Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Recombinant turnip yellow mosaic virus coat protein as a
potential nanocarrier
F.H. Tan1, J.C. Kong1, J.F. Ng1, N.B. Alitheen2, C.L. Wong2, C.Y. Yong2 and K.W. Lee1
1 School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor, Malaysia
2 Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
Keywords
capsid assembly, C-terminal modification,
IMAC, turnip yellow mosaic virus, TYMV coat
protein, virus-like particles.
Correspondence
Khai W. Lee, School of Biosciences, Faculty of
Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor, Malaysia.
E-mail: khaiwooilee@gmail.com; khaiwooi.
lee@taylors.edu.my
Foo Hou Tan and Jia Chen Kong contributed
equally to this work.
2021/2558: received 30 November 2020,
revised 17 February 2021 and accepted 19
February 2021
doi:10.1111/jam.15048
Abstract
Aims: To display a short peptide (GSRSHHHHHH) at the C-terminal end of
turnip yellow mosaic virus coat protein (TYMVc) and to study its assembly
into virus-like particles (TYMVcHis6 VLPs).
Methods and Results: In this study, recombinant TYMVcHis6 expressed in
Escherichia coli self-assembled into VLPs of approximately 30–32 nm. SDSPAGE and Western blot analysis of protein fractions from the immobilized
metal affinity chromatography (IMAC) showed that TYMVcHis6 VLPs
interacted strongly with nickel ligands in IMAC column, suggesting that the
fusion peptide is protruding out from the surface of VLPs. These VLPs are
highly stable over a wide pH range from 30 to 110 at different temperatures.
At pH 110, specifically, the VLPs remained intact up to 75°C. Additionally,
the disassembly and reassembly of TYMVcHis6 VLPs were studied in vitro.
Dynamic light scattering and transmission electron microscopy analysis
revealed that TYMVcHis6 VLPs were dissociated by 7 mol l1 urea and
2 mol l1 guanidine hydrochloride (GdnHCl) without impairing their
reassembly property.
Conclusions: A 10-residue peptide was successfully displayed on the surface of
TYMVcHis6 VLPs. This chimera demonstrated high stability under extreme
thermal conditions with varying pH and was able to dissociate and reassociate
into VLPs by chemical denaturants.
Significance and Impact of the Study: This is the first C-terminally modified
TYMVc produced in E. coli. The C-terminal tail which is exposed on the
surface can be exploited as a useful site to display multiple copies of functional
ligands. The ability of the chimeric VLPs to self-assemble after undergo
chemical denaturation indicates its potential role to serve as a nanocarrier for
use in targeted drug delivery.
Introduction
Virus-like particles (VLPs) are hollow protein container
morphologically identical to the infectious virion, but
lack their respective genomic materials (Deo et al. 2015).
In nanobiotechnology, VLPs are extensively studied as
potential nanomaterial for drug delivery and vaccine and
diagnostic assay development (Zeltins 2013). VLPs can be
modified genetically (Guillen et al. 2010; Tissot et al.
2010; Lee et al. 2011) or by chemical cross-linking (Kittelmann and Jeske 2008; Lee et al. 2011, 2012; Yan et al.
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
2015; Gan et al. 2018) to display cell-specific ligands on
the surface, thereby allowing their applications in targeted
therapy. Targeted therapy is particularly crucial for cancer
treatment, as majority of the chemotherapeutic drugs
such as daunorubicin, doxorubicin, paclitaxel and 5-fluorouracil are highly toxic to both healthy and cancerous
cells. Specific delivery of these drugs to only the cancerous cells is therefore of the utmost importance (Rohovie
et al. 2017; Senapati et al. 2018; Jin et al. 2020).
The most widely used VLP is the hepatitis B virus core
antigen (HBcAg), due to its ability to display various
1
F.H. Tan et al.
Recombinant TYMV coat protein nanocarrier
ligands through genetic modification (Choi et al. 2011;
Lee et al. 2011; Mohamed Suffian et al. 2017) and chemical cross-linking (Lee et al. 2012; Biabanikhankahdani
et al. 2017; Gan et al. 2018), which are needed for specific
interaction with the targeted host cells. Another important feature which makes HBcAg VLPs a favourable candidate for targeted therapy is their ability to be
dissembled and reassembled, thereby packaging therapeutic molecules within the VLPs, preventing unspecific
interaction of the cargo with healthy cells while delivering
therapeutic agents at high dose to the targeted cancer
cells (Suffian et al. 2018; Zhang et al. 2019; Yang et al.
2020). In addition, the VLPs can also protect the therapeutic cargo such as plasmids and small interfering RNA
(siRNA) against nuclease activity in vivo (Suffian et al.
2018). Apart from HBcAg VLPs, other VLPs such as
those of bacteriophage MS2 (Wu et al. 2005; Ashley et al.
2011), bacteriophage Qb (Pokorski et al. 2011b; Yin et al.
2016), Macrobrachium rosenbergii nodavirus (MrNV)
(Thong et al. 2019) and canine parvovirus (Gilbert et al.
2004; Singh et al. 2006) have also been explored for similar purposes.
Plant viruses generally lack ligand which interacts with
mammalian cells (Kim et al. 2018), making them highly
potential candidates for use in targeted therapy when
desired ligands are fused and displayed on the surface of
the plant VLPs. Turnip yellow mosaic virus (TYMV) is a
non-enveloped Tymovirus which infects Brassica plants.
