A series of pep-

Peptides and proteins are built from 20 kinds of amino acids, whiletides with different charges and lengths [26] and with the same amino

DNA is comprised of four types of nucleic acids. The principle of elec-acid composition but different sequences [27] were both discriminatedtrical recognition of individual amino acids is the same as that of nucleic

by using AeL nanopores. In terms of the length of peptides, it has been

acids, but the detection of 20 distinguishable signals is a tremendous

shown that AeL nanopores could resolve the signal of poly-arginine

challenge. Using various pore-forming proteins shown in Table 1, somewith a length ranging from 10 to 5 amino acids, with the resolution of

trials were reported for identifying the tiny structural difference of

one arginine [28]. Regarding the identification of single amino acids,

peptides from the constituted amino acids [10]. α-Hemolysin (αHL)

AeL nanopores succeeded in distinguishing 13 of the 20 natural amino

nanopores [11], a popular biological nanopore for oligonucleotides

acids by using polycationic carriers [29]. Besides the above studies,

Proteomics 2021, 2100070
https://doi.org/10.1002/pmic.202100070

www.proteomics-journal.com© 2021 Wiley-VCH GmbH

1 of 92 of 9several kinds of biological nanopores have been used for peptide
detection [30–34]. Nevertheless, it is still difficult to discriminate

Statement of significancebetween individual amino acids whose size ranges less than 1 nm [35,
Nanopore technology has recently had attracted attention36]. This size is generally smaller than pore size, indicating that theas the next generation of protein detection and amino acidsize-matching between the pore diameter and the amino acid is one

sequencing. Finding an appropriate nanopore that is suit-of the key requirements for discriminating between the 20 aminoable for protein/peptide sensing is a key factor for realiz-

acids.

ing it. We here focus on EXP2, the transmembrane protein

EXP2 is the transmembrane protein of a translocon from the malaria

of a translocon from malaria parasites, and investigate its

parasite Plasmodium falciparum [37] (Figure 1A,B). We have focusedpore-forming properties and capability for peptide sensing.

on EXP2 as the peptide-detectable nanopore because a translocon

Through our experiments, we revealed that EXP2 mainly

can transport a protein chain with unthreaded through the pore. The

assembled from 7 monomers with a diameter of 2.5 nm in thetranslocon called PTEX supports the malaria parasites to proliferate inlipid bilayer even without the other translocon components.

human red blood cells by exporting proteins [38]. PTEX is composed ofUsing the EXP2 nanopore, we were successful in detecting

five proteins; EXP2 constitutes the transmembrane pore (Figure S1),

poly-L-lysine (PLL) at a single-molecule level, and in size-

while the other components play a role in unfolding the luggage pro-discriminating between long PLL (Mw: 30,000–70,000) andteins. Therefore, we consider EXP2 to have a suitable pore size forshort PLL (Mw: 10,000). Our result will contribute to findingtranslocating an unfolded polypeptide. We have previously reported

the appropriate nanopore of protein/peptide detection.

that the EXP2 formed a nanopore in a lipid bilayer without the othertranslocon components [39]. Here, we evaluate its pore-forming properties and apply it in single-molecule detection to investigate its capability for peptide sensing. Through our investigations, we reveal thatpoly-L-lysine (PLL) at a single-molecule level, and in size-discriminating

EXP2 mainly forms a nanopore of 2.5 nm diameter, assembled from 7

between two individual PLL; long PLL (L-PLL, Mw: 30,000–70,000) andmonomers. Using the EXP2 nanopore, we were successful in detecting

short PLL (S-PLL, Mw: 10,000).

TA B L E 1aBiological nanopores which have been used for peptide sensing

Nanopore

Diameter[nm]aα-hemolysin

Organism

PDBID

Ref.

1.4

Staphylococcus aureus

7AHL[11-20, 35,
36]

FraC

1.6

Actinia fragacea

4TSY[21–23]

Aerolysin

1.4

Aeromonas hydrophila

5JZT[24–29]Phi-29

3.6

Bacillus virus phi29

1H5W

[30]

SPP1

3.0

Bacillus phage SPP1

2JES

[31]

MspA

1.2

Mycolicibacterium
smegmatis

1UUN

[32]

T7 connector

3.9

Escherichia phage T7

3J4A[33, 34]

Diameter of the constriction region of nanopore.

