Enhancing Solubilization of Hydrophobic ORF3 Product of HEV in Bacterial Expression System

Citation: Luo A, Yang Y, Yu Y, Xiao Y, Zhang Y. Enhancing Solubilization of Hydrophobic ORF3 Product of Hepatitis E Virus in Bacterial Expression
System. J Vaccine Immunotechnology. 2014;1(1): 5.
J Vaccine Immunotechnology
June 2014 Volume 1, Issue 1
© All rights are reserved by Zhang et al.
Enhancing Solubilization of
Hydrophobic ORF3 Product of
Hepatitis E Virus in Bacterial
Expression System
Keywords: Hepatitis E virus; HEV; ORF3; vp13; Purification;
Solubilization; Hydrophobic protein
Abstract
Hepatitis E Virus (HEV) is a major cause of acute clinical hepatitis in
developing countries. HEV genome has three open reading frames:
ORF1, ORF2 and ORF3. ORF3 encodes a small 13 kDa protein
(hereinafter called vp13). This protein has been suggested to have
multiple functions, including interference with cell signaling pathways,
viral particle release from infected cells, and viral pathogenesis.
However, the structural basis and mechanism by which the vp13 exerts
these multiple biological functions is unclear. Characterization of vp13
has been hindered by the difficulty in efficiently producing enough
hand pure vp13 proteins. This may attribute to vp13’s hydrophobic
and insoluble nature. The objective of this study was to express vp13
in prokaryotic expression system and optimize the conditions to
increase the soluble portion for vp13 purification. The ORF3 sequence
was cloned into a bacterial expression plasmid with maltose binding
protein (MBP) as a tag. The recombinant plasmid was transformed into
E. Coli BL-21 strain for protein expression. Most MBP-vp13 protein was
found in insoluble portion, making protein purification a challenge. To
increase the solubility of this recombinant protein, we optimized the
induction and processing conditions. Induction at 18°C and addition
of 1% glucose to the bacterium culture successfully led to efficient
expression of soluble recombinant MBP-vp13 fusion protein. Further,
the soluble fusion protein was easy to be purified. This enhanced
solubilization and purification of vp13 protein should facilitate further
study of vp13’s structure and function. This study provides an example
for expression and purification of hydrophobic proteins for vaccine or
immunological reagent production.
Introduction
Hepatitis E Virus (HEV) is a major cause of acute hepatitis,
prevalent in developing countries [1]. Currently, one third of the
world’s population is at risk of infection of the Hepatitis E Virus. An
estimated fourteen million symptomatic cases of HEV infection with
three hundred thousand deaths are reported annually throughout
the world, primarily in developing countries including the Indian
subcontinents, Southeast and Central Asia, the Middle East, and
Northern/Western parts of Africa [2]. Although HEV infection has
emerged as a global health threat, its relative absence in the Western
world contributes to its lack of recognition. Clinical manifestation of
HEV infection can range from asymptomatic to fulminant hepatitis
and death. Approximately 20% of infections are symptomatic in high
disease endemic areas. The death rate among pregnant women who
express symptoms can reach up to 25% and it usually occurs in their
third trimester [1]. HEV infection is also problematic for immune-
compromised individuals and organ transplant patients [3].
HEV is a small, non-enveloped, single-stranded positive-
sense RNA virus. The genome is approximately 7.2 kb, and has
a 5’ cap and a 3’-poly A tail [4]. The virus classified as a Hepevirus
in the family Hepeviridae has four genotypes yet only one serotype.
Genotypes 1 and 2 infect solely humans, while genotypes 3 and 4 also
infect pigs and other mammalian species [5]. The RNA genome of
HEV is organized into three open reading frames (ORFs), with the
nonstructural proteins encoded at the 5’ end of the genome and the
structural proteins at the 3’ end. ORF1 encodes the nonstructural
polyprotein; ORF2 encodes the major capsid protein; ORF3 encodes
a small protein, which is 114 amino acids long [6,7]. Exact function
of vp13 encoded by ORF3 is not clear, although it has been suggested
that vp13 has multiple functions including interference of cell
signaling pathways, viral particle release from infected cells and
viral pathogenesis [5]. Some studies suggested that the expression
of the intact ORF3 protein is required for the infection in vivo [6,8].
