Protein Expression and Purification 74 (2010) 129–137 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep Rapid 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, 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 July 2010 Keywords: Flavivirus Dengue virus West Nile virus Envelope Domain III Membrane chromatography Protein purification a b s t r a c t Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1–4 and WNV – New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI– TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus. 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 hemorrhagic fever and dengue shock syndrome (DHF/DSS) [1–5]. It has been estimated that more than 2.5 billion people in over 100 countries 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 illness 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 infections, including 1161 fatalities, in the USA [8]. The flavivirus genome encodes three structural proteins: the capsid protein, premembrane protein and envelope protein; and * Corresponding author. Address: Department of Microbiology, 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). Abbreviations used: DENV, dengue virus; WNV, West Nile virus; IPTG, isopropyl b-D-thiogalactoside; WP, wash profiles. seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 [9,10]. The envelope protein comprises three regions: 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 evidence 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 purification 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 common 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 order for binding to occur. This increases process time and consequently 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 Page 285 130 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 Furthermore, 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 maintained 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 chromatography method and an alternative to packed-bed chromatography [26]. Recent improvements in membrane materials and chemistries 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 carried out more rapidly at higher volumetric rates without compromising 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 advantage 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 production 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 iminodiacetic 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 extracted from virus-infected C6/36 cells using QIAamp Viral RNA Mini Kit (Qiagen, Germany). Reverse transcription was subsequently performed using SuperScript III First-Strand Synthesis System 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 comprising 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 ligated into the multiple cloning site of pET28a vector, which was downstream to the gene encoding a hexahistidine tag and a thrombin cleavage site (Novagen, Germany) (Fig. 1A). 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, pET28aDENV3rDIII, pET28aDENV4rDIII, pET28aWNV(NY)rDIII, pET28aWNV(S)rDIII, pET28aWNV(W)rDIII and pET28aWNV(K)rDIII. Subsequently, Escherichia coli (E. coli) strain BL21(DE3) (Novagen, Germany) was transformed with the pET28a-rDIII constructs. Pilot expression of rDIII protein Recombinant E. coli transformed with the pet28a-rDIII constructs were plated on Luria–Bertani (LB) agar comprising 30 lg/ 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 h in order to determine the time required for optimal expression. Protein expression Selected E. coli clones were grown in 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 OD600 of 0.6. Protein expression Table 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 CTA GCT AGC TTA AAA GGG ATG TCA TAT DENV1_reverse 50 CCG CTC GAG TTA GCT TCC CTT CTT GAA CCA 30 DENV2_forward 50 CTA GCT AGC CTC AAA GGA ATG TCA TAC 30 DENV2_reverse 50 CCG CTC GAG TTA ACT TCC TTT CTT AAA CCA 30 DENV3_forward 50 CTA GCT AGC CTC AAG GGG ATG AGC TAT 30 DENV3_reverse 50 CCG CTC GAG TTA GCT CCC CTT CTT GTA CCA 30 DENV4_forward 50 CTA GCT AGC ATC AAG GGA ATG TCA TAC 30 DENV4_reverse 50 CCG CTC GAG TTA ACT CCC TTT CCT GAA CCA 30 WNV(NY)_forward 50 CTA GCT AGC TTG AAG GGA ACA ACC TAT 30 WNV(NY)_reverse 50 CCG CTC GAG TTA GCT TCC AGA CTT GTG CCA 30 WNV(S)_forward 50 CTA GCT AGC CTG AAG GGA ACA ACA TAT 30 WNV(S)_reverse 50 CCG CTC GAG TTA GCT CCC AGA TTT GTG CCA 30 WNV(W)_forward 50 CTA GCT AGC CTG AAG GGA ACA ACA TAT 30 WNV(W)_reverse 50 CCG CTC GAG TTA GCT CCC AGA TTT GTG CCA 30 WNV(K)_forward 50 CTA GCT AGC CTG AAG GGG ACA ACT TAT 30 WNV(K)_reverse 50 CCG CTC GAG TTA ACT TCC AGA CTT GTG CCA 30 The Nhe I and Xho I restriction enzyme recognition sites are underlined and double underlined, respectively. G296–K394 G296–K394 G294–K392 G296–K394 G299–S400 G295–S396 G299–S400 G299–S400 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 131 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 lm filter {3} prior to His-tag purification using the IDA-75 MAs {4}. Flow through, wash and eluates are collected {5} for downstream analysis. was induced using mM isopropyl b-D-thiogalactoside (IPTG). At h post-induction, the bacterial cells were pelleted via centrifugation at 5000g for 45 at °C, and then resuspended in M urea containing EDTA-free protease inhibitor (Roche, Switzerland). The bacterial cells were subsequently sonicated, clarified by centrifugation (5000g for 45 at °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 L of M urea (in a 3.5 L beaker) for 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 L, the dialysis solution was replaced entirely with L solution of 25 mM Tris–HCl and 150 mM NaCl (pH 7.5) and dialyzed for another h. The protein was subsequently recovered from the dialysis tubing. 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% NaN2. 