Expression, purification and characterization of in vivo biotinylated dengue virus envelope domain III based tetravalent antigen

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Abstract

Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin–streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20 mg protein of ∼95% purity, from 1 L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be ∼85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin–enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera (n = 164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity.

Introduction

Dengue viruses (DENVs)1, of which there are four antigenically distinct serotypes (DENV-1, -2, -3 and -4) are mosquito-borne viruses of the Flaviviridae family [1]. DENV infection threatens approximately half the global population, and is endemic to over a hundred countries in the tropical and sub-tropical regions of the world [2]. Diagnosis of DENV infections, based on clinical presentation, is complicated by its similarity to that of a host of other infectious illnesses including measles, influenza, typhoid, leptospirosis, chikungunya and malaria [3]. As a result, diagnostic tests are critical in detecting DENV infections [2]. For the detection of DENV specific antibodies, most of the commercial tests use either a mixture of four inactivated DENV particles or four recombinant envelope (ectodomain) proteins representing four DENV serotypes. Invariably most of these tests fail to differentiate between the antibodies generated against various flaviviruses due to the existence of shared antigenic determinants among members of the Flaviviridae family such as Japanese encephalitis, tick-borne encephalitis and yellow fever viruses [2]. Domain III of envelope protein (EDIII) is considered to be a flavivirus species specific antigen and has been used as a diagnostic reagent [4], [5], [6], [7], [8]. To detect antibodies against all four serotypes of DENV, it is necessary to use EDIII from four DENV serotypes [4]. To obviate the need of using four DENV EDIII antigens, Hapugoda et al. had reported the use of a single chimeric tetravalent (EDIII-T) antigen containing EDIIIs from four DENV serotypes, linked by flexible peptide linkers, for developing DENV antibody indirect ELISAs [5]. In these ELISAs, EDIII-T antigen coated wells were used to allow the binding of anti-DENV antibodies present in patient serum and revealed using anti-human IgG–enzyme or IgM–enzyme conjugates [5].

An alternative format of the ELISA, which exhibits very low background signal and reduce the chances of false positivity, is based on antibody capture [9]. The basic format of this ELISA involves the use of anti-human IgM and IgG antibody sensitized wells for IgM and IgG antibody capture from human sera, respectively, followed by their detection using specific antigen [9], [10].

The specific antigen may be labeled or, if not, may be used in conjunction with an antigen-specific monoclonal antibody (mAb)–enzyme conjugate [9], [10], [11]. The availability of a labeled antigen, which not only obviates the need for an antigen-specific mAb, but also prevent the problem of epitope masking, as the mAb specific epitope on the antigen may be masked by serum antibodies [12]. Tagging of the antigen with biotin, coupled to its specific detection using streptavidin (SA)–enzyme conjugate, offers a powerful yet simple signal collection and amplification system that can be used in capture ELISAs [13].

Chemical coupling of biotin moiety to protein is a commonly used method for in vitro biotinylation of protein. Most of the chemical biotinylation methods target lysine residues of proteins and it is difficult to control precisely which residues will be modified, and it may lead to alteration in protein functionality [14], [15]. Moreover, results of chemical biotinylation process are subjected to batch to batch variation [16]. Further, during chemical biotinylation, impurities present in the protein preparation may also get biotinylated, resulting in false positivity in the assay. In vivo site-specific biotinylation of proteins eliminates these concerns.

In vivo site-specific biotinylation of recombinant protein can be achieved by expressing recombinant protein in-fusion with a short biotin acceptor peptide (BAP) [17]. BirA, the enzyme biotin ligase catalyzes the covalent attachment of biotin to a specific lysine residue of BAP fused to recombinant protein. Despite the presence of endogenous BirA in Escherichia coli, co-expression of an exogenous enzyme improves biotinylation efficiency [18]. It has also been shown that supplementation of the induction medium with biotin is required for the efficient biotinylation [14]. Since in this system, the protein is modified specifically at the BAP tag, it reduces the chances of alteration in protein properties.

This paper, shows that the EDIII-T antigen, modified to carry a biotin acceptor site, can be over-expressed and efficiently biotinylated in vivo in an E. coli host engineered to co-express recombinant BirA and purified to near homogeneity in high yields. Further, the purified, in vivo biotinylated EDIII-T (b-EDIII-T) antigen in conjunction with SA–enzyme conjugate was able to recognize mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes in antibody capture ELISA format. Finally, a pre-characterized human sera (n = 164) panel, consisting of DENV antibody positive and DENV antibody negative sera, was used for the evaluation of b-EDIII-T based DENV IgG Antibody Capture (GAC) and IgM Antibody Capture (MAC) ELISA.

Section snippets

Materials

Escherichia coli host strain DH5α was purchased from Invitrogen (Carlsbad, CA, USA). E. coli expression strain SG13009, the expression plasmid pQE60, Ni–NTA Super-flow resin, Ni–NTA His-Sorb ELISA plate and anti-His (penta-His) monoclonal antibody (mAb) were from Qiagen (Hilden, Germany). E. coli expression strain BL21(DE3), the expression plasmids pET28b, pET32a and pETDuet-1 from Novagen (Madison, WI, USA). Goat anti-human IgG (γ-chain specific), rabbit anti-human IgM (μ-chain specific),

Design, expression, purification and characterization of b-EDIII-T antigen

As the EDIII-T antigen expressed in E. coli tends to be insoluble [5], the gene encoding it was fused to the thioredoxin tag-encoding sequence, with the objective of promoting its solubility. Further, to achieve site-specific, in vivo biotinylation of this protein, BAP-encoding sequence was inserted between the two fusion partners. A 6× His tag-encoding sequence was provided to aid in purification of the expressed protein by Ni–NTA chromatography. The expression plasmid, pTrxBAP-EDIII-T

Discussion

A major objective of the current work has been to explore the feasibility of utilizing the EDIII based tetravalent antigen in antibody capture ELISA format because of several advantages of this format over antibody indirect ELISA format [9]. To this end, a strategy was devised to produce an in vivo biotinylated version of the tetravalent antigen, b-EDIII-T, with the purpose of exploiting the inherent high affinity of biotin–streptavidin interaction [18], for signal collection and amplification.

Conclusions

This study demonstrates that it is possible to obtain high levels of site-specific, enzymatic in vivo biotinylation of a chimeric tetravalent DENV antigen derived from the EDIIIs of the four DENV serotypes. In a prototype antibody capture ELISA designed to exploit the unique interaction between biotin moiety and streptavidin–enzyme conjugate, the b-EDIII-T antigen specifically recognized polyclonal mouse antibodies raised against recombinant monovalent EDIII antigens corresponding to each DENV

Acknowledgments

The authors thank Dr. D.S. Waugh, Department of Physical Chemistry, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, USA for providing plasmid pDW363 through Addgene, Inc. The authors also thank Dr. Amita Agarwal, from Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India and Dr. Raija Vainionpää, from Department of Virology, University of Turku, Turku, Finland, for providing human sera samples. This work was supported by a grant from

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