However, other studies showed that the protein is not required for
infection and virion morphogenesis in vitro [9]. HEV vp13 protein has
been suggested to interact with more than 30 human cellular proteins
[10,11]. How such a small viral protein plays so many roles in the
virus life cycle and interacts with an abundance of cellular proteins is
not well understood. The structure of vp13 protein is not known. It
is important to delineate the structural and functional relationship of
vp3 protein. However, characterization of vp13 has been hindered by
the difficulty in efficiently generating a recombinant vp13 due to the
protein’s hydrophobic nature and insolubility.
The 114 amino acids of vp13 encoded by ORF3 are highly
hydrophobic with 41 of the 114 amino acids are hydrophobic amino
acids. The objective of this study was to enhance the expression
and solubility of vp13. The hypothesis was that a recombinant
fusion protein between the ORF3 protein and a maltose binding
protein (MBP) would enhance the solubility of vp13 and that the
induction conditions could be optimized to enhance the solubility
Allyson Luo1‡
, Yonglin Yang1,2‡
, Ying Yu1
, Yueqiang
Xiao1
and Yanjin Zhang1
*
1
Molecular Virology Laboratory, VA-MD Regional College of
Veterinary Medicine and Maryland Pathogen Research Institute,
University of Maryland, College Park, MD 20742
2
Nanjing Red Cross Blood Center, Najing, Jiangsu, China
‡
Co-first authors
Address for Correspondence
Yanjin Zhang, Molecular Virology Laboratory, VA-MD Regional
College of Veterinary Medicine, University of Maryland, 8075
Greenmead Drive, College Park, MD 20742,USA, Tel: +1 301 314-
6596; Fax: +1 301 314-6855; E-mail address: zhangyj@umd.edu
Submission: 10 April 2014
Accepted: 17 June 2014
Published: 20 June 2014
Reviewed & Approved by: Dr. Guey Chuen Perng, Department
of Microbiology and Immunology, College of Medicine, National Cheng
Kung University, Taiwan
Research ArticleOpen Access
Journal of
Vaccine &
Immunotechnology
Avens Publishing Group
Inviting Innovations
Avens Publishing Group
Inviting Innovations

Citation: Luo A, Yang Y, Yu Y, Xiao Y, Zhang Y. Enhancing Solubilization of Hydrophobic ORF3 Product of Hepatitis E Virus in Bacterial
Expression System. J Vaccine Immunotechnology. 2014;1(1): 5.
J Vaccine Immunotechnology 1(1): 5 (2014) Page - 02
of the recombinant protein. The induction conditions to be adjusted
included relevant conditions such as IPTG concentration, induction
time,andadditionofglucose.Theresultsshowedthattherecombinant
MBP-vp13 fusion protein was successfully expressed as a soluble
form that was detected by a rabbit antibody specifically against vp13.
Furthermore, the recombinant MBP-vp13 fusion protein was purified
by a kit specifically designed for MBP fusion protein purification. The
results of this study can be extrapolated to other studies on vaccine
or immunological reagent production using hydrophobic proteins.
Materials and Methods
Plasmid construction: HEV ORF3 was amplified
from a pSK-E2 plasmid that contains the full-length cDNA
of the HEV genome (GenBank accession no. AF444002)
[12]. PCR was conducted using the primers H3F14
(5’-GGAATTCCATATGGGTTCGCGACCATGCG-3’) and H3R16
(5’-CGGCTCGAGTTAGCGGCGCGGCCCCAGCTGTG-3’), which
contain restriction sites for NdeI and XhoI to facilitate directional
cloning. The PCR amplified ORF3 fragment was digested with NdeI
and XhoI and cloned into the expression plasmid pVL-847 [13],
which is a prokaryotic expression plasmid containing MBP coding
sequence. The resulting plasmid was transformed into E. coli DH5α
competent cells. Miniprep DNA was prepared from the E. coli clones;
positive clones were identified with digestion of the DNA with NdeI
and XhoI. The integrity of positive clones was further confirmed by
DNA sequencing. The glycerol stock of the positive E. coli clones was
stored in a -80°C freezer until use.