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 L of PBS overnight at °C. The dialyzed purified rDIII proteins were subsequently concentrated using centrifugal concentrators (Vivaspin, Germany) with a 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. Metal affinity membrane chromatography Protein purity was verified by SDS–PAGE, performed using 12% Tris–Tricine polyacrylamide denaturing gel. The gel was subsequently stained with Coomassie blue for analysis. Western Blot was performed by transferring the proteins onto a PVDF membrane (Bio-rad, USA) by electrophoresis. The membrane was blocked for h with Tris–buffered saline/Tween 20 (TBST) containing 5% skimmed milk, at room temperature (RT). Subsequently, the membrane was incubated with 0.1 lg/ml anti-His antibody (Qiagen, Germany) at °C overnight. The membrane was then washed with TBST and subsequently incubated at RT with lg/ml antimouse 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) purification 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-terminal hexahistidine tag was performed using the Thrombin CleanCleave Kit (Sigma, USA). Cleavage efficiency was expected to be between 60% and 85%, with complete removal of the thrombin enzyme, when performed according to manufacturer’s instructions. The refolded protein samples were pre-filtered through 0.45 lm and 0.2 lm filters (Sartorius, Germany) and subsequently purified using Sartobind IDA-75 Metal Chelate Adsorber (Sartorius, Germany) unit. Each unit comprised an adsorption area of 15 layers, which was equivalent to a total of 75 cm2 of IDA metal chelate membrane. In this set-up, two IDA-75 units were connected in series with a 0.2 lm pre-filter unit, coupled with a positive displacement peristaltic pump (Cole-Parmer, USA) (Fig. 1B). Protein purification was performed according to the manufacturer’s instructions with modifications as described below. The IDA-75 MAs were first equilibrated with ml of Equilibration Buffer (0.1 M CH3COONa, 0.5 M NaCl; pH 4.5). Nickel ions were subsequently loaded onto the MAs by passing ml of Equilibration Buffer comprising 0.2 M NiSO4 through the MAs. The MAs were re-equilibrated with ml of Equilibration Buffer and ml of Loading Buffer (50 mM Na2HPO4, 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) comprising 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 M H2SO4 was passed through the MAs to unchelate the nickel ions. This was immediately followed by re-equilibration Product analysis Enzyme linked immunosorbent assay (ELISA) Indirect ELISA was performed on 96-well Multisorp plates (Nunc, USA). The rDIII protein (2.5 lg/ml) was added to each well 132 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 at 50 ll 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 performed at 37 °C. Blocking was performed using PBST containing 5% skimmed milk for half an hour. Subsequently, the plates were washed and incubated with 1:1000 dilution of respective antibodies such as anti-DENV (serotypes 1, 2, or 4) or anti-WNV(K) antisera for h. (These anti-sera were raised in mice against respective viruses.) The plates were then washed and incubated with lg/ml of anti-mouse IgG–Biotin conjugate (eBioscience, USA) for h. The plates were washed again and incubated with 1:5000 dilution of Streptavidin–HRP conjugate (Chemicon, USA) for h. After final washing, the plates were incubated for 30 with TMB One Substrate (Promega, USA), followed by 0.5 M H2SO4 (stop solution). All reagents were added at a volume of 100 ll. The resultant yellow colour was measured immediately at an absorbance of 450 nm. A B C D E F G H Fig. 2. SDS–PAGE analyses of time course protein expression of flavivirus rDIII proteins. Proteins were stained with Coomassie Blue. (A–D): DENV rDIII proteins from serotypes 1–4, respectively. (E–H): WNV (NY, S, W and K) rDIII proteins, respectively. [M]: Protein molecular weight marker (Fermentas); [T = 0–6]: Hourly profiles of bacterial lysates from prior to IPTG induction to h post-IPTG induction. Increasing levels of rDIII proteins with a molecular mass of 15 kDa were observed (indicated by arrows). L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 Immunization procedure and plaque reduction neutralization test (PRNT) Specific pathogen-free BALB/C mice were obtained from Laboratory Animal Centre (National University of Singapore). Immunization was performed according to an approved Institutional Animal Care and Use Committee protocol. Four groups of 6-weeks-old female BALB/C mice (six mice per group) were immunized intra- 133 peritoneally three times, at weeks intervals. The first two groups received 10 lg 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 wk after final immunization. Sera obtained were tested by PRNT as previously described [14]. A B C D E F G H 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 15 kDa) are indicated by arrows. 134 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 profiles demonstrated that rDIII protein expression for all four DENV serotypes and the four WNV strains was optimal at approximately 4–5 h post-IPTG induction (Fig. 2). DENV1 rDIII protein expression (Fig. 2A) was observed to be consistently lower than the expression of other rDIII proteins. As DENV1 rDIII protein comprises 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 protein yield did not improve. Furthermore, neither increased IPTG induction time nor IPTG concentration significantly improved DENV1 rDIII protein yield (data not shown). Our investigation suggested 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 Results 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 purification 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 Mean Grey Value A DEN D V1 rrDIII DEN D V2 rrDIII DEN D V3 rrDIII DEN D V4 rrDIII A age Avera E1 E3 E5 E7 E99 E111 Mean Grey Value B W WNV V(NY Y) rD DIII W WNV V(S) rDIIII W WNV V(W)) rDIIII W WNV V(K) rDIIII A Averrage E1 E3 E5 E7 E9 E11 C 50 – 40 – 30 – 25 – 20 – 15 – 10 – 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 eluates. DENV3 rDIII protein (expected molecular mass of 15 kDa) is indicated by the arrow. 