Shape3 of 9

(A)

(B)

(C)

F I G U R E 1 (A) Illustration of an EXP2 nanopore reconstituted in a lipid bilayer. (B) The top and side view of the EXP2 nanopore (PDB ID: 6E10,
previewed chains A-G by PyMOL). (C) Lipid bilayers are prepared by the droplet contact method using a microdevice, and the EXP2 nanopore isreconstituted in the lipid bilayer

2

MATERIALS AND METHODS

at 4°C. The 1 kbp double-stranded DNA (dsDNA), a part of lambda
DNA (9346–10,345: 1 kbp), was purchased (Integrated DNA Tech-

2.1

Regents and chemicals

nologies, Inc., USA), and the primers were synthesized by Fasmac Co.,
Ltd. (Kanagawa, Japan). KOD SYBR® qPCR Mix (TOYOBO Co., Ltd.,The reagents used in this study were as follows: 1, 2-diphytanoyl-

Japan) and NucleoSpin Gel and PCR Clean-up (Takara Bio Inc., Japan)

sn-glycero-3-phosphocholine (DPhPC; Avanti. Polar Lipids, Inc., USA),

were used for the amplification of 1 kbp dsDNA. The long poly-L-lysine

n-decane (Wako Pure Chemical Industries, Ltd., Japan), potassium(Mw: 30,000–70,000, Sigma-Aldrich Japan, Inc., Japan) and short poly-chloride (KCl; Nacalai Tesque, Inc., Japan), 3-morpholinopropane-1-

L-lysine (Mw: 10,000, Alamanda-polymers, Inc., USA) were dissolved

sulfonic acid (MOPS; Nacalai Tesque, Inc., Japan). Buffered electrolyte

at a concentration of 1 mM and 5 mM in ultrapure water respectively,

solutions (1 M KCl, 10 mM MOPS, pH 7.0) were prepared using ultra-

stored at –20 °C.

pure water, which was obtained from a Milli-Q system (Millipore, Billerica, MA, USA). Wild-type alpha-hemolysin (αHL; Sigma-Aldrich, St.
Louis, MO, USA, and List Biological Laboratories, Campbell, CA, USA)

2.2

Fabrication of the microdevice

was obtained as the monomer polypeptide, isolated from Staphylococcus aureus in the form of a powder and dissolved at a concentration of

The microdevice was fabricated by machining a 6.0 mm thick, 10 ×

1 mg/mL in ultrapure water. For use, samples were diluted to the desig-

10 mm polymethyl methacrylate (PMMA) plate (Mitsubishi Rayon,

nated concentration using a buffered electrolyte solution and stored

Tokyo, Japan) using a computer-aided design and computer-aided

4 of 9manufacturing with a three-dimensional modelling machine (MM-100,

Tokyo, Japan), and python 3.5 (Python Software Foundation, USA).

Modia Systems, Japan) as shown in Figure 1C. Two wells (2.0 mm diam-

Channel current measurements were conducted at 22 ± 2◦ C.

eter and 4.5 mm depth) and a chase between the wells were manufac-

Detection of double-stranded DNA. The ground side solution con-tured on the PMMA plate (Figure S2). Each well had a through-holetained dsDNA and EXP2 (Figure S3). A constant voltage of + 100 mVin the bottom and Ag/AgCl electrodes set into this hole (Figure 1C). A

was applied to the recording chamber.

polymeric film made of parylene C (polychloro-p-xylylene) with a thick-Detection of poly-L-lysine. The recoding side solution contained poly-

ness of 5 μm as shown in Figure S2 was patterned with a single pore

L-lysine and EXP2 (Figure S3). A constant voltage of + 100 mV was(100 μm diameter) using a conventional photolithography method andapplied to the recording chamber.

then fixed between PMMA films (0.2 mm thick) using an adhesive bond

Data analysis for single-molecule detection. The analysed data were(Super X, Cemedine Co., Ltd., Tokyo, Japan). The films, including theobtained from at least three different EXP2 nanopores. We definedparylene film, were inserted into the chase to separate the wells.

that dsDNA and poly-L-lysine were detected when > 0.5 nS of open
EXP2 channel currents were inhibited. The duration time was filtered to < 1 ms to exclude distinctive signals from EXP2 (the detail is

2.3
Lipid bilayer preparation and reconstitutionof the EXP2 nanopore

described in Figure S4). The bootstrap method is a method based on
the resampling of the original random sample, drawn from a population
with an unknown distribution. We used the exact bootstrap method,

