Rapid purification of recombinant dengue and West Nile virus envelopeDomain III proteins by metal affinity membrane chromatography Lik Chern Melvin Tan, Anthony Jin Shun Chua, Li Shan Liza
Trang 1Rapid purification of recombinant dengue and West Nile virus envelope
Domain III proteins by metal affinity membrane chromatography
Lik Chern Melvin Tan, Anthony Jin Shun Chua, Li Shan Liza Goh, Shu Min Pua,
Yuen Kuen Cheong, Mah Lee Ng*
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore
a r t i c l e i n f o
Article history:
Received 14 April 2010
and in revised form 22 June 2010
Available online 1 July 2010
Keywords:
Flavivirus
Dengue virus
West Nile virus
Envelope Domain III
Ó 2010 Elsevier Inc All rights reserved.
Introduction
Dengue virus (DENV) 1 and West Nile virus (WNV) belong to the
flavivirus genus in the Flaviviridae family DENV, which comprises
four antigenically distinct serotypes (1–4), causes a wide range of
diseases ranging from mild dengue fever to severe dengue
hemor-rhagic fever and dengue shock syndrome (DHF/DSS) [1–5] It has
been estimated that more than 2.5 billion people in over 100
coun-tries are at risk of dengue infection, with several hundred thousand
cases of life threatening DHF/DSS occurring every year [6] WNV
causes diseases ranging from asymptomatic infection to febrile
ill-ness and fatal encephalitis WNV comprises two lineages (1 and 2),
with most isolates which cause severe human disease belonging to
lineage 1 WNV is the pathogen responsible for the outbreak of
encephalitis which occurred in New York City in USA in 1999 [7]
To date, there have been 29,624 reported human cases of WNV
infec-tions, including 1161 fatalities, in the USA [8]
The flavivirus genome encodes three structural proteins: the
capsid protein, premembrane protein and envelope protein; and
seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 [9,10] The envelope protein comprises three re- gions: Domain I, Domain II and Domain III Domain I is the central domain, Domain II is the dimerization domain, while Domain III (DIII) is an immunoglobulin-like domain [11,12] Experimental evi- dence has shown that DIII protein is a receptor recognition and binding domain [13–16] In addition, this protein has also been demonstrated to be highly immunogenic and is able to elicit the production of neutralizing antibodies against wild-type virus [17–20] For this reason, DIII protein is an important immunogen for the development of a possible protein subunit vaccine and also
a potential diagnostic reagent for improved clinical diagnosis of flaviviral infections [21–25] In order to facilitate downstream DIII protein-based research, there is a need for a consistent and reliable method for rapid production and purification of recombinant DIII (rDIII) proteins It would be advantageous if the optimized purifica- tion process could be scaled-up easily to an industrial scale for the bioprocess industry.
Chromatography is by far the technique used most widely for high-resolution separation and analysis of proteins [26] Even though traditional packed-bed affinity chromatography is a com- mon method for the purification of recombinant proteins, there are several limitations This method requires the diffusion of target molecules to binding sites found within the pores of the resin in or- der for binding to occur This increases process time and conse- quently the amount of recovery liquid needed for elution.
1046-5928/$ - see front matter Ó 2010 Elsevier Inc All rights reserved.
doi: 10.1016/j.pep.2010.06.015
* Corresponding author Address: Department of Microbiology, 5 Science Drive 2,
Yong Loo Lin School of Medicine, National University of Singapore, Singapore
117597, Singapore Fax: +65 67766872.
E-mail address: micngml@nus.edu.sg (M.L Ng).
1 Abbreviations used: DENV, dengue virus; WNV, West Nile virus; IPTG, isopropyl
b- D -thiogalactoside; WP, wash profiles.
Contents lists available at ScienceDirect
Protein Expression and Purification
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / y p r e p
Page 285
Trang 2Furthermore, the possibility of channeling (i.e formation of flow
passages due to cracks in the packed-bed) makes quality assurance
difficult As a result, the quality of purification cannot be
main-tained consistently, especially on a larger scale For these reasons,
packed-bed chromatography could not be scaled-up easily and
readily from simple laboratory scale to industrial scale [26]
In contrast, metal affinity membrane chromatography via the
use of membrane adsorbers (MA) is seen as a superior
chromatog-raphy method and an alternative to packed-bed chromatogchromatog-raphy
[26] Recent improvements in membrane materials and
chemis-tries have generated renewed interest in applications of membrane
chromatography for bioprocessing [27] As active binding sites for
target proteins are available readily in convective through-pores in
the membrane, the flow-rate of biomolecules can be enhanced by
an external pump Thus, MA-based protein separation can be
car-ried out more rapidly at higher volumetric rates without
compro-mising its high-yield [27] MAs are versatile and can be used for
numerous applications such as purification of proteins [26,28,29] ,
monoclonal antibodies [30] , oligonucleotides [31] and viruses
[32] MAs are also able to complement high-performance liquid
chromatography (HPLC) for improved purification of proteins of
interest [33] Most importantly, as compared to traditional
packed-bed chromatography columns, MAs have the major
advan-tage of relative ease of scale-up from laboratory based pilot studies
[26,34,35]
For these reasons, metal affinity membrane chromatography
presents a more practical means for consistent and reliable
pro-duction and purification of rDIII proteins Thus, in this study, we
cloned and expressed rDIII proteins from DENV (serotypes 1–4)
and New York (NY), Sarafend (S), Wengler (W) and Kunjin (K)
strains of WNV, and explored the use of MAs [carrying
iminodiace-tic acid (IDA) groups] for the rapid purification of these proteins.