135 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 A C B 3.5 O.D. 450nm ~ 115 kD Da 2.5 1.5 0.5 DEN NV1 rDIII r E ELISA A DENV D V2 rD DIII EL LISA A -DEN AntiNV A Antibbody D DE ENV33 rDIIII ELISA Neg gativee Serrum DEN NV4 rDIII r E ELISA A Blank B k F E 0.3 O.D. 450nm 25 0.2 Da ~ 15 kD 0.2 15 0.1 0.1 05 0.0 WN NV(NY Y) rD DIII WNV W V(S) rDIII r ELIS SA E ELISA A A WNV Anti-W V(K) Antiibody y G WN NV(W W) rD DIII ELIISA Neegativ ve Seerum m WNV V(K) rDIIII E ELISA A Blannk H 00 10 Percentage Neutralization / % 90 80 70 60 ~ 15 kD Da ~ 13 kD Da 50 40 30 20 10 1:2 1:44 1:88 1:16 1:322 1:644 1:128 1:2566 1::512 1:1 10244 DEN NV1 rDIIII prootein n + Frreundd's addjuvaant WN NV(K K) rD DIII proteiin + Freun F nd's aadjuvvant Freu und'ss adjuuvan t 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 ll aliquot of 100 lg/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 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). 136 L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 concentration was varied between 0.5 mM and 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 0.45 lm and 0.2 lm filters was performed to remove cell debris. Subsequently, a series of optimization 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 stepwise 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 required for effective removal of non-specific binding of contaminating proteins. By a visual inspection of the protein profiles, protein purification using WP gave the best yield and protein purity for the DENV3 rDIII protein (Fig. 3C). Similar results were obtained for the rDIII proteins from other DENV serotypes (Fig. 3A, B and D). Likewise, optimization of WNV rDIII purification was performed and WP was found to be consistently good for the purification of WNV(NY, W and K) rDIII proteins (Fig. 3E, G and H). Factors such as protein yield and purity were taken into consideration during analyses. The purification of WNV(S) rDIII proteins required an entirely different set of wash profile, WP 6: 150–150–150–200 mM imidazole washes. Elution of the protein was performed using Elution Buffer comprising 750 mM imidazole (Fig. 3F). As the WNV(S) rDIII protein demonstrated strong retention properties in the MAs, higher concentrations of imidazole were 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 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 approximately 1–1.2 mg. As the total protein mass of cell lysate prior to purification was quantified to be approximately 42.4 mg per 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 chromatography and packed-bed chromatography, we further performed purification of DENV3 rDIII protein (representative protein) using packed-bed chromatography (Fig. 4C). The time taken for purification via the latter method was thrice longer than MA-based chromatography 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 method). Purification quality waned slightly with significant levels of rDIII proteins being eluted early during the first wash (comparison between Fig. 4C and 3C). 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 cycle time [27,38], our results supported the role of MA as a primary purification device for the rapid purification of flavivirus rDIII proteins. 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 subsequently performed. SDS–PAGE analyses demonstrated that the purified DENV and WNV rDIII proteins have a molecular mass of approximately 15 kDa (Fig. 5A and D). In addition, the DENV and WNV rDIII proteins were detected by anti-His antibody via Western Blot (Fig. 5B and E). Further confirmation of antigenic authenticity of purified DENV rDIII proteins was performed by detection using respective DENV-specific anti-sera via DENV rDIII indirect ELISA (Fig. 5C). All WNV rDIII proteins were detected using WNV(K)-specific anti-serum via WNV rDIII indirect ELISA (Fig. 5F). In addition, the identities of the DENV and WNV rDIII proteins were also confirmed by MALDI–TOF mass spectrometry analyses (Table S1). The functionality of these proteins was not compromised by our protocol. As shown in Fig. 5G, anti-sera obtained from mice immunized with DENV1 rDIII protein neutralized 50% (i.e. PRNT50) 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 levels 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. 5H). The proteins of interest can subsequently undergo HPLC grade purification procedures to ensure complete absence of uncleaved rDIII proteins, residual cleaved His-tags and possible endotoxin contamination. In addition, to evaluate if this purification strategy was applicable 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 M imidazole dissolved in Wash Buffer (Fig. S1C). Our data indicated that MA was suitable as a platform for the purification of WNV(S) recombinant 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 chromatography purification system is a versatile platform, which can be used to effectively purify a wide-spectrum of His-tagged flavivirus recombinant proteins. Conclusion The production and purification of flavivirus recombinant proteins have become increasingly popular and important for research. 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 proteins yielded promising results. Acknowledgments This work was funded by grants from the Biomedical Research Council of Singapore (R-182-000-109-305) and Exploit Technology L.C.M. Tan et al. / Protein Expression and Purification 74 (2010) 129–137 (A*Star COT fund) (R-182-000-141-592). We are grateful for the technical assistance provided by the Proteins and Proteomics Centre 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. References [1] M. Alvarez, R. Rodriguez-Roche, L. Bernardo, S. Vazquez, L. Morier, D. Gonzalez, O. Castro, G. Kouri, S.B. Halstead, M.G. Guzman, Dengue hemorrhagic fever caused by sequential dengue 1–3 virus infections over a long time interval: Havana epidermic, 2001–2002, Am. J. Trop. Hyg. 75 (2006) 1113–1117. [2] D.J. Gubler, Dengue and dengue hemorrhagic fever, Clin. Microbiol. Rev. 11 (1998) 480–496. [3] S.B. 