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Author's Personal Copy Journal of Virological Methods 235 (2016) 73–79

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High yield expression and purification of Chikungunya virus E2 recombinant protein and its evaluation for serodiagnosis Anil Verma a , Anmol Chandele c , Kaustuv Nayak c , Murali Krishna Kaja c , Arockiasamy Arulandu d , Rakesh Lodha a , Pratima Ray b,∗ a

Department of Pediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi 110029, India Department of Biotechnology, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India c ICGEB-Emory Vaccine Center, International Center for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India d Structural Biology & Computational Biology, International Center for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India b

a b s t r a c t Article history: Received 1 February 2016 Received in revised form 2 May 2016 Accepted 4 May 2016 Available online 11 May 2016 Keywords: Chikungunya Diagnostics Envelope 2 protein ELISA

Disease caused by Chikungunya virus (CHIKV) is clinically characterized by sudden-onset of fever and severe arthralgia, which may persist for weeks, months, or years after acute phase of the infection. CHIKV is spreading globally; in India it first appeared in the 1960s followed by a quiescent period and then a fullblown remergence in 2006 and sporadic persistence since then. Despite a large number of commercially available diagnostic kits for CHIKV, clinical preparedness and diagnostics suffer from sub-optimal assays. An international diagnostic laboratory survey suggested that there is a critical need for improved CHIKV diagnostics especially in the early acute phase of illness. With the recent studies indicating that a vast majority of human humoral response in CHIKV infection is directed against E2 protein, this supports strong interest to develop CHIKV E2 based serological tests. However, methods to produce large amounts of CHIKV protein are limited. Here we report cloning, expression and purification methods for obtaining a truncated 37 kDa Chikungunya E2 protein at a high yield of 65–70 mg/l. We found that this purified protein can be reliably used in ELISA and western blot to detect CHIKV specific antibodies in sera from patients who were PCR or IgM positive. Thus, using this protocol, laboratories can make large quantities of purified protein that can be potentially used in CHIKV serological analysis. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chikungunya virus (CHIKV) is a mosquito-borne single strand RNA virus of the alphavirus genus (group IV) in the family Togaviridae. CHIKV was first isolated in 1953 during an epidemic in Tanzania (Lumsden, 1955). Since then, CHIKV has emerged from being an obscure viral infection to an international threat with expanding epidemics across continents. In 2004, an outbreak that started in Kenya spread to India that resulted in an explosive epidemic involving millions of people. Although CHIKV infection is self-limiting and rarely fatal, the associated arthralgia is extremely painful and debilitating. In fact, the name ‘Chikungunya’ derives from this pain and is a Makonde word that translates to ‘disease that bends up the joints’. This acute pain typically lasts for 1–2 weeks which

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (P. Ray). http://dx.doi.org/10.1016/j.jviromet.2016.05.003 0166-0934/© 2016 Elsevier B.V. All rights reserved.

may present other symptoms such as fever, rash, myalgia and impaired ambulation. However, in a subset of individuals, joint pain and stiffness does not subside in 1–2 weeks and can last for weeks, months or years with frequent relapses. Why certain individuals have an acute episode whereas others develop chronic relapse of clinical symptoms is not known and remains a topic of intense research. Currently, there are no specific licensed prophylactic or therapeutic vaccines. Diagnostic tests are suboptimal. Generally, reverse-transcriptase PCR is often used to detect viral genome which is a gold standard for confirmed CHIKV infection (Pfeffer et al., 1997). However, at later stages i.e. at 4–5 days post illness, PCR may not necessarily be reliable when the viral titers decline. In resource limited settings, PCR is often not easily available. Conformation by viral isolation is a laborious process requiring a minimum of level 2 physical biocontainment. Alternative methods include serological tests and serum collected >7 days after onset of symptoms is typically serologic techniques are often used to detect IgM and/or IgG responses to the virus. One serologic testing method is the indirect fluorescent antibody (IFA) technique.