Materials and methods
Cell culture and virus propagation
C6/36 cells, a continuous mosquito cell line derived from Aedes
albopictus embryonic tissue were grown in L-15 medium (Sigma,
USA) supplemented with 10% heat inactivated fetal calf serum at
28 °C DENV serotypes 1–4 (Singapore isolates kindly provided by
Environmental Health Institute of Singapore) and WNV (S, W and
K) (kindly provided by Professor Edwin Westaway, Australia) were propagated in C6/36 cells at an incubation temperature of 28 °C Construction of expression plasmids
With the exception of WNV(NY), DENV and WNV RNA were tracted from virus-infected C6/36 cells using QIAamp Viral RNA Mini Kit (Qiagen, Germany) Reverse transcription was subse- quently performed using SuperScript III First-Strand Synthesis Sys- tem for RT-PCR (Invitrogen, USA) The cDNA encoding DIII region for each virus was then amplified by PCR DNA sequence encoding for DIII of WNV(NY) was amplified by PCR from a plasmid compris- ing the NY DIII DNA sequence [kindly provided by Prof Diamond (Washington University, USA)] Primers used for PCR amplification are described in Table 1 Subsequently, the PCR amplicons were purified, digested with Nhe I and Xho I restriction enzymes, and li- gated into the multiple cloning site of pET28a vector, which was downstream to the gene encoding a hexahistidine tag and a throm- bin cleavage site (Novagen, Germany) ( Fig 1 A) The clones were then sequenced to ensure absence of mutation and that the inserts were in frame for protein expression The resulting constructs were named as pET28aDENV1rDIII, pET28aDENV2rDIII, pET28aDENV3r- DIII, pET28aDENV4rDIII, pET28aWNV(NY)rDIII, pET28aWNV(S)r- DIII, pET28aWNV(W)rDIII and pET28aWNV(K)rDIII Subsequently, Escherichia coli (E coli) strain BL21(DE3) (Novagen, Germany) was transformed with the pET28a-rDIII constructs.
ex-Pilot expression of rDIII protein Recombinant E coli transformed with the pet28a-rDIII con- structs were plated on Luria–Bertani (LB) agar comprising 30lg/
ml Kanamycin and grown overnight at 30 °C Bacterial colonies were then selected and induced for rDIII protein expression at
30 °C The level of rDIII protein expression was examined over a time period of 6 h in order to determine the time required for opti- mal expression.
Protein expression Selected E coli clones were grown in 2 L of LB broth at 30 °C with swirling at 200 rpm from an absorbance OD600 of 0.1 to exponential growth phase at an OD 600 of 0.6 Protein expression
Table 1
DIII forward and reverse primers used for PCR amplification of DIII DNA sequences.
DIII forward and reverse primers Primer sequences Amino acid coordinates
DENV1_forward 50 CTA GCT AGC TTA AAA GGG ATG TCA TAT 3 0 G 296 –K 394
DENV1_reverse 50 CCG CTC GAG TTA GCT TCC CTT CTT GAA CCA 3 0
DENV2_forward 50 CTA GCT AGC CTC AAA GGA ATG TCA TAC 3 0 G 296 –K 394
DENV2_reverse 50 CCG CTC GAG TTA ACT TCC TTT CTT AAA CCA 3 0
DENV3_forward 50 CTA GCT AGC CTC AAG GGG ATG AGC TAT 3 0 G 294 –K 392
DENV3_reverse 50 CCG CTC GAG TTA GCT CCC CTT CTT GTA CCA 3 0
DENV4_forward 50 CTA GCT AGC ATC AAG GGA ATG TCA TAC 3 0 G 296 –K 394
DENV4_reverse 50 CCG CTC GAG TTA ACT CCC TTT CCT GAA CCA 3 0
WNV(NY)_forward 50 CTA GCT AGC TTG AAG GGA ACA ACC TAT 3 0 G 299 –S 400
WNV(NY)_reverse 50 CCG CTC GAG TTA GCT TCC AGA CTT GTG CCA 3 0
WNV(S)_forward 50 CTA GCT AGC CTG AAG GGA ACA ACA TAT 3 0 G 295 –S 396
WNV(S)_reverse 50 CCG CTC GAG TTA GCT CCC AGA TTT GTG CCA 3 0
WNV(W)_forward 50 CTA GCT AGC CTG AAG GGA ACA ACA TAT 3 0 G 299 –S 400
WNV(W)_reverse 50 CCG CTC GAG TTA GCT CCC AGA TTT GTG CCA 3 0
WNV(K)_forward 50 CTA GCT AGC CTG AAG GGG ACA ACT TAT 3 0 G 299 –S 400
WNV(K)_reverse 50 CCG CTC GAG TTA ACT TCC AGA CTT GTG CCA 3 0
Trang 3was induced using 1 mM isopropyl b- D -thiogalactoside (IPTG) At
4 h post-induction, the bacterial cells were pelleted via
centrifuga-tion at 5000g for 45 min at 4 °C, and then resuspended in 8 M urea
containing EDTA-free protease inhibitor (Roche, Switzerland) The
bacterial cells were subsequently sonicated, clarified by
centrifuga-tion (5000g for 45 min at 4 °C) and proteins in cell lysates refolded
by dialysis Briefly, samples were first transferred into a 3500
molecular weight cut-off SnakeSkinT Dialysis Tubing (Pierce,
USA) and dialyzed against 1 L of 6 M urea (in a 3.5 L beaker) for
6 h Subsequently, 250 ml of 25 mM Tris solution (pH 7.5) was
added to the dialysis solution every 6–12 h When the volume
reached 3 L, the dialysis solution was replaced entirely with 2 L
solution of 25 mM Tris–HCl and 150 mM NaCl (pH 7.5) and
dia-lyzed for another 6 h The protein was subsequently recovered
from the dialysis tubing.