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In: Dengue Virus: Detection, Diagnosis and Control Editor: Basak Ganim and Adam Reis ISBN 978-1-60876-398-6 © 2008 Nova Science Publishers, Inc. Chapter Dengue Envelope Domain III Protein: Properties, Production and Potential Applications in Dengue Diagnosis Lik Chern Melvin Tan and Mah Lee Ng* 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, Science Drive 2, National University of Singapore, Singapore 117597 E-mail address: micngml@nus.edu.sg Page 295 Dengue Envelope Domain III Protein: Properties, Production and Potential Applications 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 C D Lik Chern Melvin Tan and Mah Lee Ng Dengue Envelope Domain III Protein: Properties, Production and Potential Applications E F Figure 2. A-D. Antigenicity (solid bar) and hydrophilicity plots (solid line) of individual DIII proteins demonstrate the close relationship between the putative antigenic sites and the hydrophilic regions of the DIII protein of DENV to 4. These results also support the notion that the DIII protein is highly antigenic and hydrophilic in nature. E and F. A comparison of the overall antigenic indexes and hydrophilicity plots. Amino acid (a.a.) residues 30 to 37 for DENV4DIII showed significant variation in their antigenicity and polarity as compared to the other serotypes. In addition a.a. residues 45 to 50 for DENV2DIII and DENV4DIII also showed significant antigenic and polarity differences as compared to DENV1DIII and DENV3DIII proteins. Lik Chern Melvin Tan and Mah Lee Ng Antagonistic Activity of the DIII Protein The antagonistic activity of flavivirus DIII protein against flavivirus infection has been demonstrated with rDIII proteins derived from TBEV (Bhardwaj et al., 2001), WNV (Chu et al., 2005) and DENV (Chin et al., 2007; Zhang et al., 2007). The bacteria-expressed rDIII protein acts as an antagonist, competitively inhibiting entry of flaviviruses into cells in vitro. This is possibly because the binding of the rDIII proteins to the cellular surface receptors caused a decrease in the number of receptors available for the binding of the wild-type virus during viral infection. This therefore led to a reduction in viral infection of the cells. In addition, competitive inhibition was also similarly demonstrated using a recombinant DENV2 envelope protein (Chen et al., 1996; Chiu & Yang, 2003). These studies provided experimental evidence suggesting that the DIII protein is involved in host cell receptor binding. Neutralizing Epitopes on the DIII Protein Among flaviviruses, the envelope DIII protein contains critical, virus-specific neutralization epitopes. The DENV DIII protein is highly immunogenic and is able to elicit the generation of serotype-specific and DENV complex-specific neutralizing antibodies against the virus (Gromowski et al., 2008). This is consistent with studies performed on the WNV DIII protein where the DIII protein has also been determined to be an important target for neutralizing antibodies (Beasley & Barrett, 2002). For these reasons, the DIII protein is believed to be a potential candidate as a protein subunit vaccine. The antibody-mediated neutralization of flaviviruses is generally believed to occur via a “multi-hit” mechanism of neutralization. This means multiple antibodies are required to engage individual virus particles in order for neutralization to occur. Evidence for a multi-hit mechanism of neutralization has been demonstrated with DIII-specific monoclonal antibodies (Mabs) targeting two different flaviviruses: WNV and DENV2 (Gromowski & Barrett, 2007; Pierson et al., 2007). The inhibition of virus-receptor binding is believed to be due to DIIIspecific neutralizing antibodies binding to the DIII region. In a recent study by Lok and colleagues in 2008, an alternative hypothesis was proposed. The binding of the DIII-specific neutralizing antibodies to the virus caused an alteration of the spatial arrangement between the glycans on the E proteins. These changes in the structure of the viral surface were presumably responsible for inhibiting attachment to the cells (Lok et al., 2008). DENV envelope-specific Mabs are able to block virus entry into cells (Crill & Roehrig, 2001). Mabs that are specific against Domains I (IB4C-2, 4A5C-8, 2B3A-1 and 9A4D-1) and II (6B6C-1, 4G2, 4E5, 1A5D-1, 1B7 and 10A1D-2) have been shown to be able to block virus entry into Vero cells (approx. 18% to 46% blocking), but not as strongly as DIII-specific Mabs (3H5, 9A3D-8, 10A4D-2, 1A1D-2 and 9D12), which cause between 36% to 49% blocking (Crill & Roehrig, 2001). In addition, the fine mapping of neutralizing epitopes on the DIII protein on DENV2 carried out by Gromowski and Barrett in 2007, demonstrated (through the use of DIIIspecific Mabs) that amino acids K305 and P384 on the envelope protein of the DENV2 were Dengue Envelope Domain III Protein: Properties, Production and Potential Applications critical for binding. More importantly, they showed that the level of viral neutralization was associated with the relative occupancy of the Mabs on the DIII protein of the virion (i.e. degree of viral neutralization increases as antibody occupancy on the virus increases). Therefore, viral neutralization is predicted to occur once the threshold for occupancy is reached (Gromowski & Barrett, 2007). In addition, a further study by Gromowski and colleagues in 2008 demonstrated that Mabs that were type-specific may be more potent than DENV complex-specific Mabs. Complex-specific Mabs were observed to require a higher occupancy level on