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Although IFA tests have good sensitivity and specificity for CHIKV, this method requires specific material that may not be available in diagnostic laboratories worldwide. ELISA and immunochromatography test for rapid detection (RDT) are widely used all over the world. Recently, the US Centers for Disease Prevention and Control (CDC) evaluated several commercially available serologic diagnostic tests for Chikungunya, using reference samples submitted to the French Armed Forces Biomedical Research Institute (IRBA; Marseille, France) for arbovirus testing during 2005–2014. Their data revealed that these commercial kits should not be used in clinical settings, regardless of the geographic origin of the infection (Prat et al., 2014). In India, there are several diagnostic kits on the market, but a carefull evaluation of their stringency for detecting specific CHIKV infections in the febrile phase of illness is yet lacking. The genome of Chikungunya virus is 11.8 kbs and consists two open reading frames (ORF), one contains four nonstructural proteins (nsp1-4) and the other contains five structural proteins (capsid, E3, E2, 6K and E1) (Khan et al., 2002; Ross, 1956). The virus contains three structural proteins, glycosylated E1 and E2 are embedded in the viral envelope and a non-glycosylated capsid protein E3 associates with E2 during budding and formation of mature virions. Infection with other alphaviruses and CHIKV has revealed that the neutralizing antibody response is primarily directed against E2 and to a lesser extent to E1. In the case of CHIKV, E2, E3 glycoprotein, capsid and nsP3 proteins are targets of the antiCHIKV antibody response (Kam et al., 2012). Naturally-acquired IgG response to CHIKV E2 protein is dominated by specificity to a single linear epitope located at the N-terminus of the E2 glycoprotein and prominently exposed on the viral envelope. Both E1 and E2 have been major targets for the development of recombinant subunit vaccine and IgM or IgG based diagnostic assays. Therefore, development of a robust diagnostic assay using the CHIKV E protein will not only help in better diagnosis of Chikungunya fever but will also allow validation of several vaccine candidates that are currently in various phases of clinical trials. Several laboratories have generated CHIKV E2 protein using various expression systems using baculovirus, Escherichia coli or synthetic peptide, but the scope for their potential stringency in diagnostics using high yield generation methods is yet to be established (Kumar et al., 2012a,b; Tripathi et al., 2014; Verma et al., 2014). The objective of this study is to express and purify recombinant Chikungunya E2 protein, so that it can potentially be used for the development of a useful diagnostic assay. Literature search revealed that to date, there is no report on successful purification of the full-length E2 protein. Though not reported, we believe that this may be due to the inherent cysteine rich nature of the protein. The presence of a large number of cysteine residues increases the intrinsic hydrophobicity of the protein, which results in protein aggregation with very little protein in the soluble fraction. Nevertheless, we made an attempt, but failed to produce purified full length E2 protein in a bacterial expression system. Hence, we opted to truncate the protein such that we could obtain high yields of well-folded E2 protein, the results of which are presented below.

2. Subject, materials, and methods 2.1. Chikungunya virus strain The majority of circulating CHIKV strains in India belongs to the Eastern Central Southern African (ECSA) genotype (Sreekumar et al., 2010; Yergolkar et al., 2006). The African S-27Chikungunya virus strain (AF369024) used for cloning E2 was obtained from the National Institute of Virology, Pune, India. The virus was grown by passaging in mosquito cells C6/36 for one cycle followed by two