Metal affinity membrane chromatography
The refolded protein samples were pre-filtered through
0.45lm and 0.2lm filters (Sartorius, Germany) and subsequently
purified using Sartobind IDA-75 Metal Chelate Adsorber (Sartorius,
Germany) unit Each unit comprised an adsorption area of 15
lay-ers, which was equivalent to a total of 75 cm 2 of IDA metal chelate
membrane In this set-up, two IDA-75 units were connected in
ser-ies with a 0.2lm pre-filter unit, coupled with a positive
displace-ment peristaltic pump (Cole-Parmer, USA) ( Fig 1 B) Protein
purification was performed according to the manufacturer’s
instructions with modifications as described below.
The IDA-75 MAs were first equilibrated with 5 ml of
Equilibra-tion Buffer (0.1 M CH 3 COONa, 0.5 M NaCl; pH 4.5) Nickel ions
were subsequently loaded onto the MAs by passing 5 ml of
Equil-ibration Buffer comprising 0.2 M NiSO 4 through the MAs The MAs
were re-equilibrated with 5 ml of Equilibration Buffer and 5 ml of
Loading Buffer (50 mM Na 2 HPO 4 , 0.5 M NaCl; pH 8.0) sequentially
followed by loading of the protein sample The MAs were then
washed with Wash Buffer (50 mM Tris, 0.5 M NaCl; pH 8.0)
com-prising appropriate concentrations of imidazole (generally from
50 mM to 150 mM) Samples from both the flow through and wash
were collected for analyses Subsequently, the bound protein of
interest was eluted using Elution Buffer (Wash Buffer comprising
500 mM imidazole) Twelve 1.5 ml fractions of protein eluates
were collected from the elution step For regeneration of the
MAs, 10 ml of 1 M H 2 SO 4 was passed through the MAs to unchelate
the nickel ions This was immediately followed by re-equilibration
using 10 ml of Equilibration Buffer The purification procedure was then repeated for subsequent batches of purification For storage, the MAs were filled with Equilibration Buffer comprising 0.02% NaN 2 At a flow-rate of 10 ml/min, protein purification per batch may be achieved within 30 min After SDS–PAGE analysis of the purified rDIII proteins, the proteins were dialyzed against 3 L of PBS overnight at 4 °C The dialyzed purified rDIII proteins were subsequently concentrated using centrifugal concentrators (Viva- spin, Germany) with a 5 kDa cutoff Protein concentration was determined by Bradford Assay (Bio-Rad, USA) MALDI–TOF mass spectrometry analysis of the purified proteins was performed by the Proteins and Proteomics Centre of National University of Singapore.
Product analysis Protein purity was verified by SDS–PAGE, performed using 12% Tris–Tricine polyacrylamide denaturing gel The gel was subse- quently stained with Coomassie blue for analysis Western Blot was performed by transferring the proteins onto a PVDF mem- brane (Bio-rad, USA) by electrophoresis The membrane was blocked for 1 h with Tris–buffered saline/Tween 20 (TBST) contain- ing 5% skimmed milk, at room temperature (RT) Subsequently, the membrane was incubated with 0.1lg/ml anti-His antibody (Qia- gen, Germany) at 4 °C overnight The membrane was then washed with TBST and subsequently incubated at RT with 1lg/ml anti- mouse IgG–alkaline phosphatase conjugate (Millipore, USA) The membrane was washed and subsequently developed using BCIP/ NBT substrate (Millipore, USA) Packed-bed chromatography using Ni–NTA Agarose (Qiagen, Germany) was performed and optimized according to manufacturer’s instructions To allow consistent and fair comparison between both (MA-based and packed-bed) purifi- cation procedures, the cell lysate to (His-tagged protein) binding capacity ratio was maintained equal between both methods Thrombin cleavage of the rDIII protein for removal of the N-termi- nal hexahistidine tag was performed using the Thrombin CleanC- leave Kit (Sigma, USA) Cleavage efficiency was expected to be between 60% and 85%, with complete removal of the thrombin en- zyme, when performed according to manufacturer’s instructions Enzyme linked immunosorbent assay (ELISA)
Indirect ELISA was performed on 96-well Multisorp plates (Nunc, USA) The rDIII protein (2.5lg/ml) was added to each well
Fig 1 (A) Plasmid map of pET28a comprising flavivirus DIII insert (B) Purification set-up for MA-based chromatography of rDIII proteins The crude protein sample {1} is transferred via the positive displacement peristaltic pump {2} to the IDA-75 MAs The protein undergoes pre-microfiltration via 0.2 l m filter {3} prior to His-tag purification using the IDA-75 MAs {4} Flow through, wash and eluates are collected {5} for downstream analysis.