The virus has a positive-sense, single-stranded RNA genome packaged within a capsid which is made up of 180
copies of TYMV coat protein (TYMVc), arranged in an
icosahedral conformation with T = 3 symmetry (Canady
et al. 1996). During natural infection of plants by TYMV,
two types of virus particles are produced: normal virus
with packaged RNA genome and empty particles known
as natural top components (van Roon et al. 2004). The
genomic contents within TYMV can be removed and
converted to pot-like empty particles known as artificial
top components (ATCs) through repeated free–thaw
cycles (Katouzian-Safadi and Berthet-Colominas, 1983),
high pressure (Leimk€
uhler et al. 2001) or alkaline treatment (Keeling and Matthews, 1982), where such ATCs
have been deployed as a model to deliver fluorescein dye
into baby hamster kidney cells through conjugation with
transactivating transcriptional activator (TAT), a cell-penetrating peptide (CPP) (Kim et al. 2018).
The gene encoding TYMV coat protein (TYMVc) have
been cloned and expressed in E. coli, where the recombinant TYMVc self-assembled into VLPs approximately
28 nm in diameter (Powell et al. 2012). Powell et al.
(2012) have extracted the insoluble TYMVc with urea,
followed by renaturation with stepwise dialysis. It has
been demonstrated that 5–15% of the denatured TYMVc
2
was able to assemble into VLPs resembling TYMV, while
the remaining 85–95% of the TYMVc retained as precipitate due to protein misfolding. The N- and C-terminal
ends of TYMV capsid protein are exposed at the interior
and exterior of the capsid, respectively (Canady et al.
1996). These sites can be candidate sites for ligand attachment to enhance interior or exterior binding of foreign
molecules with TYMV capsid. Powell et al. (2012) have
performed N-terminal deletion on the TYMV VLPs,
where the deletion resulted in VLPs with decreased stability. To date, C-terminal modification on TYMV VLPs has
yet to be reported.
In the current study, a short peptide containing polyhistidine tag (GSRSHHHHHH) have been fused to the
C-terminal end of TYMVc and expressed in E. coli. The
recombinant TYMVc, namely, TYMVcHis6, assembled
into VLPs slightly larger than that of the wild-type TYMV
VLPs. TYMVcHis6 VLPs has been demonstrated to be
stable up to 55°C, at pH ranging from 30 to 110. The
chimeric protein interacted strongly with immobilized
metal affinity chromatography (IMAC), indicating that
the short peptide is displayed on surface of the VLPs. In
addition, the VLPs can be disassembled and reassembled
through the use of urea or guanidine hydrochloride without significant loss of the VLPs. Conjointly, the recombinant TYMV VLPs serve as a potential carrier for use in
targeted drug delivery. This is the first report depicting
the successful production of C-terminally modified
TYMVc chimeric VLPs in E. coli.
Materials and methods
Construction of the recombinant plasmid
The coding sequence of the TYMVc was amplified from
the plasmid pTYFL84 (pWt) (ATCC PVMC-61) using
ACCUZYME DNA Polymerase (Bioline Reagents Ltd,
London, UK) with forward primer (50 -AGGCCATGGAAATCGACAAA-30 )
and
reverse
primer
(50 0
TTGGATCCGGTGGAAGTGTC-3 ), respectively, for the
constructed his-tagged TYMVc. The underlined nucleotide sequences represent the recognition cutting sites for
NcoI and BamHI restriction endonucleases. The polymerase chain reaction (PCR) products and pQE-60 plasmid (Qiagen, Valencia, CA) were then digested with
NcoI and BamHI (Thermo Scientific, Waltham, MA) at
37°C for 1 h in the Tango buffer provided. The digested
PCR product was ligated to the linearized pQE-60 vector
using T4 DNA ligase (Promega, Sydney, Australia) at
4°C for 16 h, and the ligation mixture containing the
recombinant plasmid pQE60TYMVcHis6 was then transformed into E. coli strain M15 (pREP4) competent cells.
The recombinant plasmid was verified by restriction
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F.H. Tan et al.
endonuclease digestion, and the nucleotide sequences of
the insert were confirmed by DNA sequencing.
Protein expression and purification
Single bacterial colony harbouring pQE60TYMVcHis6
was inoculated into Luria Bertani (LB) broth (10 ml)
containing ampicillin (100 µg ml1) and kanamycin
(30 µg ml1) at 37°C and 240 rev min1 for overnight.
The overnight culture was then transferred into a fresh
LB broth (200 ml) supplemented with antibiotics with
continued shaking under the same conditions until
OD600nm reached 08–09. Isopropyl-b-D-thiogalactopyranoside (IPTG; 1 mmol l1) was added to the culture,
and the incubation was continued at 30°C for 18 h. After
the induction, protein expression was analysed by SDS–
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
Bacterial cells were harvested by centrifugation and
resuspended in lysis buffer (20 mmol l1 sodium phosphate, 150 mmol l1 NaCl, 02 mg ml1 lysozyme,
4 mmol l1 MgCl2, 01% (v/v) Triton X-100,
75 µg ml1 DNase I; pH 74), followed by 2-h incubation at room temperature (RT). The cells were then lysed
via sonication. Prior to purification, the lysate was centrifuged at 12 000 g at 4°C for 20 min, and the supernatant was filtered through 022-µm syringe filters
(Sartorius, G€
ottingen, Germany). Protein purification was
performed as recommended by the manufacturer using
HisTrap FF 1-ml column (GE Healthcare Life Sciences,
Buckinghamshire, UK). Briefly, protein sample (1 ml)
was loaded onto HisTrap column pre-rinsed with binding
sodium
phosphate,
buffer
(5 ml;
20 mmol l1
1
NaCl, 30 mmol l1 imidazole; pH 74).