Recombinant MBP-vp13 fusion protein expression: The
recombinant plasmid DNA was transformed into BL21 competent
cells for protein expression. The transformed BL21 cells were cultured
at a 37°C shaker for overnight at 220 rpm. The next day, a fresh 2 ml
LB broth was inoculated with the overnight culture at 1:500 dilution
and cultured for 2-3 hours at 250 rpm and 37°C. To induce fusion
protein expression, IPTG at indicated concentrations (from 0.2 to
1.0 mM) was added to the LB broth when the OD600 of the bacteria
culture reaches 0.8 and incubated further in the shaker for 3 hours.
The bacteria culture was spun down for 5 minutes at 10,000 xg to
pellet the E. coli. The pellet was resuspended in 100 µL of PBS pH7.2
with 5 mM of EDTA in 1.5 ml microfuge tubes and sonicated for
indicated time in cycles of 10 second sonication (output voltage 1000
V rms and frequency 20 KHz) and 10 second pause in a 3” diameter
cup horn (Model Q500, Qsonica Sonicator Ultrasonic Processor,
Newtown, CT). The sonicated samples were spun down for 10
minutes at 4°C and 14,000 xg. Both the supernatant and pellet from
the sonicated samples were run on SDS-PAGE (Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis), followed by Western
Blot analysis.
Todetectvp13expression,theWesternBlotmembranewasprobed
with rabbit antibody specifically against vp13 as described previously
[11] or MBP monoclonal antibody (Rockland Immunochemicals,
Inc., Gilbertsville, PA). The signal was detected by goat anti-rabbit
or anti-mouse IgGsecondary antibody conjugated with horseradish
peroxidase (Sigma-Aldrich, St. Louis, MO) and revealed using a
chemiluminescence substrate. The chemiluminescence signal was
recorded digitally using a ChemiDoc XRS imaging system (Bio-
Rad Laboratories, Hercules, CA). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology,
Inc., Dallas, TX) was also used to probe some blotting membranes
for loading control.
Purification of MBP-vp13 fusion protein: To purify
MBP-vp13 fusion protein, syringe- operated MBPTrap HP
columns were used (GE Healthcare Life Sciences, Pittsburgh,
PA) by following the manufacturer’s instructions. The syringe was
filled with binding buffer (20 mMTris-HCl, 200 mMNaCl, 1 mM
EDTA, pH7.4). The ethanol in the column was washed out with 5
column volumes (CV) of the binding buffer. Then, the column was
equilibrated with 5 CV of binding buffer at a speed of 1 ml/min. The
sample containing the fusion protein was loaded to the column by
a syringe. The column was washed with 5 CV of the binding buffer
to wash out unbound non-specific proteins. Finally, the protein
bound to the column was eluted with 5 CV of elution buffer (binding
buffer plus 10 mM maltose). The eluted fractions were combined and
concentrated up to 20-fold using Amicon Ultra-4 Centrifugal Filter
Unit (Millipore, Billerica, MA). Examination for the presence of
MBP-vp13 was done by SDS-PAGE and Coomassie blue (Bio-Rad)
staining analysis, and Western blotting with antibodies against MBP
and vp13.
Results
Construction of prokaryotic expression plasmid
encoding recombinant MBP-vp13 fusion protein: Among 114
vp13 amino acids, 41 (36%) are hydrophobic.HEV vp13 was analyzed
by a hydrophobicity prediction program available in the public
domain (http://web.expasy.org/protparam/). The hydrophobicity
index for vp13 is +0.378, suggesting the high hydrophobic nature of
vp13. The hydrophobicity index is 0.972 for the first half of the protein
and -0.216 for the second half, indicating most hydrophobicity is due
to the N-terminal half. This may explain the difficulty in efficiently
expressing a soluble full length vp13. MBP has been successfully
used as a fusion tag to express hydrophobic and insoluble proteins
[13]. In this study, vp13 was expressed as a fusion protein with MBP
by ligating the cDNA encoding vp13 that was PCR amplified from
pSK-E2 and cloned into pVL-847, which already contained the
MBP coding sequence. The integrity of the expression construct was
confirmed by restriction enzyme digest and direct DNA sequencing.