Trang 4at 50ll each and incubated overnight Washing of the plates was
performed thrice in PBS/Tween 20 (PBST) using the Columbus Plus
plate washer (Tecan, Switzerland) and all incubations were
per-formed at 37 °C Blocking was perper-formed using PBST containing
5% skimmed milk for half an hour Subsequently, the plates were
washed and incubated with 1:1000 dilution of respective
antibod-ies such as DENV (serotypes 1, 2, 3 or 4) or WNV(K)
anti-sera for 1 h (These anti-anti-sera were raised in mice against respective
viruses.) The plates were then washed and incubated with 2lg/ml
of anti-mouse IgG–Biotin conjugate (eBioscience, USA) for 1 h The plates were washed again and incubated with 1:5000 dilution of Streptavidin–HRP conjugate (Chemicon, USA) for 1 h After final washing, the plates were incubated for 30 min with TMB One Sub- strate (Promega, USA), followed by 0.5 M H 2 SO 4 (stop solution) All reagents were added at a volume of 100ll The resultant yellow colour was measured immediately at an absorbance of 450 nm.
B A
Trang 5Immunization procedure and plaque reduction neutralization test
(PRNT)
Specific pathogen-free BALB/C mice were obtained from
Labora-tory Animal Centre (National University of Singapore)
Immuniza-tion was performed according to an approved InstituImmuniza-tional Animal
Care and Use Committee protocol Four groups of 6-weeks-old
female BALB/C mice (six mice per group) were immunized
intra-peritoneally three times, at 2 weeks intervals The first two groups received 10lg DENV1 or WNV(K) rDIII protein mixed with an equal volume of complete Freund’s adjuvant (CFA) (Sigma, USA) per mice The third group received CFA mixed with equal volume
of PBS The fourth group received PBS only Subsequent boosts were performed using incomplete Freund’s adjuvant in place of CFA Mice were sacrificed 1 wk after final immunization Sera ob- tained were tested by PRNT as previously described [14]
D C
F E
H G
Fig 3 SDS–PAGE analyses of the purification profiles for MA-based purification of rDIII proteins of DENV serotypes 1–4 (A–D, respectively) and WNV (NY, S, W and K) (E–H, respectively) Proteins were stained with Coomassie Blue [M]: Protein molecular weight marker (Fermentas); [FT]: Flow through; [125(A), 125(B), 125(C) and 150] or [100(A), 100(B), 100(C) and 125] or [150(A), 150(B), 150(C) and 200]: Imidazole washes; [E1–E11]: Protein eluates DENV and WNV rDIII proteins (expected molecular mass of
Trang 6Results and discussion
To date, the expression of rDIII proteins has been performed in
various hosts, such as bacteria [14,23,25,36] , yeast [22] and in the
leaves of tobacco plants [37] By up-scaling the protein expression
process using a bioreactor and enhancement of the culture media
from LB broth to Terrific broth, the yield of bacteria-expressed rDIII
protein improved tremendously [25] Generally, purification of
rDIII proteins from crude bacterial lysate was performed using
packed-bed (or resin-based) chromatography [14,23,25,36,37] To
the best of our knowledge, this is the first optimized MA-based
purification procedure that has been described for rapid
purifica-tion of DENV and WNV rDIII proteins.