Lipid bilayers were prepared using the device produced by micro-

which availed of the entire space of resamples. In the exact bootstrap

fabrication (Figure 1C). Lipid bilayers can be simultaneously formed

method, accuracy verification is possible when the sample number isin this device by the droplet contact method (Figure 1C) [40, 41]. In

over 30 [42]. In this study, our bootstrap procedure took 120 samples

this method, the two lipid monolayers contact each other and form

randomly from the primary common translocation data with 65,536

lipid bilayers on a parylene C film that separates two chambers. Lipid

replacements and calculated the mean for these samples. Three hun-bilayers were formed as follows: each chamber of the device was filled

dred samples were randomly extracted from the bootstrapped data towith n-decane (0.5 μL) containing the lipid composition of DPhPC

produce the scatter plot of duration time versus current blocking con-

(10 mg/mL). Wild-type EXP2 monomers were expressed by E. coli

ductance.

following our previous protocol [39] and were dissolved at a concentration of 760 nM in 20% glycerol, and stored at −80◦ C. The buffer
solution (4.7 μL) containing EXP2 (final concentration 80 nM) was

2.5

Preparation of 1 kbp double-stranded DNA

poured into one chamber which was connected to the ground terminal.
The buffer solution (4.7 μL) without EXP2 was poured into another1 kbp dsDNA, a part of lambda DNA (9346–10,345: 1 kbp), was pur-chamber which was connected to the recoding terminal (Figure 1C).

chased and amplified using PCR. First, 0.01 ng/mL template DNA (45In this study, the buffer solution was the same for both droplets (1μL), and 10 μM each of either the forward or reverse primer (9 μL,

M KCl and 10 mM MOPS (pH 7.0)). Within a few minutes of adding

respectively), and ultrapure water (162 μL) were mixed in a 1.5 mL DNA

the solutions, lipid bilayers formed and EXP2 created nanopores fromlow-bind tube. Subsequently, 25 μL of each solution was poured into a

the poured chamber by reconstitution in the lipid bilayers. If the lipid

PCR tube. The PCR reaction solution contained KOD SYBR qPCR Mix

bilayers ruptured during this process, they were recreated by tracing(half of the total volume, 25 μL). PCR was carried out using a Ther-

at the interface of the droplet with a hydrophobic stick.

mal Cycler to amplify the 1 kbp dsDNA by the following production
steps: preheat at 98◦ C for 2 min; 40 cycles of 98◦ C for 10 s, 57◦ C for
10 s, 68◦ C for 1 min. Then, the amplified dsDNA was purified by Nucle-

2.4
Channel current measurement and data
analysisoSpin Gel and PCR Clean-up. The concentration of purified dsDNA wasconfirmed by the absorbance at 260 nm using the NanoDrop 2000c(Thermo Scientific).

The channel current was monitored using an Axonpatch 200B amplifier (Molecular Devices, USA) or a Pico patch-clamp amplifier (Tecella,
Foothill Ranch, CA, USA). The signals were detected using a 10 kHz

3RESULTS AND DISCUSSION

low-pass Bessel filter at a sampling frequency of 50 kHz in Axonpatch 200B and an 8 kHz low-pass Bessel filter at a sampling frequency of 40 kHz in Pico patch-clamp. A constant voltage of + 100 mV

3.1
Current signal classification for theevaluation of pore-forming properties

was applied to the recording chamber, and the other chamber was
grounded. The recorded data from Axonpatch 200B were acquired

The pore-forming properties of EXP2 were examined by channel cur-with Clampex 9.0 software (Molecular Devices, USA) through a Digi-rent measurements using our lipid bilayer system (Figure 1C) [43–46].

data 1440A analog-to-digital converter (Molecular Devices, USA).

As shown in Figure 2A,B, step-like signals and the other different

Data were analysed using Clampfit 11.1 (Molecular Devices, USA),

shapes of current signals were observed under + 100 mV. Following our

Excel (Microsoft, Washington, USA), Origin pro 8.5J (Light Stone,

proposed current signal classification for the different signal shapes,

5 of 9

(A)

(C)

(B)

(D)

(E)