Majority of recombinant MBP-vp13 fusion protein in
E. coli are insoluble: The fusion protein expression plasmid was
transformed into BL21 competent E. coli. IPTG 1 mM was used to
induce the protein expression. Whole protein from the bacterial cells
was subjected to SDS-PAGE and Western blotting with vp13 antibody
to check the protein expression. Among seven colonies selected, two
had high level expression of MBP-vp13 protein (Figure 1).
Knowing the fusion protein was expressed in E. coli, we checked
for its solubility. After IPTG induction, the E. coli pellet was sonicated
to release the protein. After sonication, the sample was separated
by centrifugation into soluble fraction (supernatant) and insoluble
fraction (pellet). Both fractions were run on SDS-PAGE followed by
Western Blot analysis with anti-vp13 specific antibody. Surprisingly,
the majority of the fusion protein, which reacted with the anti-vp13
specific antibody, was in the insoluble fraction, only a minor fraction
of the target protein was detected in the soluble fraction (Figure 2A).
Optimization of recombinant MBP-vp13 fusion protein
expression: To enhance the protein expression level and to increase
its solubility, several conditions were tested, including concentration
ISSN: 2377-6668

of IPTG, induction time and temperature. IPTG concentration from
0.2 to 1.0 mM was first tested (Figure 2B). It was found that IPTG at 0.2
mM induced 53-fold increase in MBP-vp13 fusion protein expression
when compared to mock-induction control, after normalization with
GAPDH. Therefore, 0.2 mM IPTG was chosen for further studies.
Next, different induction temperatures were tested. It was found
that the protein can be expressed in culture at 18°C and there was
minimum difference in solubility of the protein, ranging in relative
levels of vp13 from 1 to 1.4-fold (Figure 3A). Majority of the vp13
fusion protein was still in pellet. So 18°C was selected for further
studies to enhance the protein solubility. Induction time was then
tested to find the optimal time of induction for the protein expression
(Figure 3B). The protein level was similar from 1.5 h to 5.5 h at 18°C
with 0.2 mM IPTG induction. Thus, three hour induction was selected
for further studies of this protein.
Optimization of MBP-vp13 fusion protein extraction
from E. coli: To increase the level of MBP-vp13 in the soluble
fraction, time duration of sonication was tested. At a constant power,
duration of sonication ranged from 1 to 8 minutes in cycles of 10
second sonication and 10 second pause. Along with the increase of
sonication time, MBP-vp13 level in the soluble fraction significantly
increased while the protein level in the insoluble fraction decreased
(Figure 4A, 4B). In vp13 blotting, the relative levels of MBP-vp13 in
supernatant were 1 to 2.5 for the duration of sonication from 1 to
8 minutes (Figure 4A). In MBP blotting, the relative levels of MBP-
vp13 in supernatant were 1 to 2.3 for the duration of sonication from
1 to 8 minutes (Figure 4B). It appeared that 3-minute sonication was
enough to cause release of most soluble MBP-vp13, especially shown
in blotting with MBP antibody (Figure 4B). The result suggests that
the duration of sonication time needs to be optimized to increase the
release of the protein into the soluble fraction.
Enhancing solubility of MBP-vp13 fusion protein:
Glucose and ethanol were tested for their effect on overall protein
expression with particular focus on the level of protein in the soluble
fraction. Glucose represses induction of the lac promoter by lactose,
which is present in most rich media including LB [14]. Addition of
glucose at 1% and ethanol at 3% significantly increased the portion
of MBP-vp13 in the soluble fraction to 2 and 1.5-fold, respectively
(Figure 5). Addition of ethanol at 5% caused reduction of the fusion
protein level, indicating a delicate concentration range is hard to
optimize and its use should be avoided in this case. So 1% glucose was
selected for further work on MBP-vp13 expression and purification.