150 mmol l
The weakly bound proteins were then washed off with
washing buffer (20 ml; 20 mmol l1 sodium phosphate,
150 mmol l1 NaCl, 40 mmol l1 imidazole; pH 74).
The bound protein was then eluted with elution buffer
(5 ml; 20 mmol l1 sodium phosphate, 150 mmol l1
NaCl, 500 mmol l1 imidazole; pH 74). The eluted protein was further dialysed overnight against sodium phossodium
phosphate,
phate
buffer
(20 mmol l1
1
150 mmol l NaCl; pH 74).
Recombinant TYMV coat protein nanocarrier
as the amount of TYMVcHis6 protein in relation to the
total amount of protein in the eluted fraction. The
amount of total protein in cell lysate was analysed using
Bradford assay (Bradford 1976), whereas the expression
yield of the target protein was calculated based on the
relative amount of TYMVcHis6 obtained from the ImageJ
software analysis.
For Western blotting, proteins on the gel were electrotransferred onto nitrocellulose membrane and blocked
with blocking solution (10% (w/v) skimmed milk,
20 mmol l1 Tris, 150 mmol l1 NaCl; pH 76) at RT
for 1 h. The membrane was then rinsed with TBST
(20 mmol l1 Tris, 150 mmol l1 NaCl; pH 76, 01%
(v/v) Tween 20) and incubated with mouse anti-histidine
tag antibody (1 : 500 dilution, MCA1396; Bio-Rad) at
RT for 1 h. The membrane was then washed three times
with TBST buffer and incubated with enhanced chemiluminescence (ECL) horseradish peroxidase-linked sheep
anti-mouse antibody (1 : 5000 dilutions, NA931; GE
Healthcare Life Sciences) at RT for 1 h. The membrane
was again washed three times with TBST and developed
with Pierce DAB Substrate Kit (Thermo Scientific).
Dynamic light scattering analysis
The homogeneity and size of the VLPs were determined
by Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK). Purified TYMVcHis6 (03 mg ml1) was
filtered (022 µm) and loaded into quartz sample cells.
The sample was illuminated with a miniature solid-state
laser of 50 mW power at 532 nm wavelength. The hydrodynamic radius (Rh) of the VLPs was determined via the
Stokes–Einstein autocorrelation function: Rh = kBT/
6pgDT, where kB is Boltzmann’s constant, T is the absolute temperature in Kelvin, g is the solvent viscosity and
DT is diffusion coefficient.
Transmission electron microscopy (TEM)
TYMVcHis6 sample (15 µl; 03 mg ml1) was adsorbed
onto carbon-coated grids (Agar Scientific, Essex, UK),
negatively stained with 2% (w/v) uranyl acetate and visualized under transmission electron microscope (LIBRA
120; Carl Zeiss AG, Oberkochen, Germany).
SDS-PAGE and Western blotting
The protein samples were mixed with 29 Laemmli sample buffer (Bio-Rad, Laboratories, Hercules, CA), boiled
and electrophoresed on 12% SDS-PAGE. After SDSPAGE, the proteins were stained with Coomassie Brilliant
Blue R-250. The amount of the purified TYMVcHis6 protein (21-kDa band) was measured with the ImageJ software (NIH, Bethesda, MD), where the purity is defined
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
pH and thermal stability of TYMVcHis6
TYMVcHis6 samples (03 mg ml1) were prepared at pH
30 (20 mmol l1 sodium acetate, 150 mmol l1 NaCl),
58 (20 mmol l1 sodium phosphate, 150 mmol l1
NaCl),
74
(20 mmol l1
sodium
phosphate,
1
NaCl) and 110 (10 mmol l1 sodium
150 mmol l
bicarbonate, 150 mmol l1 NaCl). The samples at each
3
F.H. Tan et al.
Recombinant TYMV coat protein nanocarrier
pH were then incubated at 25, 35, 37, 45, 55, 65 and
75°C for 3 h. The samples were then analysed with
dynamic light scattering (DLS) at their respective temperatures.
Dissociation and reassociation of TYMVcHis6 VLPs
TYMVcHis6 samples (03 mg ml1) were prepared under
pH 74 and mixed with urea (10 mol l1) or guanidine
(6 mol l1) stock solutions to make up sample mixture
containing 0, 1, 2, 3, 4, 5, 6 and 7 mol l1 of urea and 0, 1,
2, 3, 4 and 5 mol l1 of guanidine hydrochloride. Chemically treated samples were then analysed with DLS to
obtain the dissociation profile of the TYMVcHis6 VLPs.
Complete dissociation of TYMVcHis6 particles was
confirmed through DLS analysis prior to the reassociation
steps. The reassociation of the dissociated TYMVcHis6
VLPs was achieved through complete dialysis against
sodium phosphate buffer (20 mmol l1 sodium phosphate, 150 mmol l1 NaCl; pH 74) without denaturant.
Results
Cloning and expression of TYMVcHis6
The amplified TYMVc coding region was purified and
ligated into pQE60 vector. Protein expression of the
recombinant plasmid, pQE60TYMVcHis6 is regulated by
T5 promoter transcription system (Bujard et al. 1987).