F I G U R E 2 Typical current-time traces of EXP2 at + 100 mV, which are classified into (A) stable pore formation, and (B) unstable pore
formation. (C) The ratio of stable and unstable pore formation, calculated from the channel current analysis. (D) The average of the lifetime, whichis the length of time that the pore is open with a plateau current state. (E) Histogram of the current conductance for the initial step increase fromthe 0 A (baseline)

we classified the current signals into four types; step, square-top,

ically calculated using the current conductance of the step signals andmulti-level, and erratic (Figure S5) [47–49]. The detailed definition ofHille’s equation as follows:

this signal classification is described in Figure S5. To evaluate the poreforming activity of EXP2, we here defined step and square-top as signals relating to the formation of stable pores. Multi-level and erratic are(𝜋r ) 𝜌
R = l+
2𝜋r2(1)considered to correlate with unstable pore formation. In the channel

Here, r is the pore radius, l is the pore length (13 nm for the EXP2), ρcurrent measurements, the ratio of stable signals was 35% (Figure 2C).

is the solvent resistivity, and R is the resistance of the pore. R is calcu-

We have previously reported that the other pore-forming proteins

lated as V/I, where I is the current through the pore and V is the applied

showed a higher stable signal ratio, of at least around 60% [50]. Hence,voltage between two chambers. The open channel conductance was

optimization of the experimental conditions of the EXP2 nanopore isdefined as the amplitude of the initial step signals from the baseline.

required in order to increase the formation of stable pores. As shown

As shown in Figure 2E, the histogram of the pore conductance of EXP2

in Table S1, EXP2 has lots of charged amino acids, so we attempted

showed Gaussian distribution with a peak at 3.8 nS, which corresponds

to stabilize the nanopores by reducing the energy of the electric field.
As predicted, the appearance ratio of stable signals increased to 56%

to a pore diameter of 2.5 nm.
Next, we mathematically estimated the number of assembled

when decreasing the applied voltage from + 100 to + 50 mV (Fig-monomers from the step signals as follows [51]:(ure 2C, the detailed result of signal classification is shown in Figure S6).
In terms of the lifetime, which is the time of the pore-opening plateau

d = a

1
−1
sin (𝜋∕n))(2)state (Figure S7), it was also improved by the optimization of theapplied voltage, resulting in about eight-fold prolongation (Figure 2D).

where d is the pore diameter, a is the width of the EXP2 monomerfrom the PDB (1.77 nm for EXP2, PDB ID: 6E10), and n is the numberof assembled monomers [50]. With the substitution of calculated pore

3.2
Analysis of the pore size and the number ofassembled EXP2 monomers

diameter for d, the assembling monomer number was calculated to be
7 monomers for the peak value of the histogram. Considering the wide
distribution of the conductance, it may possible that the EXP2 formed

Although cryo-electron microscopy observations showed the rigid

polydisperse-sized nanopores assembled from 5 to 9 monomers,

structure of EXP2 as a part of the translocon [38], it is necessary towith the 7-mer nanopore most frequently observed. This result wasinvestigate the dynamic pore size of EXP2 in the lipid bilayer without

consistent with the in vivo condition that the EXP2 nanopore is com-the other translocon components. The size of the pore can be theoret-bined with other translocon components [38].

6 of 9F I G U R E 3 Results of the single-molecule detection using the EXP2 nanopore at the applied voltage of +100 mV. (A) The typical step signal of
the EXP2 nanopore without any additional molecules. (B-D) The typical current-time traces of EXP2 with (b) dsDNA, (C) L-PLL, and (D) S-PLL at theconcentration of 200 nM, 1μM, and 1 μM. (E-G) The event frequency as a function of the concentration of (E) dsDNA, (F) L-PLL, and (G) S-PLL. (H)Each typical blocking signal and the scatter plot of duration time versus current blocking conductance. The bootstrapped data were used to
produce the scatter plot (N is the number of EXP2 nanopores and n is the number of blocking signals)

3.3Single-molecule detection using the EXP2
nanoporethat of S-PLL. The scatter plot could distinguish the detection signals ofeach PLL from the EXP2 noise-like signals. PLL forms differing randomcoil structures in aqueous solution depending on its length [55], result-

The optimized + 50 mV was not suitable for the molecular detect-ing in different blocking conductance (Figure 3H). Regarding the dura-

ing experiments (Figure S8), probably owing to the insufficient elec-tion time, it is considered to reflect the time for the target molecule totrical force to get the target molecule captured into the nanopore, sotranslocate through the nanopore. Thus, L-PLL showed a longer dura-

we conducted the following experiments at + 100 mV. Single-strandedtion time than that of S-PLL owing to its molecular length. In otherDNA is able to pass through conventional biological nanopores such aswords, the EXP2 nanopore had the resolution to distinguish the differ-