passages in Vero-76 cell line using standard protocols (Wikan et al., 2012). 2.2. Viral RNA extraction Genomic viral RNA was extracted from 140 ␮l of CHIKV infected Vero cell culture supernatant using QIA Amp Viral RNA mini kit (QIAGEN, Germany) according to the manufacturer’s protocol. The RNA was eluted from the QIA spin columns in a final volume of 60 ␮l of elution buffer and stored at −80 ◦ C (Ray et al., 2012). 2.3. Construction of recombinant expression vector and purification of E2 protein 2.3.1. Cloning of E2 gene The partial E2 gene that corresponds to amino acid residues 46-327 was generated by PCR with gene specific cloning primers. CHIKV viral RNA was used for cDNA synthesis (High Capacity cDNA synthesis kit - Invitrogen, USA). This was then further amplified for E2 with High fidelity Fusion Taq Polymerase (NEB, England) using gene specific forward (5 -CCATGGTAT TGGA GATGG AAC-3 ) and reverse (5 -GTCGACGACCCTAGTGA AGGC-3 ) cloning primers for 3 min at 98 ◦ C, 35 cycles of 30 s at 98 ◦ C, 30 s at 58 ◦ C and 1 min at 72 ◦ C, followed by final elongation 5 min at 72 ◦ C. The PCR product was analyzed on 2% agarose gel and cloned into the cloning vector pGEMT (Promega, Germany). The colonies were screened by blue/white screening method and the pGEMT-E2 clones were confirmed by sequencing with T7 promoter and SP6 universal primer (ABI). The pGEMT-E2 plasmids were then subcloned into the BamHI/XhoI sites of pET28b+ expression vectors (Novagen, Germany) and transformed into DH5␣ strain of E. coli. The E2pET28b+ plasmid was reconfirmed with restriction digestion and transformed into BL-21 (DE3) strain of E. coli and made 20% glycerol stock for further use. 2.3.2. E2 protein expression For the expression of recombinant E2 protein (rE2 protein), transformed E. coli was grown overnight at 37 ◦ C in the presence of 50 ␮g/ml kanamycin. This overnight culture was then inoculated in 50 ml L.B medium and incubated 37 ◦ C under shaking conditions till the bacterial culture O.D reached 0.4-0.6. E2 expression was then induced by adding iso-propyl thio-␤-d-glycosidase (IPTG) at a 1 mM concentration for 4 h followed by centrifugation at 6000g (Sorvall, Thermo scientific) for 15 min. The cell pellets were stored in −80 ◦ C till further processing. 2.3.3. Cell lysis and inclusion body preparation The frozen cell pellet (induced) was thawed and resuspended in cell lysis buffer (1:20, w/v) containing lysozyme and incubated at RT with continuous stirring. The cell suspension was disrupted by sonication (Sonics, USA) for 10 min in ice with 5 s on/off pulses after which the lysate was centrifuged at 10,000g for 20 min at 4 ◦ C to recover the inclusion body pellet. The inclusion bodies were washed 6 times with wash buffer containing 2 M urea and 1% Triton X 100 and 4 times with wash buffer without urea and TX100. The clean Inclusion Body (IB) pellet was stored in aliquots at −80 ◦ C till further use. 2.3.4. Protein purification The recombinant E2 protein was purified by affinity chromatography using Ni2+ −NTA column (gravity flow column, Bio-Rad) in denaturing conditions using standard methods. Briefly, the IB washed pellet was resuspended in binding buffer with 6 M guinidium-Cl (1:20, w/v) and incubated at RT for 1 h on a shaker followed by centrifugation at 10,000g for 45 min at 4 ◦ C. The supernatant was filtered with 0.45-␮m syringe filter (Millipore, USA)

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Fig. 1. Sequence analysis shows truncation of E2 protein at N and C terminus. The sequence alignment of E2 full length protein and partial E2 protein shows deletion of 8 cysteine residues and intact 9 cysteine residues with truncated protein both denoted with (*). In-silico analyses of the B cell epitopes are shown in blue text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. These structures show predicted protein folding of the E2 protein of CHIKV. (A) The actual protein folding of the E2 protein (Voss et al., 2010). (B) The folding pattern of partial E2 protein by PyMol software.