In this study, we cloned the DIII gene into pET28a expression
vector and chemically induced the expression of rDIII proteins in
recombinant E coli with IPTG SDS–PAGE analyses of the protein
profiles demonstrated that rDIII protein expression for all four DENV serotypes and the four WNV strains was optimal at approx- imately 4–5 h post-IPTG induction ( Fig 2 ) DENV1 rDIII protein expression ( Fig 2 A) was observed to be consistently lower than the expression of other rDIII proteins As DENV1 rDIII protein com- prises a higher percentage of rare codons compared to that from other serotypes, we investigated if DENV1 rDIII protein expression may be improved via the use of recombinant E coli strain BL21(DE3) Rosetta, for codon optimization Unexpectedly, the pro- tein yield did not improve Furthermore, neither increased IPTG induction time nor IPTG concentration significantly improved DENV1 rDIII protein yield (data not shown) Our investigation sug- gested that lower DENV1 rDIII protein yield was not significantly influenced by codon usage or by the duration and concentration
of IPTG induction Overall, there was no observable difference in the expression of DENV and WNV rDIII proteins when the IPTG
9
E11
E1 1
1
D D D D A
W W W W A
DEN DEN DEN DEN Avera
WNV WNV WNV WNV Aver
V1 r V2 r V3 r V4 r age
V(NY V(S) V(W) V(K) rage
rDIII rDIII rDIII rDIII
Y) rD rDII ) rDI rDII
DIII II III II
Fig 4 (A and B) Quantitative densitometry analyses of protein eluates (E1, E3, E5, E7, E9 and E11) of DENV (serotypes 1–4) and WNV (NY, S, W and K) rDIII proteins (A) DENV rDIII elution profile (B) WNV rDIII elution profile Average protein concentrations for DENV (serotypes 1–4) or WNV (NY, S, W and K) rDIII proteins at various elution steps are represented by the solid line (C) SDS–PAGE analysis of the purification profile for DENV3 rDIII protein purification via packed-bed metal affinity chromatography Proteins were stained with Coomassie Blue [M]: Protein molecular weight marker (Fermentas); [FT]: Flow through; [20(A), 20(B)]: Imidazole washes; [E1–E11]: Protein
Trang 70 0 20 0 40 0 60 0 0 0 00
1:2
DEN WN Freu
1:4
NV1 NV(K und's
4
rDII K) rD
B
E
8
otein rotei t
1:32
d's ad nd's a
2
djuva adjuv
1:64
ant vant
C
0.0 0 0.1 0 0.2 0
:512
0 5 1 5 2 5 3 5
A
0 05 1 15 2 25 3
A
1:1
DEN E
-DEN
NV(NY ELIS
WNV
4
rDIII A
NV A
Y) rD SA
body
WNV E
ibody
H
V2 rD LISA
V(S) r ELISA
y
DIII
Neg
rDIII A
e Ser
NV(W ELI
ve Se
3 rDII SA
rum
W) rD ISA
erum
~ ~
Blank
WNV E
Blan
Da
Da
NV4 r ELISA
k
V(K) ELISA
nk
rDIII A
rDIII A
I
Fig 5 Characterization of DENV and WNV rDIII proteins (A and D) SDS–PAGE analyses of purified DENV rDIII and WNV rDIII proteins, respectively Proteins were stained with Coomassie Blue [M]: Protein molecular weight marker (Fermentas); Lanes 1–4: rDIII proteins for DENV (serotypes 1–4) or WNV (NY, S, W and K) Each lane was loaded with 10 l l aliquot of 100 l g/ml rDIII protein (B and E) His-Tag Western Blot analyses of purified DENV and WNV rDIII proteins, respectively [M]: Protein molecular weight marker (Fermentas); Lanes 1–4: rDIII proteins for DENV (serotypes 1–4) or WNV (NY, S, W and K); [N]: Bacterial lysate negative control (C and F) Indirect ELISA detection of DENV and WNV rDIII proteins, respectively Wells were coated with individual rDIII proteins and each assay was carried out in triplicates The proteins were detected using respective anti-DENV or anti-WNV(K) anti-sera and validated against negative controls and blanks Asterisks (*) indicate that the results were statistically significant according to Student’s T-test (p-values < 0.05) (G) Plaque neutralization assay of DENV1 and WNV(K) with murine polyclonal antibodies raised against the DENV1 and WNV(K) rDIII proteins, respectively Standard deviation is represented by the error bar (H) Thrombin cleavage of DENV3 rDIII protein carried out at 2 h and overnight (O/N) durations Proteins were stained with Coomassie Blue for SDS–PAGE analysis [M]: Protein molecular weight marker (Fermentas) The reaction yielded two products (uncleaved and cleaved DENV3 rDIII proteins, approximately 15 and 13 kDa, respectively).
Trang 8concentration was varied between 0.5 mM and 3 mM (data not
shown) Furthermore, we observed that protein expression by the
pET28a vector was tightly regulated with little or no protein
expression prior to IPTG induction ( Fig 2 ).
After bacterial lysis by sonication and subsequent protein
refolding, microfiltration using 0.45lm and 0.2lm filters was
performed to remove cell debris Subsequently, a series of
optimi-zation experiments for protein purification using IDA-75 MA was
performed Bacterial cell lysate from the DENV3 rDIII protein
expression was used for this optimization process Using a
step-wise increase in the concentration of imidazole (50–75–100–
125–150 mM) in the Wash Buffer (4.5 ml per wash), the protein
profiles analysed according to SDS–PAGE showed that acceptable
protein purity was obtained by the 5th wash (150 mM imidazole
wash) (data not shown) The proteins were eluted using 500 mM
imidazole wash Higher concentrations of imidazole wash did not
have any significant improvement on protein elution (data not
shown) Other wash profiles (WP) evaluated included: WP 1:
50–50–50–50–150; WP 2: 125–150; WP 3: 50–125–150; WP 4:
100–100–100–125; and WP 5: 125–125–125–150 mM imidazole
washes We observed that at least 18 ml of Wash Buffer was
re-quired for effective removal of non-specific binding of
contami-nating proteins By a visual inspection of the protein profiles,
protein purification using WP 5 gave the best yield and protein
purity for the DENV3 rDIII protein ( Fig 3 C) Similar results were
obtained for the rDIII proteins from other DENV serotypes
( Fig 3 A, B and D).