Figure 1: Induction of MBP-vp13 expression in E. coli at 37°C with IPTG 1
mM for 3 hours. Seven colonies were selected to inoculate LB at 2 ml each
and cultured for IPTG induction. Colony 1 without induction was included
as a control. The bacteria were collected and pellets were dissolved in the
Laemmli sample buffer. The total proteins were separated in SDS-PAGE and
blotted with rabbit anti-vp13 antibody.
Figure 2: Most of MBP-vp13 protein expressed is in insoluble portion.
A. Detection of MBP-vp13 in supernatant and pellet after sonication and
centrifugation. Four colonies were selected to inoculate LB culture, cultured
at 37°C, and induced with 1 mM IPTG for 3 h. Pellets were resuspended in
PBS and sonicated. The samples were centrifuged to separate supernatant
and pellet. Western blotting with rabbit anti-vp13 antibody was conducted.
B. Different IPTG concentrations in induction tested. Supernatant samples
were used for the Western blotting with antibodies against vp13 and GAPDH.
IPTG concentrations at 0 (control lane), 0.2, 0.4, 0.6, 0.8, and 1.0 mM were
tested. The bacteria samples at equal volumes from different concentrations
of IPTG were sonicated and analyzed by SDS-PAGE and Western blotting.
Densitometry analysis of the bands was done with Image Lab (Bio-Rad) and
relative levels of MBP-vp13 after normalization with GAPDH are shown below
the images.
Figure 3: Optimization of induction temperature and time for MBP-vp13
expression: A. Effect of temperature on vp13 solubility. The bacterium
culture was induced with 0.2 mM IPTG for 3 h. Western blotting with rabbit
anti-vp13 antibody was conducted. Densitometry analysis of the bands was
done and relative levels of MBP-vp13 are shown below the image. B. Effect
of induction time of MBP-vp13 solubility in BL21 cells at IPTG 0.2 mM, 180
rpm and 18°C. Western blotting with antibody against vp13 was conducted.
Figure 4: Optimization of sonication time for vp13 release. Western
blotting was done with antibodies against vp13 (A) and MBP (B). The bacteria
were cultured at 18°C and 180 rpm, and induced with IPTG 0.2 mM. Total
time in minutes of sonication is listed above the image. Sonication was done
as cycles of 10 seconds pulse and 10 seconds pause. Densitometry analysis
of the bands was done with Image Lab (Bio-Rad) and relative levels of MBP-
vp13 in supernatant are shown below the images.
ISSN: 2377-6668

Purification of recombinant MBP-vp13 fusion protein:
To purify the fusion protein, a GE MBPTrap HP column was used by
following the manufacturer’s instruction. The soluble fraction after
sonication of the bacteria containing the fusion protein was applied to
the column. After washing away the unbound non-specific proteins,
the fusion protein was eluted. The eluted proteins were examined by
SDS-PAGE and Coomassie blue staining. A band at ~55 kDa and
some minor bands below it appeared in the gel (Figure 6A). The size of
the band matches the expected size of recombinant MBP-vp13 fusion
protein, indicating that the elusions contains the soluble MBP-vp13
and that purification by this simple one-step method appeared to be
pureasonlysinglemajorbandwasobserved.Toconfirmthespecificity
of the band, we ran Western blot analysis with antibodies against both
vp13 and MBP, showing a single major band (Figure 6B). The data
suggested that MBP-vp13 fusion protein can be readily purified by
the column and that the process from expression to extraction yields
intact MBP-vp13 fusion protein. Densitometry analysis of the images
was done and approximate calculations were conducted taking into
consideration of loading amount and total volume of each sample in
the purification process. It appeared that the amount of MBP-vp13
in preloading soluble portion is 27% of total MBP-vp13 in the whole
culture harvested (Figure 6c), which indicates that at least more than
a quarter of the protein was release into soluble portion in the initial
process. The soluble vp13 portion was significantly higher than the
approximate 7% in the initial optimization (Figure 3A). The total
amount of eluted MBP-vp13 is 44% of the total soluble vp13 in the
preloading sample. The results suggest that the purification process
is efficient, yet further optimization is needed to improve recovery by
avoiding overloading the MBPTrap HP column.