Fig. 3. (A) Agarose gel analysis showing the amplified product of the E2 gene of CHIKV: lane1, DNA molecular weight marker (base pairs); lane2, amplified product of the E2 partial gene (846 bps); lane3, amplified product of the E2 full length gene (1260 bps) and lane4, negative control. (B) SDS-PAGE analysis showing expression of recombinant E2 protein (37 kDa) of CHIKV in bacterial host: lane1, molecular weight marker (kDa); lane2, uninduced cell lysate of E2 protein; lane3, induced cell lysate of E2 protein; lane4, purified recombinant E2 protein. (C) Western blot analysis of the purified recombinant E2 protein, lane1, molecular weight marker (kDa); lane2, E2 protein.

and then passed through a Ni2+ −NTA column for protein binding. The column was washed with 10 column volumes (CV) of washing buffer for the removal of unbound or nonspecifically bound proteins. The rE2 protein eluted from the column by passing through the elution buffer containing 6 M urea and 350 mM immidazole. 2.3.5. Protein refolding and protein desalting The purified rE2 protein was refolded by the rapid dilution method. All eluted fractions of affinity purified rE2 protein were pooled and dialyzed against immidazole, then added in a drop wise manner to refolding buffer containing 0.4 M l-arginine, with

reduced and oxidized glutathione. This refolding suspension was kept at 4 ◦ C for 14–16 h with gentle stirring. After incubation, the solution was centrifuged at 10,000g for 30 min at of 4 ◦ C to remove any precipitated protein and the supernatant was concentrated by using the ultrafilteration unit (Stirred Cells − Amicon, Millipore, USA) with 10 kDa cut-off membrane (Millipore, USA). The refolded protein was passed through a PD-10 desalting column (GE Healthcare, Sweden) for buffer exchange or desalting, and then further concentrated with Amicon Ultra-15 centrifugal filter (Millipore, USA) by centrifugation at 4000g. The final concentrated protein was stored in aliquots at −80 ◦ C.



Fig. 4. Immunoblot analysis of the recombinant E2 protein of CHIKV: (A) titrations of CHIKV confirmed convalescent phase pooled positive and negative sera samples at 1:500, 1:1000, 1:2000, 1:4000, and 1:6000 dilutions. (B) Recombinant E2 protein detection with 10 individual CHIKV paired (acute and convalescent phase), seven positive (CHIKV-1 to CHIKV-7) and three negative (CHIKV-8 to CHIKV-10) sera samples at 1:2000 dilution. (A-acute phase sera; C-convalescent phase sera).

2.4. SDS PAGE analysis of recombinant E2 protein Uninduced and induced culture lysate of the recombinant clones and affinity purified His-tag rE2 proteins were separated on a 10% SDS polyacrylamide gel and the protein was visualized by staining with coomassie brilliant blue R-250.

developed with OPD substrate and the color reaction was stopped with 2 N HCl. The optical density (OD) was read at 490 nm with a reference filter at 650 nm using a bio-rad Elisa plate reader.

3. Results 2.5. Western blot analysis of recombinant E2 protein Purified His-tag rE2 protein was separated on a 10% SDSpolyacrylamide gel and then transferred to a poly vinylidene fluoride membrane (MDI, India). The membrane was blocked with 5% NFM in 1X PBS containing 0.05% tween-20 (PBST) for overnight ◦ at 4 C. After blocking, the membrane was washed four times with 1X PBS containing 0.1% tween-20 (PBST). The membrane was probed with anti-His − antibody or human sera (see section 2.6) at 1:3000 and 1:500-1:6000 dilutions respectively, for 1 h at room temperature. The membrane was washed four times with 1X PBST and incubated for 1 h at RT with goat-anti mouse IgG, HRP(1:7500) or anti-human IgG (Fc specific) HRP conjugated antibody (1:8000), conjugates were diluted in 1X PBS(T-0.05%) containing 5% NFM. To visualize the antibodies bound to protein, ECL luminal kit was used as enzyme substrates. The blots were developed on X-ray films with developer and fixer solution. 2.6. Subject and serum samples Paired serum/plasma from 10 febrile patients confirmed to have CHIKV infection either by RT-PCR or by IgM ELISA were collected from an earlier study (Ray et al., 2012) at All India Institute of Medical Sciences (AIIMS), New Delhi. The study was reviewed and approved by the AIIMS human ethics committee. All these 10 paired samples {seven positive (CHIKV-1 to CHIKV-7) and three negative (CHIKV-8 to CHIKV-10)} were pooled and used as a positive and negative control for western blot assay.