the virion than type-specific Mabs, therefore accounts for the observed lowered effectiveness in viral neutralization. Furthermore, Mabs that are cross-reactive against the DENV serotypes have also been identified. The DIII-specific Mabs, mAB4E11 and 9F12, have been shown to be able to cross-neutralize all DENV serotypes with varying effectiveness (Lisova et al., 2007; Rajamanonmani et al., 2009). DIII Protein as a Potential Protein Subunit Vaccine Currently, there is no commercially available vaccine against DENV infection. Efforts to develop a suitable vaccine against DENV have focused mainly on live attenuated vaccines, followed by other approaches such as protein subunit, vectored and DNA vaccines (Whitehead et al., 2007). As the DIII protein is able to elicit the generation of neutralizing antibodies against the wild-type DENV, it is therefore a potential candidate as a protein subunit vaccine (Table 1). Table 1. Recent studies on DENV rDIII production and rDIII-based vaccine 2008 Protein(s) Tested DENV4rDIII Animal Model Balb/C mice Bernard o et al. 2008 DENV2rDIII Macaca fascicularis monkeys Immunization of monkeys with DENV2rDIII proteins resulted in the generation of neutralizing antibodies against DENV 2. Etemad et al. 2008 Tetravalent DENV(14)rDIII Balb/C mice Tetravalent rDIII protein was expressed in yeast, purified and administered into mice. Neutralizing antibodies against all four DENV serotypes were detected. Authors Year Babu et al. Results DENV4rDIII protein was produced and administered into mice as an antigenadjuvant mix. Different adjuvants such as Freund’s Complete Adjuvant, Montanide ISA 720 or Alum were used. The vaccination resulted in generation of neutralizing antibodies against DENV 4. 10 Lik Chern Melvin Tan and Mah Lee Ng Sim et al. 2008 DENV2rDIII Balb/C or C57BL/6 mice DENV2rDIII protein was produced and administered via the mucosal route into mice. Neutralizing antibodies was generated against DENV 2. Chen et al. 2007 Tetravalent DENV(14)rDIII Balb/C mice Tetravalent rDIII protein was produced as a single protein. Mice administered with the protein was protected against DENV 1,2 and (80% protection). Only 18% of the mice were protected against DENV viral challenge. Chin et al. 2007 DENV1rDIII and DENV2rDIII Balb/C mice DENV1rDIII and DENV2rDIII protein were expressed in a bacterial system. The proteins generated neutralizing antibodies in mice, and can block viral entry into cells in vitro. Saejung et al. 2007 DENV2rDIII C3H mice DENV2rDIII protein was expressed in plants. The protein was subsequently purified and administered into mice. Neutralizing antibodies against DENV was generated in mice. Zhang et al. 2007 DENV2rDIII Balb/C mice DENV2rDIII protein was expressed in high levels using a bacterial system. The protein was refolded, purified and then administered into mice. Neutralizing antibodies against DENV was generated. Hermida et al. 2006 DENV2rDIII Macaca fascicularis monkeys DENV2rDIII protein was expressed and used to immunize monkeys. Protection from viraemia during DENV viral challenge was observed. Saejung et al. 2006 DENV2rDIII Rat DENV2rDIII protein was expressed using bacterial system, refolded and purified. The protein generated in mice anti-DENV2rDIII antibodies. One major challenge to DENV vaccine development is the potential development of antibody-dependent enhancement (ADE) of virus replication, which is believed to cause DHF and DSS (Halstead, 1988). ADE occurs when heterotypic non-neutralizing antibodies present in the host bind to the DENV particle during a subsequent heterotypic infection but cannot neutralize the virus. Instead this complex attaches to the Fcγ receptors (FcγR) on the Dengue Envelope Domain III Protein: Properties, Production and Potential Applications 11 circulating monocytes. This therefore facilitates the infection of FcγR cell types in the body, which are normally not readily infected in the absence of a non-neutralizing antibody. Therefore this leads to an increase in viral infection, leading to the potential development of a more severe disease (Guzman & Kouri, 2002; Whitehead et al., 2007). In addition, immunization against one dengue serotype induces life-long immunity against the homologous serotype and short-lived immunity against the other serotypes. Put together, it is widely believed that for a DENV vaccine to be effective, it must comprise neutralizing epitopes from all four serotypes (tetravalent) (Halstead, 1988; Whitehead et al., 2007). Presently, DIII protein immunization in animal has demonstrated promising results. In these studies, a variety of parameters affecting DIII protein immunogenicity has been researched. These parameters include: antigen combination - monovalent, bivalent, or tetravalent rDIII, type of animal model used, type of adjuvant used and the route of administration (Table 1). On the whole, rDIII protein immunization generated satisfactory levels of antibodies that are neutralizing against the virus. This observation is consistent for DENV and WNV rDIII proteins expressed from E. coli (Babu et al., 2008; Chin et al., 2007; Chu et al., 2005 & 2007; Martina et al., 2008). Bacteria-expressed DENV rDIII proteins generally elicit significant levels of homotypic DIII-specific neutralizing antibodies against the homologous wild-type DENV, with reduced levels of heterotypic neutralizing antibodies. The tobacco mosaic virus-based expression of rDIII protein in plants could also generate rDIII proteins that induced neutralizing antibodies against the DENV (Saejung et al., 2007). In addition, tetravalent DIII protein vaccination studies have been performed with some degree of success. The rDIII proteins from all four DENV serotypes were constructed in tandem and expressed as a single fusion protein (Chen et al., 2007). Immunization studies in mice with this tetravalent rDIII protein failed to generate equal