The translated TYMVc is fused with a polyhistidine tag at
the C-terminal end. Escherichia coli M15 cells harbouring
the recombinant plasmid produced an extra band in
SDS-PAGE of about 21 kDa upon induction with IPTG,
which corresponds to the calculated mass of TYMVcHis6
(2123 kDa). The protein band was detected by anti-His
antibody in the Western blot analysis, thereby confirming
successful expression of the recombinant protein (Fig. 1).
Quantitation of the relative TYMVcHis6 protein band
intensity revealed that the recombinant protein was about
17% of the total protein of its host cells. The yield of soluble recombinant protein was therefore calculated to be
around 8 mg ml1 of culture.
M
1
2
M
1
2
kDa
55
40
35
25
Figure 1 Expression of TYMVcHis6 (left: SDS-PAGE; right: Western
blot). Induction of TYMVcHis6 expression was done at 1 mmol l1
IPTG. Lane M: Molecular weight marker; lane 1: total protein before
induction; lane 2: total protein after induction. Arrow indicates protein band which corresponds to TYMVcHis6.
was finally eluted with an excess amount of imidazole
(500 mmol l1) (Fig. 2a, lane 9–13). Quantitation of the
relative TYMVcHis6 protein band intensity with ImageJ
revealed that the purity of the recombinant protein was
approximately 87%. Analysis of the purified protein with
Western blotting against anti-histidine antibody showed a
single protein band of approximately 21 kDa, indicating
successful purification of TYMVcHis6 protein (Fig. 2b).
Purified TYMVcHis6 assembled into empty VLPs
To investigate if TYMVcHis6 assembles into VLPs, the
purified protein was diluted to 03 mg ml1 and analysed
with DLS. A single peak was obtained with DLS at
32 nm, with a polydispersity index of 023 and a mass
percentage above 99%. This suggests that the recombinant protein self-assembled into particles of approximately 32 nm in diameter. TEM analysis provided
further confirmation that the purified TYMVcHis6 indeed
self-assembled into spherical nanoparticles with the diameter of about 32 nm (Fig. 3), which is in well agreement
with the size determined by DLS.
Purification of TYMVcHis6
Dissociation of TYMVcHis6 particles in urea and
guanidine hydrochloride
To purify the TYMVcHis6 from crude lysate, the crude
lysate was applied to IMAC. The target band intensity
reduced significantly upon application through IMAC column, indicating that most of the TYMVcHis6 protein has
bound to the IMAC column (Fig. 2a, lane 1–2). During
the washing step, a small amount of TYMVcHis6 along
with some weakly bound proteins were gradually removed
from the column (Fig. 2a, lane 3–8). The purified protein
In order to ascertain the stability of TYMVcHis6 particles
against chemical denaturants, purified TYMVcHis6 particles were incubated in 0–70 mol l1 of urea or 0–
50 mol l1 of guanidine hydrochloride. Figure 4 shows
that TYMVcHis6 particles were dissociated from around
35 nm to <1 nm hydrodynamic diameter according to
DLS, in the presence of 70 mol l1 urea or 20 mol l1
guanidine hydrochloride.
4
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
F.H. Tan et al.
Recombinant TYMV coat protein nanocarrier
(a)
kDa
(b)
M
1
2
3
4
5
6
7
8
9
10
11
12
13
kDa
M
1
M
2
55
40
40
35
30
25
20
25
15
Figure 2 Purification of TYMVcHis6. (a) Purification profile of TYMVcHis6 with IMAC. Crude lysate (1 ml) was applied onto HisTrap FF column
and washed with 20 ml of washing buffer. The targeted protein was eluted with 5 ml of elution buffer. Lane M: Molecular weight marker; lane
1: feedstock; lane 2: flow through; lane 3–8: washing fraction (first 3 and last 3 fractions; 1 ml per fraction); lane 9–13: elution fraction (1 ml
per fraction). (b) SDS-PAGE and Western blot of the purified recombinant TYMVcHis6 protein. Lane M: Molecular weight marker; lane 1: SDSPAGE the of purified recombinant TYMVcHis6 protein; lane 2: Western blot of the purified recombinant TYMVcHis6 protein. Arrow indicates protein band which corresponds to TYMVcHis6.
(a) 50
Particle Size (nm)
40
30
20
10
50 nm
Reassociation of denatured TYMVcHis6 particles
To find out if the denatured TYMVcHis6 particles were
able to refold and assemble back to its icosahedral structure, samples after incubation in 7 mol l1 urea and
2 mol l1 guanidine hydrochloride were dialysed, concentrated and visualized with TEM. The icosahedral structure
of TYMVcHis6 particles was observed, indicating the
reassembly of TYMVcHis6 VLPs (Fig. 5).
Heat treatment on TYMVcHis6 particles
Heat treated TYMVcHis6 particles were analysed with
DLS. The results revealed that the TYMVcHis6 VLPs
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
0
5
6
3
4
2
Urea Concentration (mol l-1)
1
7
8
(b) 50
40
Particle Size (nm)
Figure 3 Transmission electron microscopic analysis of TYMVcHis6.
Arrows indicate several empty capsids formed from TYMVcHis6 protein, where the internal cavity of the particles was stained dark. Scale
bar: 50 nm.