3.1. Structure analysis of Chikungunya E2 protein Analysis of CHIKV E2 protein was performed using PyMol software. The full length E2 protein contains 17 cysteine residues. We envisioned that these increased cysteine residues could lead to protein aggregation. Therefore, we took a two pronged approach, wherein we cloned the full-length CHIKV E2 protein and separately removed 45 amino acids from the N and C termini to make a truncated protein. This resulted in the deletion of 8 cysteine residues and the recombinant protein with 9 cysteine residues remaining intact that are denoted with (*) in Fig. 1. Fig. 2(A) shows the E2 protein in blue and the truncated regions are in green. The cysteine residues are shown as blue circles, disulphide bonds as yellow circles and the red circles denote oxygen atoms that form disulphide bonds. Fig. 2(B) shows the predicted structure after the 45 amino acid truncation where in N-terminus and some at C-terminus. We also analysed the full-length and the truncated E2 sequence through B cell epitope prediction algorithms such as Bepipred linear epitope prediction available on immunepitope.org. Truncation of the E2 protein resulted in the loss of 3 and 2 linear epitopes from both the N and C terminus respectively. Epitopes that were available in the truncated protein are marked in blue (Fig. 1). As predicted, the majority of the full-length E2 protein aggregated with very low yield in solution. Even though there was a predicted loss of five B cell epitopes we chose to express the truncated (46-327aa) protein.

2.7. Development of indirect Chikungunya specific ELISA assay

3.2. Cloning of Chikungunya virus E2 gene

For CHIKV specific IgM or IgG ELISA assay, 96 − well microtitre plates with high binding capacity (Costar) were coated 5 ␮g/ml His-tag purified rE2 protein diluted in 0.1 M carbonate-bicarbonate buffer (pH-9.2) at 4 ◦ C overnight. Before use, the plates were washed with 1X PBS containing 0.1% tween 20 (PBST) and blocked with 1X PBS containing 10% FBS for 2 h at RT followed by washing four times with PBST. Subsequently, the Chikungunya positive/negative serum samples were diluted in sample dilution buffer and added to the respective wells and incubated at RT for 1 h under shaker conditions followed by 1 h incubation with biotinylated goat anti-human IgM or IgG. The plates were washed and then

RNA was extracted from CHIKV and cDNA was made using random hexamers. The E2 gene was amplified using gene specific primers as illustrated in the methods section. Based on the structural analysis, we took two approaches to clone the E2 gene: partial region and full length. Two separate single bands at 846 base pairs and 1260 base pairs were obtained as shown in Fig. 3(A) for partial E2 gene and full-length respectively. The bands were gel eluted and cloned into pGEMT-Easy using T/A overhangs as described in the methods section which was followed by subcloning into the BamH1/Xho1 sites of the PET28b+ vector and the ligated product was transformed into BL21 (DE3) strain of E. coli.

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Fig. 5. ELISA analysis of CHIKV IgG (A) and IgM (B) antibodies in acute and convalescent phase paired sera from ten patients against recombinant E2 protein and formalin fixed virus.

3.3. Expression and purification of recombinant 37 kDa E2 protein The 37 kDa truncated E2 protein (rE2) was induced during log phase growth with 1 mM IPTG for 4 h at 37 ◦ C. A clear band was observed after induction that corresponded to 37 kDa on an 10% SDS-PAGE gel as shown in lane 2 of Fig. 3(B). After induction, the rE2 protein as purified showed and a single band corresponding to 37 kDa devoid of any bacterial impurities was observed (Fig. 3B, lane 3). In order to confirm that the 37 kDa band observed on the SDS PAGE gel was rE2 protein, a western blot hybridization was performed using anti-His tag antibody. A band at 37 kDa that correspond to the His tag-E2 fusion protein was detected − suggestive of a successful cloning and expression of E2 protein {Fig. 3(C)}.