Discussion
Characterization and understanding of vp13 have been hindered
by the difficulty in efficiently generating a soluble recombinant
vp13. This could be due to the fact that vp13 is a highly hydrophobic
and insoluble protein. In this study, the expression of recombinant
MBP-vp13 fusion protein was optimized, including IPTG level and
glucose addition. The fusion protein was efficiently extracted to
increase soluble portion. A significant portion of the fusion protein
was present in the soluble rather than the insoluble fraction after the
optimizations. The intact fusion protein was successfully purified by a
simple one-step affinity purification process.
In order to study biological function of vp13 as well as its structure
and function relationship, a cDNA encoding a fusion protein
between maltose binding protein (MBP) and vp13 was engineered.
The reason MBP was chosen as a fusion partner for vp13 was due to
MBP’s hydrophilic nature. Furthermore, MBP has been successfully
used by other researchers to generate fusion proteins when the target
protein is insoluble and hydrophobic [15,16]. Yet, initial attempts to
efficiently express the fusion protein in soluble form met very limited
success. Therefore, a series of conditions were optimized, including
IPTG concentration and addition of glucose to enhance protein
expression level, as well as sonication time to increase the portion of
soluble fusion protein. The results suggested that 0.2 mM IPTG and
1% glucose led to a high level expression of the soluble fusion protein.
When the combination of optimized conditions was established, the
fusion protein was produced with increased solubility. Further, the
MBP tag facilitated the purification of the recombinant protein with
an MBPTrap HP column, which was specifically designed for the
purification of MBP fusion proteins.
To the best of our knowledge, there have been considerable
hurdles in attempt to efficiently express the recombinant vp13 due to
the hydrophobic and insoluble nature of this protein. The creation of
the plasmid encoding MBP-vp13 fusion protein and the development
of a process to express the fusion protein as well as the convenient
purification method should assist further study of the biology of vp13.
The optimized conditions in this study can also be a good reference
for others to express and purify proteins that have similar difficulty in
soluble expression.
The availability of pure vp13 fusion protein can facilitate the
protein’s structural analysis, which in turn can help delineate its
biological function. For example, a larger quantity of pure MBP-vp13
could be used for protein crystallization. Protein crystallization has
playedavitalroleinunderstandingstructureandfunctionrelationship
for many proteins. Hopefully, the materials generated and the process
Figure 5: Effect of glucose and ethanol in induction of MBP-vp13 expression
as soluble protein. The bacteria were cultured at 18ᵒC and 180 rpm, and
induced with IPTG 0.2 mM for 3 h. Western blotting was done with antibodies
against vp13.Densitometry analysis of the bands was done with Image Lab
(Bio-Rad) and relative levels of MBP-vp13 in supernatant are shown below
the images.
Figure 6: Purification of MBP-vp13 protein using MBP column. A.
Coomassie Blue staining of purified MBP-vp13 in SDS-PAGE. Total protein
was included as a control. MW: molecular weight marker. 1E: elusion of first
column in purification, loading 10 µl (a total of 5 ml). 1Ed: dilution of first
column elusion at 1:5. 2E: elusion of second column in purification, loading
10 µl (a total of 5 ml). 2Ed: dilution of second column elusion at 1:5. PT: pass
through during column purification, 10 µl. PL: pre-loading sample, loading
10 µl (a total of 8 ml). TP: total protein of the lysate of 100 µl culture (a
total culture of 400 ml). B. Western blotting of the purified MBP-vp13 protein
with antibodies against vp13 and MBP. Loading volumes are the same as in
“A”. C. Densitometry analysis of the bands in “B” and calculation of relative
percentage of MBP-vp13 level in each step of the purification compared with
total vp13 in culture harvested. Loading volumes and total volumes were
taken into consideration.
ISSN: 2377-6668

developed in this study will accelerate the understanding of vp13 in
its roles in viral replication and pathogenesis. Further understanding
the structure and function of vp13 may also facilitate anti-HEV drug
development.
More importantly, the methods used in this study to optimize
the expression and solubilization of vp13 can be extrapolated to
other studies on hydrophobic proteins. Expression and purification
of such proteins for vaccines or other immunological reagents can
be optimized similarly to enhance the portion of soluble proteins.