Likewise, optimization of WNV rDIII purification was performed
and WP 4 was found to be consistently good for the purification of
WNV(NY, W and K) rDIII proteins ( Fig 3 E, G and H) Factors such as
protein yield and purity were taken into consideration during
anal-yses The purification of WNV(S) rDIII proteins required an entirely
different set of wash profile, WP 6: 150–150–150–200 mM
imidaz-ole washes Elution of the protein was performed using Elution
Buffer comprising 750 mM imidazole ( Fig 3 F) As the WNV(S) rDIII
protein demonstrated strong retention properties in the MAs,
high-er concentrations of imidazole whigh-ere required for both its wash and
elution buffers.
In addition, quantitative densitometry analyses of the elution
profiles for DENV and WNV rDIII proteins demonstrated that
majority of the proteins were eluted within the 1st five elutions
(E1–E5) ( Fig 4 ), with the exception of WNV(NY) rDIII protein.
Using individually optimized purification protocols, the WNV(NY)
rDIII protein generally retained better in the MA, with majority
of the protein eluted only by E11 ( Fig 3E and 4B ).
Generally, per 1 L batch of E coli culture, approximately
1.5–2 mg of purified recombinant protein was obtained, with the
exception of DENV1 rDIII protein, which gave a yield of
approxi-mately 1–1.2 mg As the total protein mass of cell lysate prior to
purification was quantified to be approximately 42.4 mg per 1 L
culture, therefore, the proportion of rDIII protein in the lysate
was estimated to be approximately 5% This implied that MAs were
able to effectively remove 695% contaminating proteins that were
present in the lysate.
In order to draw a comparison between MA-based
chromatog-raphy and packed-bed chromatogchromatog-raphy, we further performed
purification of DENV3 rDIII protein (representative protein) using
packed-bed chromatography ( Fig 4 C) The time taken for
purifica-tion via the latter method was thrice longer than MA-based
chro-matography as the speed of buffer passing through the resin
depended solely on gravity as opposed to positive displacement
pressure using a pump (used in the MA-based purification
meth-od) Purification quality waned slightly with significant levels of
rDIII proteins being eluted early during the first wash (comparison
between Fig 4C and 3 C) This implied that retention of His-tagged
rDIII protein was weaker in the resin than in the MAs.
Thus, as MAs are superior to traditional chromatography in time-yield performance, flow-independent capacity and short cy- cle time [27,38] , our results supported the role of MA as a primary purification device for the rapid purification of flavivirus rDIII pro- teins We propose that HPLC purification by size exclusion could be used subsequently to complement this MA purification method to achieve higher protein purity.
Characterization of purified recombinant proteins was quently performed SDS–PAGE analyses demonstrated that the purified DENV and WNV rDIII proteins have a molecular mass of approximately 15 kDa ( Fig 5 A and D) In addition, the DENV and WNV rDIII proteins were detected by anti-His antibody via Western Blot ( Fig 5 B and E) Further confirmation of antigenic authenticity
subse-of purified DENV rDIII proteins was performed by detection using respective DENV-specific anti-sera via DENV rDIII indirect ELISA ( Fig 5 C) All WNV rDIII proteins were detected using WNV(K)-spe- cific anti-serum via WNV rDIII indirect ELISA ( Fig 5 F) In addition, the identities of the DENV and WNV rDIII proteins were also con- firmed by MALDI–TOF mass spectrometry analyses (Table S1) The functionality of these proteins was not compromised by our protocol As shown in Fig 5 G, anti-sera obtained from mice immu- nized with DENV1 rDIII protein neutralized 50% (i.e PRNT 50 ) of DENV1 at a serum dilution of 1:64 WNV(K) rDIII-specific anti-sera neutralized 50% of WNV(K) at a dilution between 1:16 and 1:32 Neutralizing antibodies were similarly generated and tested with the other DENV and WNV rDIII proteins (data not shown) Further work is in progress to elucidate the homologous and heterologous (i.e same serotype and across different serotypes) neutralizing lev- els of anti-sera obtained from rDIII-immunized mice To improve the suitability of these proteins as potential vaccine candidates,
we further demonstrated that the N-terminal hexahistidine tag could be removed via thrombin cleavage ( Fig 5 H) The proteins
of interest can subsequently undergo HPLC grade purification cedures to ensure complete absence of uncleaved rDIII proteins, residual cleaved His-tags and possible endotoxin contamination.
pro-In addition, to evaluate if this purification strategy was ble to other flavivirus proteins, the WNV(S) recombinant capsid protein was expressed (Fig S1A and B) and effectively purified via membrane chromatography using a strategy of WP 7: 750–750 mM imidazole washes and eluted using 1 M imidazole dissolved in Wash Buffer (Fig S1C) Our data indicated that MA was suitable as a platform for the purification of WNV(S) recombi- nant capsid protein The identity of WNV(S) recombinant capsid protein was further confirmed via Western Blot with anti-His antibody (Fig S1D) and MALDI–TOF mass spectrometry (Fig S1E) Collectively, our results demonstrated that membrane chromatog- raphy purification system is a versatile platform, which can be used
applica-to effectively purify a wide-spectrum of His-tagged flavivirus recombinant proteins.
