Expression and Purification of Recombinant Envelope (rE) Protein of Dengue Virus in Escherichia coli BL21(DE3) with Computational Analysis

Dengue hemorrhagic fever (DHF), caused by the dengue virus (DENV), has become an endemic issue in tropical and subtropical regions, making it a significant global health challenge. One molecular biology approach to DHF prevention is through vaccination. This study aims to evaluate the heterologous expression of the recombinant E (rE) protein of DENV using the Escherichia coli expression system with the pET-15b vector. The DENV envelope protein (E) is a crucial target for vaccine development due to its ability to elicit neutralizing antibodies (NAbs). This study optimized the gene sequence encoding the rE protein, followed by in silico testing of protein characteristics and structure modeling. The results showed that the rE protein had an instability index, aliphatic index, and isoelectric point (pI) of 32.14, 75.08, and 7.17, respectively. Ramachandran plot analysis revealed that 95.4 of amino acid residues were in allowed regions, with 4.7 in disallowed regions, indicating accurate protein modeling for rE. The in silico analysis demonstrated that the rE protein had a stable and high-quality structure. The rEgene was subsequently inserted into the pET-15b vector and expressed in the E. coli BL21(DE3) host system. Positive E. coli colonies carrying the rE gene were induced with 1 mM IPTG, and SDS-PAGE analyzed the expression. The rE protein was purified using a Ni-NTA column and further analyzed by SDS-PAGE and western blotting. The results confirmed the successful insertion of the recombinant pET-15b-rE plasmid into E. coli BL21(DE3), with the rE protein, approximately 50.68 kDa in size, successfully expressed, as shown by the SDS-PAGE analysis with a band in the 50-60 kDa range. In conclusion, this study successfully achieved the expression and purification of the recombinant DENV envelope (rE) protein in E. coli BL21(DE3). Copyright © 2025 by Indonesian Journal of Pharmacy (IJP).