Chikungunya E2 Protein Produced in E. coli and HEK293-T Cells—Comparison of Their Performances in ELISA

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)—a prokaryotic system unable to produce post-translational modifications—or in HEK-293T mammalian cells—a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches

“…43−45 However, on comparing the performance of the two CHIKV E2 antigens, a recent study found that both the prokaryotically and eukaryotically expressed E2 proteins demonstrated similar performance for indirect ELISA using anti-CHIKV E2 IgG antibodies. 45 Since recombinant proteins expressed in the prokaryotic system are much simpler, faster, cost-effective, and usually obtained in higher quantities when compared to proteins expressed in the eukaryotic system, we attempted cloning, expression, and purification of the fulllength CHIKV E2 protein in E. coli in our study.…”

“…Multiple studies have reported cloning, expression, and purification of CHIKV E2 proteins using the prokaryotic expression system. ,,,− However, almost all of the previously reported studies have described expression and purification of the truncated E2 protein. Additionally, few studies have demonstrated expression and purification of the CHIKV E2 protein using the baculovirus-based insect cell expression system and HEK-293T mammalian cells, which allow post-translational modifications like glycosylation. − However, on comparing the performance of the two CHIKV E2 antigens, a recent study found that both the prokaryotically and eukaryotically expressed E2 proteins demonstrated similar performance for indirect ELISA using anti-CHIKV E2 IgG antibodies . Since recombinant proteins expressed in the prokaryotic system are much simpler, faster, cost-effective, and usually obtained in higher quantities when compared to proteins expressed in the eukaryotic system, we attempted cloning, expression, and purification of the full-length CHIKV E2 protein in E.…”

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya

“…Several studies have shown the usefulness of the CHIKV E2 for the serological diagnosis of chikungunya, using prokaryotic and eukaryotic expression systems [55,[60][61][62]. Recently, a study using recombinant CHIKV E2 antigens, produced in both prokaryotic and eukaryotic expression systems, showed a strong reaction to anti-CHIKV IgG antibodies, with an accuracy higher than 90% [62]. An assay for chikungunya diagnosis, using recombinant multi-epitope proteins expressed in plants, reported a good quality expression and confirmation by Western-blotting [63].…”

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein

“…Various approaches are used to test CHIKV infection, including reverse transcriptase polymerase chain reaction (RT-PCR), [7][8][9][10][11] real-time RT-PCR, [12][13][14][15] reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), 16 and enzyme-linked immunosorbent assay (ELISA) (IgM/IgG antibodies). 17,18 RT-PCR is often used to detect the viral genome, which is a gold standard for confirmed CHIKV infection; 19 however, in resource-limited settings, PCR is often not easily available. One serologic testing method is the indirect fluorescent antibody (IFA) technique.…”