Conclusion The production and purification of flavivirus recombinant pro- teins have become increasingly popular and important for re- search This is especially relevant for DENV and WNV rDIII proteins because their good immunogenic properties make them suitable as protein subunit vaccine candidates [39] and reagents for DENV and WNV diagnosis [40,41] Our attempt to use the industrial scaled-down model, IDA-75 MA, for routine purification
of medically important biologics such as DENV and WNV rDIII teins yielded promising results.
pro-Acknowledgments This work was funded by grants from the Biomedical Research Council of Singapore (R-182-000-109-305) and Exploit Technology
Trang 9(A*Star COT fund) (R-182-000-141-592) We are grateful for the
technical assistance provided by the Proteins and Proteomics
Cen-tre of NUS for Mass Spectrometry analyses, and Samuel Chiang for
constructive discussions.
Appendix A Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.pep.2010.06.015
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Trang 10In: Dengue Virus: Detection, Diagnosis and Control ISBN 978-1-60876-398-6
Editor: Basak Ganim and Adam Reis © 2008 Nova Science Publishers, Inc
Chapter 3
Dengue Envelope Domain III Protein:
Properties, Production and Potential
Applications in Dengue Diagnosis
Dept of Microbiology, National University of Singapore, Singapore
Abstract
Dengue virus (DENV) is a positive-sense, single-stranded RNA virus belonging to
the Flaviviridae family It causes dengue fever in humans and in some cases, progresses
to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which result in
mortality The DENV comprises four antigenically distinct serotypes (1 to 4) The
envelope (E) protein of the virus comprises three Domains - I, II and III The Domain III
(DIII) protein has been demonstrated to be involved in host recognition More
importantly, the DIII protein has been shown to be highly immunogenic, and is able to
elicit the generation of neutralizing antibodies against the wild-type virus itself For this
reason, the DIII protein is believed to be a potential candidate as a protein subunit vaccine
and as a diagnostic reagent for dengue serology In this review, we discuss the distinct
biological properties of the DIII protein, issues relating to its production and the
prospects for a DIII protein- based diagnostic assay
* Corresponding author: Mah Lee Ng
Flavivirology Laboratory, Department of Microbiology,
5 Science Drive 2, National University of Singapore, Singapore 117597
E-mail address: micngml@nus.edu.sg
Page 295
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2
Introduction
Dengue virus (DENV), a member of the genus flavivirus within the family Flaviviridae,
causes a wide range of diseases from mild dengue fever (DF) to severe dengue hemorrhagic
fever and dengue shock syndrome (DHF/DSS) (Alvarez et al., 2006; Gubler, 1998; Halstead,
1988; Malavige, 2004) It has been estimated that more than 2.5 million people in over 100 countries are at risk of dengue infection, with several hundred thousand cases of life threatening DHF/DSS occurring every year (Gubler, 2002) Other members of the
Flaviviridae family include the Japanese encephalitis virus (JEV), tick-borne encephalitis
virus (TBEV) and the West Nile virus (WNV) etc
DENV comprises four antigenically distinct serotypes (1 to 4) Its viral genome encodes for three structural proteins: the capsid protein, the premembrane protein and the envelope glycoprotein; and 7 non-structural proteins: the NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (Clyde, 2006; Mackenzie 2004) The envelope protein comprises 3 regions: Domain I, Domain II and Domain III The Domain I is the central domain, the Domain II is the dimerization and fusion domain, while the Domain III (DIII) is an immunoglobulin-like
receptor binding domain (Mukhopadhyay, 2005; Rey et al., 1995)
Experimental evidences have shown that the DIII protein is a receptor binding domain
(Bhardwaj et al., 2001; Chin et al., 2007; Chu et al., 2005; Zhang et al., 2007) In addition, it
has also been demonstrated to be highly immunogenic and able to elicit the production of
neutralizing antibodies against the wild-type virus (Gromowski et al., 2008) For this reason,
the DIII protein is an important immunogen for the development of a prospective protein subunit vaccine and also a prospective diagnostic reagent for the improved clinical diagnosis
of dengue infections Although to date, there is no DIII protein-based diagnostic assay available commercially, many in-house tests have demonstrated the possibility of using the DIII protein as a reagent to serologically detect and differentiate between serotypes of dengue infection
In this review, we discuss the recent advances in the understanding of the dengue DIII protein We also examine the issues pertaining to the expression and purification of recombinant DIII (rDIII) fusion proteins and discuss the prospects for its incorporation as a reagent for dengue diagnosis
Properties of the Dengue DIII Protein
Structural Studies on the Flavivirus Envelope and DIII Proteins
A huge step forward in the field of flavivirus research was made when the structure of the
flavivirus major envelope protein was first determined in TBEV (Rey et al., 1995) The
Domain I of the TBEV envelope protein consists of three segments, which span across residues 1-51, 137-189 and 285-302 (according to the TBEV envelope amino acid sequence) The Domain II comprises 2 segments, ranging between residues 52-136 and 190-284 The DIII consists only 1 segment that span across residues 303-395, and is located C-terminal to Domains I and II Furthermore, the DIII consists mainly of β-barrels that project
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perpendicularly to the viral surface For this reason, the accessibility of the DIII protein on the flaviviral envelope supports its role as a receptor binding protein and as an immunogenic