Immunoblot with convalescent pooled sera obtained from CHIK PCR or IgM ELISA positive patients (n = 7) at different dilutions (1:500-1:6000), showed a specific band corresponding to 37 kDa. A similar blot that was developed with the pooled sera from CHIKV negative cases (n = 3) did not detect any such band. Of note, the titration of the pooled CHIKV positive sera showed that rE2 protein could be detected up to 1:6000 dilutions {Fig. 4(A)}. Because a pooled sera does not allow us to estimate the approximate frequency of positive patient sera that would detect CHIKV rE2 protein, we developed western blots separately with each of the 10 individual serum pair (acute and convalescent) that were used to make the pool. All of the positive samples detected E2 protein albeit at different levels of sensitivity {Fig. 4(B)}.



3.4. Functional analysis of cloned E2 protein We evaluated whether the recombinant E2 protein could be used to detect CHIKV specific antibodies in an indirect Elisa as robustly as formalin fixed Chikungunya virus. An indirect ELISA standardized in the lab was performed to detect CHIKV specific IgG and IgM {Fig. 5(A and B)}. We observed recombinant E2 protein showing robust IgM and IgG antibody response as compared to formalin fixed virus. Our results suggest that recombinant E2 protein has strong diagnostic potential and merits further studies.

4. Discussion The goal of this study was to clone, express and purify the truncated recombinant E2 protein of Chikungunya virus and evaluate its use in a standard diagnostic ELISA or a rapid strip test. Herein, we report that we have expressed a recombinant truncated (AA46327) CHIKV E2 protein in E. coli that successfully detects anti CHIKV IgG and IgM comparable to formalin fixed virus. Our literature search revealed that there was no full length protein cloned by any laboratory. However, our initial attempts failed to do so either. Further structural analysis of Chikungunya E2 protein with Pymol software and published atomic crystal structure Li et al. (2010) and Voss et al. (2010) revealed that Chikungunya E2 full-length protein has 17 cysteine residues and contains six disulfide bonds. Due to unusually high number of cysteine residues of the E2 protein, the protein is highly hydrophobic that probably results in aggregation during refolding process. Therefore, we modified our approach to clone and express a truncated E2 protein that retained most of the B cell epitopes. During the process of developing this protocol we realized that there is very little information available on the roadblocks in cloning and expression of cysteinerich proteins from Chikungunya virus. With this report, we hope to fill these gaps in knowledge. Though the expression of recombinant proteins in a bacterial system has the caveat that it does not support post-translational modifications such as glycosylation (Sarmientos et al., 1989; Makrides, 1996), it is still the preferred method of expression because of rapid bacterial growth, stability, high yield of the expressed protein, easy to operate and cost effective. The cloning, expression and purification of CHIKV E2 protein also reported by Tripathi et al. (2014) and Voss et al. (2010); was carried out in E. coli and insect cells (Drosophila melanogaster Schneider 2) respectively. Tripathi et al. (2014) described the use of diverse culture media (LB medium, terrific broth medium and terrific broth modified medium) and methods (shake flask culture, batch and fed batch fermentations culture) to achieve a high protein yield. Voss et al. (2010) reported structural analysis of CHIKV envelopes polyproteins as well as proteins. Our study describes a detailed method of high yield expression and protein production by using shake flask culture with regular LB medium, which is the standard protocol followed for protein production in E. coli. In contrast to this report, our methodology is cost effective and uses a conventional protein expression method that can be translated in resource constraint laboratories. Initially we tried different protein refolding method i.e. step gradient method, on column refolding with urea, followed by rapid dilution method. Unfortunately, all of these methods resulted in protein aggregation. This we speculated was due to exposed hydrophobic surface. Addition of arginine significantly suppresses protein aggregation (Das et al., 2007) and presence of both reduced and oxidized glutathione facilitates correct di-sulfide bonding of cysteine residue allowing protein folding. By following this method our protein yield was ∼65–70 mg per liter of culture, as compared to the previously reported yield of ∼8.5 mg per liter of culture (Tripathi et al., 2014). Thus, combining two approaches, one

