In Silico Design and Expression of Dengue Virus Type 2 Envelope Domain III in Escherichia coli BL21 (DE3) for Specific Diagnostics

Abstract

Dengue virus type 2 (DENV-2) is a mosquito-borne virus of global public health concern. Routine diagnostic tests often target whole envelope glycoproteins that suffer from cross-reactivity. The envelope domain III (EDIII) protein contains subtype virus-specific 
epitopes, offering improved specificity over complete envelope. This study evaluated a modified DENV-2 EDIII expression in E. coli 
BL21 (DE3) using a codon-optimized construct. AlphaFold2 Algorithms were utilized to predict its 3D structure and identify 
potential antigenic epitopes. The construct was stable (instability index <40), soluble and antigenic (>0.5score, respectively) containi
ng three key linear epitopes; CTGKFKVVKEIAETQH (302–317aa), SPCKIPFEIMDLEKR (331–345aa), and QLKLNWFKKGSS (386-396aa). Optimal expression occurred at 30°C and 4 hours post induction, producing a distinct 14 kDa band on SDS-PAGE consistent with the expected molecular weight of target EDIII protein. Overall, these findings demonstrate that in silico protein design with low-temperature bacterial expression can produce recombinant proteins suitable for specific diagnostics.