protein (Rey et al., 1995)
The study on the envelope glycoprotein of other flaviviruses ensued Advancement in this
field included the determination of the structure of DENV2 envelope protein (Kuhn et al., 2002; Modis et al., 2003; Zhang et al., 2003), WNV envelope protein (Kanai et al., 2006; Mukhopadhyay et al., 2003), DENV3 envelope protein (Modis et al., 2005); DENV4 DIII protein (Volk et al., 2007); immature WNV (Zhang et al., 2007) and DENV (Yu et al., 2008), and the precursor membrane-envelope protein complex (Li et al., 2008) Several of these
studies were key to the prediction of the viral envelope membrane fusion mechanism
It is believed that DENV enter cells via receptor-mediated endocytosis The association
of the envelope protein with the cell-surface receptor, possibly through the DIII protein,
allows the endosomal uptake of the virus particle into the cell (Modis et al., 2004) In a study
by Modis and colleagues in 2003, the structure of the DENV2 envelope glycoprotein was analyzed in the presence or absence of a detergent n-octyl-β-D-glucoside during crystallization This led to the finding of a hydrophobic pocket lined by residues that influence pH threshold for viral fusion This pocket lay in the “hinge” region of the envelope
protein (Rey et al., 1995), thus supporting the notion that a fusogenic conformational change
may be triggered by the acidic environment of endosomes after flaviviruses enter cells by
receptor-mediated endocytosis (Modis et al., 2003; Rey, 1995; Zhang et al., 2004)
Furthermore, the binding of the detergent denoted the pocket as a potential site for
small-molecule fusion inhibitors (Modis et al., 2004)
By obtaining the structural information of the viral surface envelope glycoproteins at the atomic level, these findings can facilitate the better understanding of the molecular interactions that occur between the viral surface proteins and their receptors The elucidation
of the three dimensional structure of the envelope protein can also enhance the future development of anti-viral drugs that potentially bind to specific target sites on the envelope protein (i.e DIII region or the ligand binding pocket) and cause the virus to lose its infectivity
(Modis et al., 2004; Rey, 2003)
Bioinformatics Analysis of the DIII Protein
Multiple sequence analyses of the amino acid sequences performed on the envelope protein of flaviviruses led to the direct mapping of the DIII region (according to the TBEV
structure) onto other members of the Flaviviridae family (Rey et al., 1995) We have also
previously performed amino acid sequence alignment of the DIII protein across the
Flaviviridae family (Chu et al., 2005) In these analyses, we reported that there are significant
differences in the homology of the DIII amino acid sequences across flaviviruses
In this review, the amino acid sequences of the DIII protein of all four DENV serotypes are aligned and evaluated according to their percentage amino acid identities and similarities (Figure 1A) Based on these analyses, we observed that the DENV1 and DENV3 DIII proteins share the closest homology (according to their amino acid sequence identities and similarities), while the DENV3 and DENV4 DIII proteins show the greatest differences in
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4
amino acid sequences (Figure 1B) The amino acid identity for the DIII proteins ranges from 47.5% to 69.7% across the serotypes In addition, the phylogram analysis further lends credence to the observation that the DENV1 and DENV3 DIII proteins are most closely related according to their evolutionary relationship (Figure 1C) Taken together, the large differences in the amino acid sequences of the DIII proteins across the serotypes may dramatically affect the conformation of the DIII protein structure As a result, these differences are therefore expected to contribute significantly to the differential sero-cross-reactivity of the antibodies across the dengue serotypes, due to exposure of the adaptive immune system to varying DIII antigenic epitopes
Figure 1 A Multiple sequence alignment of the amino acid sequences of the DENV DIII protein serotypes 1
to 4 The DENV strain and accession numbers used in this analysis are the DENV1 (NC_001477), DENV2 (ABO28784), DENV3 (DQ675533) and DENV4 (NP_073286), which can be obtained from the NCBI database Conserved regions are indicated with a “*” and highlighted in yellow In this evaluation, significant differences in the amino acid sequences are observed across the serotypes B Comparison between the amino acid sequences demonstrated that percentage identity and similarity across the DENV serotypes range between 47.5% to 69.7% and 69.7% to 87.9% respectively The DIII of DENV1 and DENV3 showed the closest homology while the homology between the DIII of DENV 3 and DENV 4 showed the greatest differences in amino acid identity and similarity C A phylogram analysis based on the DIII region shows the evolutionary relationship among the DENV serotypes According to this prediction based on the DIII amino acid sequences, DENV serotypes 1 and 3 are suggested to be closest in their evolutionary relationship as compared to the other serotypes
In order to predict specific amino acids that may contribute to the differential antigenicity
on the DIII proteins, we performed another analysis based on the DIII antigenicity and hydrophilicity plots (Figure 2A - D) These analyses suggested that the DIII protein is largely
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a hydrophilic protein with no membrane spanning region The antigenicity of the DIII protein
is closely correlated to its hydrophilic sites In addition, we notice significant variations in the antigenic index at various amino acid residues (30 to 37) and (45 to 50) on the DIII protein across the four DENV serotypes (Figure 2E - F) The significance of these variations is yet to
be determined
A
B