αHL and MspA [52, 53], whereas double-stranded DNA cannot passence in molecular weight between L-PLL and S-PLL.

and clogs the pore vestibules owing to the size mismatch between

We here compared the polypeptide sensing ability of EXP2 with αHL

dsDNA and pore diameter. EXP2 mainly formed nanopores with a

as a common biological nanopore for sensing applications. It is revealed

diameter of 2.5 nm, which is close to the diameter of dsDNA. There-that the frequency of the detection events through the EXP2 nanopore

fore, we first attempted to detect dsDNA with a length of 1 kbp using

was higher than that of the αHL nanopore (Figure 4A-C) at the samethe EXP2 nanopore. In the channel current measurements, blocking

1 μM L-PLL. As shown in Figure 4D,E, the properties of the nanopores

signals that reflect the detection of dsDNA were observed at con-such as electrostatic potential mapping and shape are significantly dif-

centrations 50, 100, and 200 nM (Figure 3B), and the frequency offerent between EXP2 and αHL, resulting in the difference in event fre-

blocking events increased with an increase in dsDNA concentrationquency.

(Figure 3E). Although EXP2 has noise-like signals in the pore-opening
plateau states (Figure 3A), probably owing to the fluctuation of its Cterminus charged amino acids, the dsDNA detection signals could still

4

CONCLUSION

be discriminated in the scatter plot of duration time versus blocking
conductance (Figure 3H).

We examined the pore-forming properties of EXP2, the transmem-

Next, we attempted to detect poly-L-lysine (PLL) to check the capa-brane protein of a translocon from a malaria parasite, through channel

bility of EXP2 as a biological nanopore for polypeptide sensing. The

current measurements. The results reveal that EXP2 forms a nanopore

channel current measurement was conducted using two individual PLL,

mainly assembled from seven monomers with a diameter of 2.5 nm

long-PLL (L-PLL) with molecular weight ranging from 30,000 to 70,000,

in the lipid bilayer. These data were consistent with in vivo condi-

and short-PLL (S-PLL) with a molecular weight of 10,000. The block-tions where EXP2 forms a nanopore with other translocon compo-

ing signals were observed at concentrations 250, 500, and 1000 nM ofnents. Using the EXP2 nanopore allowed us to detect dsDNA with aeach PLL (Figure 3C,D), and the blocking frequency depended on thelength of 1 kbp and PLL at a single-molecule level. Regarding polypep-

concentration of each molecule (Figure 3F,G). Depending on the dif-tide sensing, the EXP2 nanopore has the resolution to distinguish theference of the total charge [54], L-PLL showed a larger frequency thandifference of molecular weight between L-PLL (Mw: 30,000–70,000)

7 of 9

(C)

(A)

(B)

(D)

(E)

F I G U R E 4 Typical detection signals of L-PLL at the concentration of 1 μM through (A) αHL and (B) EXP2 at + 100 mV. (C) Analysis of thefrequency of the blocking signals reflecting the detection of L-PLL by each nanopore. The calculated frequency of 1 μM L-PLL detection was 0.86 ±0.19 s–1 for αHL, and 151 ± 19 s–1 for EXP2. (D-E) The three dimensional model and the electrostatic potential map of (D) αHL nanopore and (E)
EXP2 nanopore at neutral pH condition. The electrostatic potential was calculated through the APBS tool in PyMOL software.

and S-PLL (Mw: 10,000). Analysis of detection event frequency indicated that EXP2 is more suitable to detect polycationic peptides thanthe αHL nanopore. Nanopore sensing has recently been postulated asa promising technology in mass spectrometry [56], especially peptide
mass analysis [23]. Our data supports the potential of the translocon
nanopore in such chemical and biological sensing applications.
ACKNOWLEDGMENTS
This work was partly supported by Grant-in-Aid for Scientific Research(KAKENHI) (Grant Nos. 19H00901 and 21K19786) from the Ministryof Education, Culture, Sport, Science and Technology (MEXT), Japan.
With thanks to A. Cooney for language editing.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
All data is included in this published article and its Supplementary
Information file.
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SUPPORTING INFORMATION
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onlinehttps://doi.org/10.1002/pmic.202100070 in the Supporting Information section at the end of the article.

How to cite this article: Miyagi, M., Takiguchi, S., Hakamada,K., Yohda, M., & Kawano, R. (2021). Single polypeptide
detection using a translocon EXP2 nanopore. Proteomics,
e2100070. https://doi.org/10.1002/pmic.202100070