immune response against the wild-type DENV. It was reported that efficacy of the immunogen was 70 % protective in mice against DENV1, and viral challenge, but only 18 % protective against DENV3. In another study, a chimeric tetravalent rDIII protein that was constructed and expressed using the yeast expression system was reported to be able to generate neutralizing antibodies against all four DENV serotypes (Etemad et al., 2008). With regards to type of animal model used for DIII protein vaccination studies, the use of mice breeds such as Balb/C or C57BL/6 have been widely reported (Babu et al., 2008; Sim et al., 2008). Furthermore, the use of other animal models such as rats or monkeys has also been reported (Bernardo et al., 2008; Hermida et al., 2006; Saejung et al., 2006). For example, Macaca fascicularis monkeys immunized with a DENV rDIII fusion protein (DIII fused with P64K protein from Neisseria meningitidis) generated an anamnestic protective antibody response against the wild-type DENV (Bernardo et al., 2008). Unlike live attenuated vaccines, protein subunit vaccines need to be adjuvanted in order to elicit a suitably good immune response (Whitehead et al., 2007). It has been observed that rDIII protein mixed with either complete/incomplete Freund’s Adjuvant or Montanide ISA 720 adjuvants elicited polyclonal antibodies (in mice) with higher neutralizing efficacies (PRNT90 of 1:128) as compared to the neutralizing antibodies generated using an rDIII protein-Alum mix (PRNT90 of 1:64). Cell-mediated immune responses were also varied according to the type of adjuvant used (Babu et al., 2008). In addition, the immunization of 12 Lik Chern Melvin Tan and Mah Lee Ng mice via mucosal administration of a recombinant Lactococcus lactis strain expressing the rDIII protein was also shown to be capable of eliciting the generation of neutralizing antibodies against the wild-type DENV (Sim et al., 2008). Production of the rDIII Protein rDIII Protein Expression Currently, there is a need for the production of cost effective and safe rDIII protein related biologics for the development of protein subunit vaccines or diagnostic reagents. For these purposes, the recombinant proteins produced must maintain their biological activity (i.e., generate neutralizing antibodies against wild-type virus or able to bind to anti-DENV antibodies found in patient serum). rDIII proteins may be expressed using various hosts, such as bacteria, yeast and even in the leaves of tobacco plants (Etemed et al., 2008; Saejung et al., 2007; Tripathi et al., 2008). Escherichia coli is by far the most commonly used host for the production of rDIII proteins. In general, the gene of interest (i.e. the DIII gene) is first cloned into expression vectors, such as the pET28a vector or pET30a (Novagen). Following that, the E. coli is transformed with the recombinant plasmids for protein expression. For high-yield protein production, the BL21(DE3) or its derivatives is the strain of choice. It has the advantage of being deficient in both lon and ompT proteases and it is also highly compatible with the T7 lacO promoter system (Graslund et al., 2008). Vectors encoding resistance to antibiotics such as Kanamycin, as in the case of the pET28a vector, are widely used for the antibiotic selection of recombinant clones. This is to ensure that the majority of the culture consists of recombinant E. coli clones that carry the required vector for protein expression. Using T7 systems, protein expression can be induced either with the chemical inducer isopropyl-β-Dthiogalactoside (IPTG) or by manipulating carbon sources during E. coli growth (autoinduction) (Studier, 2005). Protein expression is often induced at mid-log phase of the growth curve to ensure maximal yield while circumventing problems associated with cells going into the stationary phase, i.e. induction of proteases (Chin et al., 2007; Graslund et al., 2008). Small scale pilot expression is widely used as a predictive tool to determine which of the derivative clones comparatively produce a better yield of the protein of interest. It is also generally a platform for optimizing conditions required for establishing the best parameters for a large-scale production of recombinant proteins such as the rDIII proteins (Graslund et al., 2008). Parameters such as the type of culture media used, type and duration of induction, incubation temperature and concentration of the chemical inducers (such as IPTG) should be tested. With regards to rDIII protein production, we have observed that rDIII protein expression is optimal between to hours after induction by IPTG, at 30˚C, and there is generally no difference in induction for IPTG concentrations between 0.5mM to 3mM (unpublished data). This observation is universal for all four serotypes of DENV rDIII proteins. The up-scaling process of rDIII protein production may be performed by replacing commonly used batch cultivation in shake flasks (Babu et al., 2008; Chin et al., 2007; Dengue Envelope Domain III Protein: Properties, Production and Potential Applications 13 Pattnaik et al., 2007; Tripathi et al., 2008; Zhang et al., 2007) to fed-batch or continuous batch cultivation in a bioreactor (Tripathi et al., 2008), thereby tremendously increasing the protein yield. Additionally, the enhancement of the culture media used, i.e. from Luria Bertani broth to Terrific broth, experimentally improved DENV4rDIII protein yield (Tripathi et al., 2008). By incorporating the rDIII gene of DENV2 into a tobacco mosaic virus-based vector (TocJ), the DENV2rDIII protein can be effectively expressed in the leaves of Nicotiana benthamiana plants (Saejung et al, 2007). The DENV2rDIII protein after extraction and subsequent purification, was detectable by enzyme-linked immunosorbent assay (ELISA), and was able to illicit the generation of neutralizing antibodies in mice against the wild-type