Vol 7, Issue 5, 2014 ISSN - 0974-2441 SMALL SCALE EXPRESSION, SOLUBILIZATION, AND CHARACTERIZATION OF CHIKUNGUNYA VIRUS STRUCTURAL PROTEINS NAMRATA DUDHA 1 , JYOTI RANA 1 , REEMA GABRANI 1 , AMITA GUPTA 2 , VIJAY K CHAUDHARY 3 , SANJAY GUPTA 1 * 1 Department of Biotechnology, Center for Emerging Disease, Jaypee Institute of Information Technology, Noida - 201 307, Uttar Pradesh, India. 2 Department of Microbiology, University of Delhi South Campus, New Delhi - 110 021, India. 3 Department of Biochemistry, University of Delhi South Campus, New Delhi - 110 021, India. Email: sjay1908@gmail.com Received: 25 August 2014, Revised and Accepted: 13 September 2014 ABSTRACT Objective: The severity and spread of Chikungunya fever in absence of effective antiviral therapy presents a serious public health threat. The present investigation aims to generate soluble and purified viral structural proteins that can be utilized to facilitate generation of reagents for development of both diagnostic and therapeutic measures. Methods: Bacterial expression system was used for optimization of expression and solubilization of structural proteins of CHIKV (Capsid, 6K and envelope proteins 1-3 [E1, E2 and E3]) as fusions with large (GST) and small (His and Strep) tags on a single platform. Affinity chromatography was used for small scale purification of viral proteins. Result: The effect of different tags, inducer concentrations, temperatures and duration of induction on solubilization of proteins has been optimized and small scale purification of all the structural proteins has been attempted. Utility of these solubilized proteins has been shown by analyzing the interaction of E2 with all the structural proteins using pull down assay. Conclusion: Small scale purification of all five structural proteins and ectodomains of envelope proteins E1 and E2 has been standardized. The data and reagents generated can be utilized for large scale purification and studying CHIKV biology. Keywords: Chikungunya virus, Protein expression, Solubilization, Purification. INTRODUCTION Chikungunya virus (CHIKV), a member of the genus Alphavirus and family Togaviridae has re-emerged as a major threat to public health by being responsible for the largest epidemics among Alphaviruses, infecting around 1.4-6 million people across different regions of the world [1-6]. CHIKV, a ~70-100 nm diameter wide enveloped virus, has a single-stranded, positive-sense RNA genome approximately 11.8 kb long. The viral genome bears a resemblance to host mRNAs, as it possesses 5฀ and 3฀ untranslated regions, with a 5฀ terminal methylguanylate cap and a 3฀ terminal polyadenylate tail. The genome is comprised of two distinct open reading frames: The 5฀ two-thirds of the genome, encodes the nonstructural polyprotein (nsP1-nsP2- nsP3-nsP4), and the remaining 3฀ one-third, encodes the structural polyprotein (capsid-E3-E2-6K-E1) [7]. Each viral protein performs specific functions during various stages of the viral life cycle. The nonstructural proteins of CHIKV are essential for viral replication while structural proteins (capsid, E1 and E2) form the virion structure and are majorly involved in receptor identification, fusion with cell membrane and elicitation of antigenic response in host cells [8-14]. Capsid, after autoproteolytic cleavage by its protease activity, forms viral particles and packages the viral genomic RNA [15]. The E1 and E2 proteins control viral entry into host cells: E1 mediates virus fusion to cell membranes in low pH conditions [16,17], while E2 interacts with a cellular receptor [18,19]. Small proteins E3 and 6k do not form the part of CHIKV structure. E3 is responsible for the proper localization of the structural polyprotein and its cleavage from E2 is essential for spike maturation [20,21]. 6K is a classified viroporin that has been shown to be involved in membrane permeabilization which facilitates virus budding [22,23]. Re-emergence of CHIKV has been especially attributed microevolution of its genome that has facilitated in the increased vector specificity of the virus [24]. Since CHIKV has emerged from a neglected mosquito-borne tropical disease to a viral infection of epidemic scope; there is an urgent need for the development and acquisition of reagents which will facilitate understanding of disease mechanism, detection, and effective treatment of CHIKV infection. In this direction, efforts are being focused towards purification and studying proteins of CHIKV. The present work investigates the effects of different tags, inducer concentrations, temperatures, and duration of induction on solubilization of structural proteins of CHIKV followed by their small scale purification and characterization. METHODS Construction of plasmids expressing CHIKV structural proteins The constructs for protein expression and pull-down were generated by amplifying genes from clones in TOPO vectors [25] encoding capsid, E3, E2, 6K, and E1 genes, using forward and reverse primers compatible for cloning in prokaryotic expression vectors pGEX-4T3 glutathione S-transferase (GST tag), pCAK (Strep tag) and pLTA (His tag). The polymerase chain reaction was performed and the purified amplicons were either digested with restriction enzymes (for cloning in pGEX-4T3 vector) or treated with T4 DNA polymerase and dTTP to generate BsaI compatible ends (for cloning in pCAK/pLTA vectors)and ligated into corresponding vectors as explained earlier by the authors [26-28]. Expression and solubilization of CHIKV fusion proteins Escherichia coli BL21 (DE3) cells transformed with individual recombinant vectors (plasmids pGEX-4T3, pLTA, and pCAK containing CHIKV structural genes) having N-terminal fusion tags were cultured in LB medium supplemented with appropriate antibiotic (100 µg/ml ampicillin for pGEX-4T3 and pLTA vectors; 30 µg/ml kanamycin for pCAK vector) at 37°C overnight with shaking at 220 rpm. The secondary cultures were induced at 0.3 OD 600 at different conditions of temperature, induction time, and inducer concentration (Table 1). Research Article