The soluble proteins are always welcomed due to their proper
conformation and function similar to native counterparts.
References
1. Kamar N, Dalton HR, Abravanel F, Izopet J (2014) Hepatitis E virus infection.
Clin Microbiol Rev 27: 116-138.
2. Aggarwal R (2010) The global prevalence of hepatitis E virus infection and
susceptibility: a systemic review. World Health Organization.
3. Geng Y, Yang J, Huang W, Harrison TJ, Zhou Y, et al. (2013) Virus host
protein interaction network analysis reveals that the HEV ORF3 protein may
interrupt the blood coagulation process. PLoS One 8: e56320.
4. Emerson S, Anderson D, Arankalle VA, Meng XJ, Purdy M, Schlauder
GG, Tsarev S, (2004) Hepevirus, in: Fauquest CM, Mayo MA, Maniloff J,
Desselberger U, Ball LA (Editors) Virus Taxonomy, VIIIth report of the ICTV.
Elseiver/Academic Press, London, UK.
5. Ahmad I, Holla RP, Jameel S (2011) Molecular virology of hepatitis E virus.
Virus Res 161: 47-58.
6. Graff J, Nguyen H, Yu C, Elkins WR, St Claire M, et al. (2005) The open
reading frame 3 gene of hepatitis E virus contains a cis-reactive element and
encodes a protein required for infection of macaques. J Virol 79: 6680-6689.
7. Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, et al. (1991)
Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length
viral genome. Virology 185: 120-131.
8. Huang YW, Opriessnig T, Halbur PG, Meng XJ (2007) Initiation at the third in-
frame AUG codon of open reading frame 3 of the hepatitis E virus is essential
for viral infectivity in vivo. J Virol 81: 3018-3026.
9. Emerson SU, Nguyen H, Torian U, Purcell RH (2006) ORF3 protein of
hepatitis E virus is not required for replication, virion assembly, or infection of
hepatoma cells in vitro. J Virol 80: 10457-10464.
10. Geng Y, Yang J, Huang W, Harrison TJ, et al. (2013) Virus host protein
interaction network analysis reveals that the HEV ORF3 protein may interrupt
the blood coagulation process. PLoS One 8: e56320.
11. Kannan H, Fan S, Patel D, Bossis I, Zhang YJ (2009) The hepatitis E virus
open reading frame 3 product interacts with microtubules and interferes with
their dynamics. J Virol 83: 6375-6382.
12. Emerson SU, Zhang M, Meng XJ, Nguyen H, St Claire M, et al. (2001)
Recombinant hepatitis E virus genomes infectious for primates: importance
of capping and discovery of a cis-reactive element. Proc Natl Acad Sci U S A
98: 15270-15275.
13. Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, et al. (2007) A
cyclic-di-GMP receptor required for bacterial exopolysaccharide production.
Mol Microbiol 65: 1474-1484.
14. EMBL (2009) Protein Expression and Purification Core FacilityProtein
Expression E.coli. European Molecular Biology Laboratory.
15. Hewitt SN, Choi R, Kelley A, Crowther GJ, Napuli AJ, et al. (2011) Expression
of proteins in Escherichia coli as fusions with maltose-binding protein to
rescue non-expressed targets in a high-throughput protein-expression and
purification pipeline. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:
1006-1009.
16. Sorensen HP, Mortensen KK (2005) Soluble expression of recombinant
proteins in the cytoplasm of Escherichia coli. Microb cell Fact 4: 1.
We thank Dr. Suzanne Emerson in NIH and Dr. X-J Meng at Virginia
Tech University for some plasmid and antibody used in this study.
This study was partially supported by University of Maryland.
Acknowledgements
ISSN: 2377-6668
Copyright: © 2014 Luo A, et al. This is an open access article distributed
under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited.

Enhancing Solubilization of Hydrophobic ORF3 Product of HEV in Bacterial Expression System

More Related Content

What's hot (19)

Viewers also liked (14)

Similar to Enhancing Solubilization of Hydrophobic ORF3 Product of HEV in Bacterial Expression System (20)

Enhancing Solubilization of Hydrophobic ORF3 Product of HEV in Bacterial Expression System