virus. This is the first time the DENV rDIII protein expression is reported to have been successfully performed on plant hosts. In addition, rDIII proteins can also be expressed using the mammalian protein expression system. Taking advantage of the high expression potential of the methylotrophic yeast Pichia pastoris, a chimeric tetravalent DIII protein was successfully expressed at high concentrations (Etemad et al., 2008). The advantages of using Pichia sp. for protein expression are that this yeast has a high growth rate, able to grow on simple inexpensive media, and is suitable for small scale pilot expression that can be scaled up to industrial size rDIII Protein Purification To facilitate the purification of rDIII proteins, the proteins are commonly produced as fusion proteins that comprise of the DIII protein fused with an affinity tag, such as the hexahistidine tag (Chin et al., 2007; Pattnaik et al., 2007; Uhlen et al., 1992; Zhang et al., 2007). The advantages of using a hexahistidine tag are manifold. Firstly, hexahistidine-tagged proteins can be purified by immobilized metal-ion affinity chromatography (IMAC) by the means of a relatively simple protocol (Arnau et al., 2006). Secondly, hexahistidine tags rarely affect the characteristics of the protein. Lastly, the hexahistidine tag is relatively small and generally does not alter the solubility of the protein of interest (Graslund et al., 2008). In general, IMAC purification procedures are relatively straightforward (Arnau et al., 2006; Gaberc-Porekar & Menart, 2001). The processed lysate is first loaded onto the IMAC column. The protein of interest binds to the column via its affinity tag and is subsequently washed with a buffer comprising intermediate concentrations of imidazole. This “washing” step removes contaminating proteins from the column. Following that, the recombinant protein is eluted with a higher concentration of imidazole (i.e. 200mM to 500mM imidazole). Normally, trace amounts of bacterial proteins co-purify with the recombinant protein. The SlyD protein, which comprise multihistidine residues, and other proteins such as GroES, Fur, CA, RplB, DnaJ, GroEL and DnaK that are found in E. coli, are common contaminants of the IMAC purified proteins (Bolanos-Garcia & Davis, 2006; Howell et al., 2006). There are several factors that may adversely affect the binding of the recombinant proteins to the IMAC column. Parameters such as pH of buffer, the presence of chelators such as EDTA, or high concentration of imidazole or DTT must be considered for successful IMAC protein purification (Graslund et al., 2008). 14 Lik Chern Melvin Tan and Mah Lee Ng After the preliminary purification using IMAC, the purity level of these rDIII proteins can be further enhanced by size exclusion methods using the high-performance liquid chromatography (HPLC). In order to determine if the purified protein is the protein of interest, Western blot using affinity tag-specific and protein-specific antibodies may be performed to identify the affinity tag and also the protein of interest, respectively (Chin et al., 2007; Chu et al., 2007; Pattnaik et al., 2007; Tripathi et al., 2008). As an additional confirmation procedure, the purified protein of interest can be further identified and confirmed by mass spectrometry. Figure describes the possible production workflow for laboratory-based production of DIII protein as reagents for downstream research. Figure 3. Production workflow of bacterially expressed DENV rDIII proteins. Dengue Envelope Domain III Protein: Properties, Production and Potential Applications 15 Potential Applications of rDIII Protein in Dengue Diagnosis Currently, dengue diagnosis is based on serology, virus isolation and RNA detection (Malavige et al., 2004). Five serological tests are available for the diagnosis of dengue infection: hemagglutination inhibition test, complement fixation test, plaque reduction neutralization test, IgM antibody capture ELISA (MAC-ELISA) and indirect IgG ELISA (IgG-ELISA) (De Paula & Fonseca, 2004; Samuel & Tyagi, 2006). Because of its high sensitivity and ease of use, the ELISA platform is widely used as surveillance tool to detect and differentiate between primary and secondary infections in patient serum. As IgM antibody titres in primary infections are significantly higher than in secondary infections, this difference can be distinguished by MAC-ELISA. Furthermore, by using specific Mabs, the specificity of IgG-ELISA can be improved for definite serotyping of dengue infection (Samuel & Tyagi, 2006). As discussed earlier, the range of amino acid identity and similarity across the DENV serotypes to DIII proteins varies between 47.5% to 69.7% and 69.7% to 87.9% respectively. Therefore, these differences in amino acid sequences result in conformational differences between the proteins. As a consequence, this could lead to the differential detection of antibodies specific to different DENV serotypes. To date, studies have shown that the rDIII proteins have the ability to detect for anti-DENV antibodies in patient serum (Pattnaik et al., 2007; Tripathi et al., 2008). However, the ability of the rDIII proteins to accurately detect and differentiate between the serotypes of DENV infection in patient serum remains to be elucidated. Many in-house rDIII protein-based assays have been developed for research purposes. Examples of these in-house assays are as follows: IgG-ELISA performed using DENV4rDIII protein coated immuno-plate was used for the detection of increasing DIII protein-specific antibodies in mice that seroconverted following DENV4rDIII protein immunization (Babu et al., 2008). IgG-ELISA was similarly performed using bivalent rDIII antigens (from DENV2 and 4) for detection of anti-DIII antibodies in mice serum (Khanam et al., 2007). Dot blot analysis of DENV4rDIII protein was shown to be sensitive to immuno-detection using dengue patient sera (Pattnaik et al., 2007; Tripathi et al., 2008). ELISA plates coated with individual rDIII proteins (from the four DENV serotypes) can be used for the determination of the dissociation constant of various Mabs for the rDIII antigens. In addition, tetravalent rDIII proteins could also be used for the detection of antibodies against DENV via an IgGELISA platform (Chen et al., 2007) or a dot blot method (or dipstick ELISA method) (Tripathi et al., 2007). Based on these findings, the rDIII protein demonstrates great potential in being produced as diagnostic reagents for the development of serological tools such as ELISA and rapid dipstick tests. 