deletion of 8 cysteine residues, and other is rapid dilution method of protein refolding in the presence of L-arginine, enhanced the yield of recombinant CHIKV E2 protein significantly and reproducibly. Additionally, cloning the protein in a pET28b+ vector allow the addition of Histidine tag that facilitates the nickel affinity purification method of the protein, thereby making it cost effective. The reports of Kumar et al. (2012a,b) described the immunogenicity of CHIKV E2 protein. Our findings corroborated these studies to reveal that CHIKV E2 protein has strong antigenicity and can be used for detecting very low levels of CHIKV specific antibody responses. The recombinant CHIKV E2 protein based ELISA or western blot assay has numerous advantages over similar experiment using formalin fixed viral antigen, such as no biohazard associated with the production of CHIKV rE2 protein in E.coli. 5. Conclusion This report describes a cloning, expression and purification method for obtaining a truncated 37 kDa Chikungunya E2 protein at a high yield of 65–70 mg/l. This protein can be used for detection of both CHIKV specific IgM and IgG antibodies in sera of CHIKV confirmed patients. The detection of CHIKV specific IgG and IgM antibodies by ELISA with this rE2 protein was remarkably comparable, if not better than formalin fixed virus. Thus, using this protocol, laboratories can make large quantities of purified recombinant E2 protein that can be potentially used in CHIKV serological analysis. Conflict of interests None. Acknowledgements We acknowledge the Department of Biotechnology (DBT), Government of India for providing financial support to P.R. (Grant nos. 102/IFD/SAN/PR2493/2007 and BT/MB/Indo-US/VAP/06/2013) to carry out this work. We also acknowledge Dr. V.K. Paul, department chair for providing the facility. References Das, U., Hariprasad, G., Ethayathulla, A.S., Manral, P., Das, T.K., Pasha, S., Mann, A., Ganguli, M., Verma, A.K., Bhat, R., Chandrayan, S.K., Ahmed, S., Sharma, S., Kaur, P., Singh, T.P., Srinivasan, A., 2007. Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key. PLoS One 2 (11), e1176. Kam, Y.W., Lee, W.W., Simarmata, D., Harjanto, S., Teng, T.S., Tolou, H., Chow, A., Lin, R.T., Leo, Y.S., Rénia, L., Ng, L.F., 2012. Longitudinal analysis of the human antibody response to chikungunya virus infection: implications for serodiagnosis and vaccine development. J. Virol. 86 (23), http://dx.doi.org/10. 1128/JVI.01780-12, 13005–15. Khan, A.H., Morita, K., Parquet Md Mdel, C., Hasebe, F., Mathenge, E.G., Igarashi, A., 2002. Complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site. J. Gen. Virol. 83, 3075–3084. Kumar, J., Khan, M., Gupta, G., Bhoopati, M., Lakshmana Rao, P.V., Parida, M., 2012a. Production, characterization, and application of monoclonal antibodies specific to recombinant (E2) structural protein in antigen-capture ELISA for clinical diagnosis of chikungunya virus. Viral Immunol. 25 (2), 153–160, http://dx.doi. org/10.1089/vim.2011.0068. Kumar, M., Sudeep, A.B., Arankalle, V.A., 2012b. Evaluation of recombinant E2 protein-based and whole virus inactivated candidate vaccines against chikungunya virus. Vaccine 30 (43), 6142–6149, http://dx.doi.org/10.1016/j. vaccine.2012.07.072. Li, L., Jose, J., Xiang, Y., Kuhn, R.J., Rossmann, M.G., 2010. Structural changes of envelope proteins during alphavirus fusion. Nature 468 (7324), 705–708, http://dx.doi.org/10.1038/nature09546. Lumsden, W.H., 1955. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53: II. General description and epidemiology. Trans. R. Soc. Trop. Med. Hyg. 49 (1), 33–57. Makrides, S.C., 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60 (3), 512–538. Pfeffer, M., Proebster, B., Kinney, R.M., Kaaden, O.R., 1997. Genus-specific detection of alphavirus by a semi-nested reverse transcription polymerase chain reaction. Am. J. Trop. Med. Hyg. 57 (6), 709–718.

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