0
30
20
10
0
0
1
2
3
4
5
6
GdnHCI Concentration (mol l-1)
Figure 4 Dissociation profile of TYMVcHis6 VLPs. DLS analysis on
TYMVcHis6 in presence of (a) 0, 1, 2, 3, 4, 5, 6 and 7 mol l1 urea
and (b) 0, 1, 2, 3, 4 and 5 mol l1 of guanidine hydrochloride.
GdnHCl: guanidine hydrochloride.
5
F.H. Tan et al.
Recombinant TYMV coat protein nanocarrier
(a)
(b)
50 nm
50 nm
Figure 5 Electron micrographs showing the reassociated TYMVcHis6 VLPs. (a) VLPs after removal of urea. (b) VLPs after removal of guanidine
hydrochloride. Arrow indicates several reassociated TYMVcHis6 VLPs. Scale bar: 50 nm.
50
Particle Size (nm)
40
30
20
10
0
0
10
20
50
30
40
Temperature (°C)
60
70
80
Figure 6 Heat treatment profile of TYMVcHis6 particles under different pH conditions. TYMVcHis6 particles were diluted to a concentration of 03 mg ml1 and incubated in temperatures ranging from 25
to 75°C, at pH 30 ( ), 58 ( ), 74 ( ) and 110 ( ) for 3 h.
dissociated at 65°C at pH 30 and 75°C at pH 58 and
74 (Fig. 6). Interestingly, at pH 110, the size of TYMVcHis6 particles remained around 30 nm up to even 75°C.
Discussion
Virus-like particles are highly potential candidates which
can be used to deliver a wide variety of chemotherapeutics, owing to their biocompatibility and biodegradability
(Zhao et al. 2011). VLPs are well known for their capability to self-assemble into homogeneous nanoparticles with
relatively large cavity that can be utilized to package therapeutic molecules while having outer surfaces that can be
modified to display different epitopes and ligands via
genetic engineering or chemical cross-linking (Mateu
2011; Zhao et al. 2011; Pokorski et al. 2011a; Lucon et al.
2012; Tang et al. 2016). The VLPs of plant viruses such
6
as tobacco mosaic virus and cowpea mosaic virus have
been used for the delivery of platinum-based drugs such
as cisplatin, phenanthriplatin (Franke et al. 2018; Vernekar et al. 2018) and mitoxantrone (Lam et al. 2018).
Although the recombinant VLPs of the TYMV produced
in E. coli has been reported (Powell et al. 2012), alteration on the C-terminal end of the recombinant TYMVc
which is exposed on the viral surface has yet been performed.
In the current study, a 10-amino acid residue peptide
containing a polyhistidine tag was fused to the carboxyl
end of the TYMVc and produced in E. coli. The recombinant protein displaying polyhistidine tag, namely, TYMVcHis6, self-assembled into VLPs of approximately 30–
32 nm in diameter. Powell et al. (2012) reported that the
wild-type TYMVc produced in E. coli assembled into
VLPs of around 28 nm in diameter, similar to that of the
native virion. TYMVcHis6 formed slightly larger VLPs,
probably due to the addition of the fusion peptide. It is
known that the C-terminal end of the TYMVc is exposed
on surface of the virion (Canady et al. 1996; Shin et al.
2013). TYMVcHis6 has demonstrated a strong interaction
with IMAC, indicating the successful display of this
fusion peptide on the surface of the VLPs. Taken
together, the slight increase in the size of the VLPs is
most likely due to the extension of this linear fusion peptide on the surface of the VLPs.
Various modifications at the N- and C-terminal
regions have been performed on TYMV virion (Bransom
et al. 1995; Powell et al. 2012; Shin et al. 2013; Chae
et al. 2016). Genetic manipulation at the C-terminal
region of TYMVc often resulted in a less stable virion
with poor genomic RNA packaging ability (Bransom
et al. 1995) and compromised virion capsid assembly
(Shin et al. 2013). Unlike virion, recombinant TYMV
VLPs does not package viral RNA. Hence, the C-terminal
modification of TYMVc could have different effects on
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F.H. Tan et al.
the stability of VLPs. Powell et al. (2012) have performed
N-terminal deletions up to 26 amino acids on the recombinant TYMVc, where longer deletion resulted in compromised VLPs formation. In this study, the capability of
the recombinant TYMV VLPs to harbour additional short
peptide at the C-terminal end has been assessed. Polyhistidine tag was used as a model, mainly for the ease of
detection and purification. The results show that the
purified TYMVcHis6 readily assembles into slightly larger
VLPs, suggesting that the VLPs can be used to display
other short peptides, including various CPP such as TAT
(Baoum et al. 2012), penetratin (Nielsen et al. 2014),
MAP (Wada et al. 2013) and melittin (Hou et al. 2013),
which can then be deployed for targeted therapy.
Apart from its capability to display additional short
peptide, TYMVcHis6 VLPs readily disassemble and
reassemble with the use or denaturants. Powell et al.
(2012) used urea to extract TYMVc (wild-type and deletion mutants) from the insoluble fraction of cell lysate.
Wild-type TYMVc, being the most stable construct compared with the mutants, have lost 85–95% of the proteins
when urea was dialysed to below 1 mol l1. In this study,
soluble TYMVcHis6 VLPs were denatured using urea or
guanidine HCl. The recombinant protein was able to
reassemble back to VLPs when dialysed against buffer
with no denaturant, without visible loss of protein due to
precipitation, further justifying its potential usage as a
vehicle for packaging and targeted delivery of therapeutic
agents.