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[...]... presumably responsible for < /b> inhibiting attachment to the cells (Lok et al., 2008) DENV envelope-< /b> specific Mabs are able to block virus < /b> entry into cells (Crill & Roehrig, 2001) Mabs that are specific against Domains I (IB4C-2, 4A5C-8, 2B3 A-1 and < /b> 9A4D-1) and < /b> II ( 6B6 C-1, 4G2, 4E5, 1A5D-1, 1B7 and < /b> 10A1D-2) have been shown to be able to block virus < /b> entry into Vero cells (approx 18% to 46% blocking), but not... premembrane protein and < /b> the envelope < /b> glycoprotein; and < /b> 7 non-structural proteins: the NS1, NS2a, NS 2b, NS3, NS4a, NS 4b and < /b> NS5 (Clyde, 2006; Mackenzie 2004) The envelope < /b> protein comprises 3 regions: Domain < /b> I, Domain < /b> II and < /b> Domain < /b> III < /b> The Domain < /b> I is the central domain,< /b> the Domain < /b> II is the dimerization and < /b> fusion domain,< /b> while the Domain < /b> III < /b> (DIII) is an immunoglobulin-like receptor binding domain.< /b> .. possible production workflow for < /b> laboratory-based production of DIII protein as reagents for < /b> downstream research Figure 3 Production workflow of bacterially expressed DENV rDIII proteins Dengue < /b> Envelope < /b> Domain < /b> III < /b> Protein: Properties, Production and < /b> Potential Applications 15 Potential Applications of rDIII Protein in Dengue < /b> Diagnosis < /b> Currently, dengue < /b> diagnosis < /b> is based on serology, virus < /b> isolation and.< /b> .. (1996) Demonstration of binding of dengue < /b> virus < /b> envelope < /b> protein to target cells J Virol 70, 8765-8772 Chin, J.F.L., Chu, J.J.H & Ng, M.L (2007) The envelope < /b> glycoprotein domain < /b> III < /b> of dengue < /b> virus < /b> serotypes 1 and < /b> 2 inhibit virus < /b> entry Microbes and < /b> Infect 9, 1-6 Chiu, M.W & Yang, Y.L (2003) Blocking the dengue < /b> virus < /b> 2 infections on BHK-21 cells with purified recombinant dengue < /b> virus < /b> 2 E protein expressed... in the understanding of the dengue < /b> DIII protein We also examine the issues pertaining to the expression and < /b> purification of recombinant DIII (rDIII) fusion proteins and < /b> discuss the prospects for < /b> its incorporation as a reagent for < /b> dengue < /b> diagnosis < /b> Properties of the Dengue < /b> DIII Protein Structural Studies on the Flavivirus Envelope < /b> and < /b> DIII Proteins A huge step forward in the field of flavivirus research... recombinant soluble dengue < /b> virus < /b> 2 envelope < /b> domain < /b> III < /b> protein production in Escherichia coli trxB and < /b> gor double mutant J Biosci Bioeng 102, 333-339 Sim, A.C.N., Lin, W., Tan, G.K.X., Sim, M.S.T., Chow, V.T.K & Alonso, S (2008) Induction of neutralizing antibodies against dengue < /b> virus < /b> type 2 upon mucosal administration of a recombinant Lactococcus lactis strain expressing envelope < /b> domain < /b> III < /b> antigen.< /b> .. induction by IPTG, at 30˚C, and < /b> there is generally no difference in induction for < /b> IPTG concentrations between 0.5mM to 3mM (unpublished data) This observation is universal for < /b> all four serotypes of DENV rDIII proteins The up-scaling process of rDIII protein production may be performed by replacing commonly used batch cultivation in shake flasks (Babu et al., 2008; Chin et al., 2007; Dengue < /b> Envelope < /b> Domain < /b> III.< /b> .. and < /b> DENV complex-specific neutralizing antibodies against the virus < /b> (Gromowski et al., 2008) This is consistent with studies performed on the WNV DIII protein where the DIII protein has also been determined to be an important target for < /b> neutralizing antibodies (Beasley & Barrett, 2002) For < /b> these reasons, the DIII protein is believed to be a potential candidate as a protein subunit vaccine The antibody-mediated... envelope < /b> protein domain < /b> III < /b> of dengue < /b> 2 virus < /b> J Virol 82, 8828-8837 Gubler, D.J (1998) Dengue < /b> and < /b> dengue < /b> hemorrhagic fever Clin Microbiol Rev 11, 480-496 Gubler, D.J (2002) Epidemic dengue/< /b> dengue hemorrhagic fever as a public health, social and < /b> economic problem in the 21st century Trends Microbiol 10,100-103 Guzman, M.G & Kouri, G (2002) Dengue:< /b> an update Lancet Infect Dis 2, 33-42 Halstead, S .B (1988) Pathogenesis... increasing DIII protein-specific antibodies in mice that seroconverted following DENV4rDIII protein immunization (Babu et al., 2008) IgG-ELISA was similarly performed using < /b> bivalent rDIII antigens (from DENV2 and < /b> 4) for < /b> detection of anti-DIII antibodies in mice serum (Khanam et al., 2007) Dot blot analysis of DENV4rDIII protein was shown to be sensitive to immuno-detection using < /b> dengue < /b> patient sera (Pattnaik . 2010 Available online 1 July 2010 Keywords: Flavivirus Dengue virus West Nile virus Envelope Domain III Membrane chromatography Protein purification abstract Arthropod-borne flaviviruses such as dengue virus. 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. and envelope protein; and seven non-structural proteins: NS1, NS2A, NS 2B, NS3, NS4A, NS 4B and NS5 [9,10]. The envelope protein comprises three re- gions: Domain I, Domain II and Domain III. Domain