Optimized high-yield expression of envelope glycoprotein domain III from dengue virus serotypes 1 to 4.

Dengue virus (DENV) envelope glycoprotein Domain III (EDIII) is critical for viral entry, highly immunogenic, and induces robust neutralizing antibody response. It is a prominent candidate for designing subunit-based vaccines and can also be harnessed as an antigenic bait for isolation of neutralizing human mAbs. Here, we describe an optimized method for high-yield expression of recombinant domain EDIII protein from DENV serotypes 1 to 4 in different Escherichia coli (E. coli) expression strains. The DENV EDIII proteins show differential expression patterns in tested E. coli expression strains. The structural integrity of the purified and refolded proteins is further validated using the Circular Dichroism (CD) spectroscopy and Fourier Transform Infrared (FTIR) spectroscopic analysis. The functional validation of the purified refolded DENV EDIII proteins through Enzyme-linked Immunosorbent Assay (ELISA) and co-immunoprecipitation (Co-IP) exhibits efficient binding with a well-characterized humanized neutralizing mAb 513. Further, we compared the potency of purified EDIII in blocking viral through competitive inhibition assay. Our study highlights that a universal expression system may not be an ideal approach for all DENV EDIII protein expression.