The thermal stability of empty TYMV capsid was
found to be higher compared with infectious virion, in
which the empty capsid was able to withstand up to
835°C at neutral pH (Virudachalam et al. 1985). Another
research carried out later discovered that the TYMV capsid was disrupted at temperature of about 65°C, while
the coat protein subunit denatures at 85°C, with evidence
from differential scanning microcalorimetry and electron
microscopy (Mutombo et al. 1993). Virudachalam et al.
(1985) suggested that the capsid stability decreased at
lower pH, possibly due to the repulsion between the three
histidine residues within the TYMVc, as the side chain of
histidine gets protonated, and in this current study, additional six histidine residues per coat protein were fused
and displayed on the outer surface of the VLPs. Interestingly, the thermal stability of TYMVcHis6 decreased at
lower pH and increased at higher pH, of which the VLPs
remained intact even at 75°C as indicated by DLS. Overall, TYMVcHis6 VLPs demonstrated stability across pH
range of 30–110, up to 55°C.
In summary, TYMV VLPs can display additional short
peptide on the surface of VLPs when fused to the C-terminal end of TYMVc. The resulting recombinant protein,
TYMVcHis6, self-assembled into robust and thermal
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Recombinant TYMV coat protein nanocarrier
stable VLPs that can be dissociated and reassociated to
package therapeutic agents. While the polyhistidine tag
can be replaced with other CPP for different applications,
TYMVcHis6 VLPs can be readily used to immobilize anticancer drug on the polyhistidine tag through zinc ion
and nitrilotriacetic acid for controlled drug delivery (Biabanikhankahdani et al. 2017). The potential use of TYMV
VLPs as a vaccine development platform can also be
explored in the future, provided that the TYMV VLPs
can harbour longer peptide at its C-terminal region.
Acknowledgements
This study was supported by the Fundamental Research
Grant Scheme (FRGS; Ministry of Higher Education
Malaysia) #FRGS/1/2013/SG06/TAYLOR/03/1 and Taylor’s
Internal Research Grant Scheme–Emerging Research Funding Scheme (TIRGS-ERFS) #TRGS/ERFS/1/2018/SBS/039
from Taylor’s University, Malaysia. Foo Hou Tan and Jia
Chen Kong were funded by the Tutorship Scheme from
the School of Biosciences, Taylor’s University.
Conflict of Interest
The authors declare no conflict of interest.
Author contributions
Foo Hou Tan and Jia Chen Kong carried out the experiment. Foo Hou Tan and Jia Chen Kong wrote the manuscript with support from Chuan Loo Wong and Chean
Yeah Yong. Jeck Fei Ng, Noorjahan Banu Alitheen and
Khai Wooi Lee involved in supervision, editing and
review process.
References
Ashley, C.E., Carnes, E.C., Phillips, G.K., Durfee, P.N., Buley,
M.D., Lino, C.A., Padilla, D.P., Phillips, B. et al. (2011)
Cell-specific delivery of diverse cargos by bacteriophage
MS2 virus-like particles. ACS Nano 5, 5729–5745.
Baoum, A., Ovcharenko, D. and Berkland, C. (2012) Calcium
condensed cell penetrating peptide complexes offer highly
efficient, low toxicity gene silencing. Int J Pharm 427,
134–142.
Biabanikhankahdani, R., Bayat, S., Ho, K.L., Alitheen, N.B.M.
and Tan, W.S. (2017) A simple add-and-display method
for immobilisation of cancer drug on His-tagged virus-like
nanoparticles for controlled drug delivery. Sci Rep 7, 5303.
Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem 72,
248–254.
7
Recombinant TYMV coat protein nanocarrier
Bransom, K.L., Weiland, J.J., Tsai, C.H. and Dreher, T.W.
(1995) Coding density of the turnip yellow mosaic virus
genome: roles of the overlapping coat protein and p206readthrough coding regions. Virology 206, 403–412.
Bujard, H., Gentz, R., Lanzer, M., Stueber, D., Mueller, M.,
Ibrahimi, I., Haeuptle, M.T. and Dobberstein, B. (1987) A
T5 promoter-based transcription-translation system for
the analysis of proteins in vitro and in vivo. Methods
Enzymol 155, 416–433. https://doi.org/10.1016/0076-6879
(87)55028-5
Canady, M.A., Larson, S.B., Day, J. and McPherson, A. (1996)
Crystal structure of turnip yellow mosaic virus. Nat Struct
Biol 3, 284–289.
Chae, K.-H., Kim, D. and Cho, T.-J. (2016) N-terminal
extension of coat protein of turnip yellow mosaic virus
has variable effects on replication, RNA packaging, and
virion assembly depending on the inserted sequence. J
Bacteriol Virol 46, 13–21.
Choi, K.M., Choi, S.H., Jeon, H., Kim, I.S. and Ahn, H.J.
(2011) Chimeric capsid protein as a nanocarrier for
siRNA delivery: stability and cellular uptake of
encapsulated siRNA. ACS Nano 5, 8690–8699.
Deo, V.K., Kato, T. and Park, E.Y. (2015) Chimeric virus-like
particles made using GAG and M1 capsid proteins
providing dual drug delivery and vaccination platform.
Mol Pharm 12, 839–845.
Franke, C.E., Czapar, A.E., Patel, R.B. and Steinmetz, N.F.
(2018) Tobacco mosaic virus-delivered cisplatin restores
efficacy in platinum-resistant ovarian cancer cells. Mol
Pharm 15, 2922–2931.
Gan, B.K., Yong, C.Y., Ho, K.L., Omar, A.R., Alitheen, N.B.
and Tan, W.S. (2018) Targeted delivery of cell penetrating
peptide virus-like nanoparticles to skin cancer cells. Sci
Rep 8, 8499. https://doi.org/10.1038/s41598-018-26749-y
Gilbert, L., Toivola, J., Lehtom€aki, E., Donaldson, L., K€apyl€a,
P., Vuento, M. and Oker-Blom, C. (2004) Assembly of
fluorescent chimeric virus-like particles of canine
parvovirus in insect cells. Biochem Biophys Res Commun
313, 878–887.
Guillen, G., Aguilar, J.C., Due~
nas, S., Hermida, L., Guzman,
M.G., Penton, E., Iglesias, E., Junco, J. et al. (2010) Virus-like
particles as vaccine antigens and adjuvants: application to
chronic disease, cancer immunotherapy and infectious
disease preventive strategies. Procedia Vaccinolgy 2, 128–133.
Hou, K.K., Pan, H., Lanza, G.M. and Wickline, S.A. (2013)
Melittin derived peptides for nanoparticle based siRNA
transfection. Biomaterials 34, 3110–3119.
Jin, K.-T., Lu, Z.-B., Chen, J.-Y., Liu, Y.-Y., Lan, H.-R., Dong,
H.-Y., Yang, F., Zhao, Y.-Y. et al. (2020) Recent trends in
nanocarrier-based targeted chemotherapy: selective
delivery of anticancer drugs for effective lung, colon,
cervical, and breast cancer treatment. J Nanomater 2020,
1–14. https://doi.org/10.1155/2020/9184284
Katouzian-Safadi, M. and Berthet-Colominas, C. (1983)
Evidence for the presence of a hole in the capsid of turnip
8
F.H. Tan et al.
yellow mosaic virus after RNA release by freezing and
thawing. Eur J Biochem 137, 47–55.
Keeling, J. and Matthews, R.E.F. (1982) Mechanism for release
of RNA from turnip yellow mosaic virus at high pH.
Virology 119, 214–218.
Kim, D., Lee, Y., Dreher, T.W. and Cho, T.-J. (2018) Empty
Turnip yellow mosaic virus capsids as delivery vehicles to
mammalian cells. Virus Res 252, 13–21.
Kittelmann, K. and Jeske, H. (2008) Disassembly of African
cassava mosaic virus. J Gen Virol 89, 2029–2036.
Lam, P., Lin, R. and Steinmetz, N. (2018) Delivery of
mitoxantrone using a plant virus-based nanoparticle for the
treatment of glioblastomas. J Mater Chem B 6, 5888–5895.
Lee, K.W., Tey, B.T., Ho, K.L. and Tan, W.S. (2011) Delivery
of chimeric hepatitis B core particles into liver cells. J Appl
Microbiol 112, 119–131.
Lee, K.W., Tey, B.T., Ho, K.L., Tejo, B.A. and Tan, W.S.
(2012) Nanoglue: an alternative way to display cellinternalizing peptide at the spikes of hepatitis B virus core
nanoparticles for cell-targeting delivery. Mol Pharm 9,
2415–2423.
Leimk€
uhler, M., Goldbeck, A., Lechner, M.D., Adrian, M.,
Michels, B. and Witz, J. (2001) The formation of empty
shells upon pressure induced decapsidation of turnip
yellow mosaic virus. Arch Virol 146, 653–667.
Lucon, J., Qazi, S., Uchida, M., Bedwell, G.J., LaFrance, B.,
Prevelige, P.E. and Douglas, T. (2012) Use of the interior
cavity of the P22 capsid for site-specific initiation of
atom-transfer radical polymerization with high-density
cargo loading. Nat Chem 4, 781–788.
Mateu, M.G. (2011) Virus engineering: functionalization and
stabilization. Protein Eng Des Sel 24, 53–63.
Mohamed Suffian, I.F.B., Wang, J.-T.-W., Hodgins, N.O.,
Klippstein, R., Garcia-Maya, M., Brown, P., Nishimura, Y.,
Heidari, H. et al. (2017) Engineering hepatitis B virus core
particles for targeting HER2 receptors in vitro and in vivo.
Biomaterials 120, 126–138.
Mutombo, K., Michels, B., Ott, H., Cerf, R. and Witz, J.
(1993) The thermal stability and decapsidation mechanism
of tymoviruses: a differential calorimetric study. Biochimie
75, 667–674.
Nielsen, E.J.B., Yoshida, S., Kamei, N., Iwamae, R., Khafagy,
E.-S., Olsen, J., Rahbek, U.L., Pedersen, B.L. et al. (2014)
In vivo proof of concept of oral insulin delivery based on
a co-administration strategy with the cell-penetrating
peptide penetratin. J Control Release 189, 19–24.
Pokorski, J.K., Breitenkamp, K., Liepold, L.O., Qazi, S. and
Finn, M.G. (2011a) Functional virus-based polymerprotein nanoparticles by atom transfer radical
polymerization. J Am Chem Soc 133, 9242–9245.
Pokorski, J.K., Hovlid, M.L. and Finn, M.G. (2011b) Cell
targeting with hybrid Qb virus-like particles displaying
epidermal growth factor. ChemBioChem 12, 2441–2447.
Powell, J.D., Barbar, E. and Dreher, T.W. (2012) Turnip
yellow mosaic virus forms infectious particles without the
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
F.H. Tan et al.
native beta-annulus structure and flexible coat protein Nterminus. Virology 422, 165–173.
Rohovie, M.J., Nagasawa, M. and Swartz, J.R. (2017) Viruslike particles: next-generation nanoparticles for targeted
therapeutic delivery. Bioeng Transl Med 2, 43–57.
van Roon, A.-M.-M., Bink, H.H.J., Plaisier, J.R., Pleij, C.W.A.,
Abrahams, J.P. and Pannu, N.S. (2004) Crystal structure
of an empty capsid of turnip yellow mosaic virus. J Mol
Biol 341, 1205–1214.
Senapati, S., Mahanta, A.K., Kumar, S. and Maiti, P. (2018)
Controlled drug delivery vehicles for cancer treatment and
their performance. Signal Transduction Targeted Ther 3, 7.
Shin, H.-I., Chae, K.-H. and Cho, T.-J. (2013) Modification of
turnip yellow mosaic virus coat protein and its effect on
virion assembly. BMB Rep 46, 495–500.
Singh, P., Destito, G., Schneemann, A. and Manchester, M.
(2006) Canine parvovirus-like particles, a novel
nanomaterial for tumor targeting. J Nanobiotechnol 4, 2-2.
Suffian, I.F.M., Wang, J.T.W., Faruqu, F.N., Benitez, J.,
Nishimura, Y., Ogino, C., Kondo, A. and Al-Jamal, K.T.
(2018) Engineering human epidermal growth receptor 2targeting hepatitis B virus core nanoparticles for siRNA
delivery in vitro and in vivo. ACS Appl Nano Mater 1,
3269–3282.
Tang, S., Xuan, B., Ye, X., Huang, Z. and Qian, Z. (2016) A
modular vaccine development platform based on sortasemediated site-specific tagging of antigens onto virus-like
particles. Sci Rep 6, 25741.
Thong, Q.X., Biabanikhankahdani, R., Ho, K.L., Alitheen, N.B.
and Tan, W.S. (2019) Thermally-responsive virus-like
particle for targeted delivery of cancer drug. Sci Rep 9,
3945.
Tissot, A.C., Renhofa, R., Schmitz, N., Cielens, I., Meijerink,
E., Ose, V., Jennings, G.T., Saudan, P. et al. (2010)
Versatile virus-like particle carrier for epitope based
vaccines. PLoS One 5, 3–10.
Vernekar, A.A., Berger, G., Czapar, A.E., Veliz, F.A., Wang,
D.I., Steinmetz, N.F. and Lippard, S.J. (2018) Speciation
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
Recombinant TYMV coat protein nanocarrier
of phenanthriplatin and its analogs in the core of tobacco
mosaic virus. J Am Chem Soc 140, 4279–4287.
Virudachalam, R., Low, P.S., Argos, P. and Markley, J.L.
(1985) Turnip yellow mosaic virus and its capsid have
thermal stabilities with opposite pH dependence: studies
by differential scanning calorimetry and 31P nuclear
magnetic resonance spectroscopy. Virology 146, 213–220.
Wada, S.-I., Hashimoto, Y., Kawai, Y., Miyata, K., Tsuda, H.,
Nakagawa, O. and Urata, H. (2013) Effect of Ala
replacement with Aib in amphipathic cell-penetrating
peptide on oligonucleotide delivery into cells. Bioorg Med
Chem 21, 7669–7673.
Wu, M., Sherwin, T., Brown, W.L. and Stockley, P.G. (2005)
Delivery of antisense oligonucleotides to leukemia cells by
RNA bacteriophage capsids. Nanomedicine: Nanotechnol
Biol Med 1, 67–76.
Yan, D., Wei, Y.Q., Guo, H.C. and Sun, S.Q. (2015) The
application of virus-like particles as vaccines and
biological vehicles. Appl Microbiol Biotechnol 99,
10415–10432.
Yang, J., Zhang, Q., Liu, Y., Zhang, X., Shan, W., Ye, S., Zhou, X.,
Ge, Y. et al. (2020) Nanoparticle-based co-delivery of siRNA
and paclitaxel for dual-targeting of glioblastoma.
Nanomedicine (London, England) 15, 1391–1409.
Yin, Z., Dulaney, S., McKay, C.S., Baniel, C., Kaczanowska, K.,
Ramadan, S., Finn, M.G. and Huang, X. (2016) Chemical
synthesis of GM2 glycans, bioconjugation with
bacteriophage Qb, and the induction of anticancer
antibodies. ChemBioChem 17, 174–180.
Zeltins, A. (2013) Construction and characterization of viruslike particles: a review. Mol Biotechnol 53, 92–107.
Zhang, Q., Xu, D., Guo, Q., Shan, W., Yang, J., Lin, T., Ye, S.,
Zhou, X. et al. (2019) Theranostic quercetin nanoparticle for
treatment of hepatic fibrosis. Bioconjug Chem 30, 2939–2946.
Zhao, Q., Chen, W., Chen, Y., Zhang, L., Zhang, J. and
Zhang, Z. (2011) Self-assembled virus-like particles from
rotavirus structural protein VP6 for targeted drug delivery.
Bioconjug Chem 22, 346–352.
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