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 has become an endemic problem in countries with tropical and subtropical climates, thus categorizing it as a global challenge. One molecular biology approach for DHF prevention is through vaccination. A protein-based recombinant vaccine approach can be employed by utilizing the Envelope protein (E) of the Dengue Virus, as this protein is an ideal target as a vaccine candidate. This study optimized the gene sequence of recombinant Envelope (rE) coding for the recombinant Envelope protein (rE), followed by in silico testing of protein characteristics and structure modeling. The obtained results revealed that the rE protein exhibited instability index, aliphatic index, and isoelectric point values of 32.14, 75.08, and 7.17, respectively. The Ramachandran plot analysis indicated that 95.4% of amino acid residues were within the allowed region, while 4.7% were within the disallowed region, demonstrating the accuracy of the in silico protein modeling for rE. Consequently, the in silico testing results demonstrated that the rE protein possessed a stable and high-quality structure. The rE gene was then inserted into the pET-15b vector plasmid for subsequent expression using the Escherichia coli BL21(DE3) expression host system. Positive Escherichia coli colonies carrying the rE gene were induced with 1 mM IPTG. The expression results were analyzed using SDS-PAGE, followed by purification using a Ni-NTA column, and further analyzed by SDS-PAGE and western blot. The research findings demonstrated the successful insertion of the recombinant pET-15b-rE plasmid into E. coli BL21(DE3). The rE protein, with a size of 50.68 kDa, was successfully expressed in Escherichia coli BL21(DE3), as evidenced by the SDS-PAGE analysis showing a band within the 50-60 kDa range. In conclusion, this study successfully achieved the expression and purification of the recombinant Envelope protein (rE) of Dengue Virus in Escherichia coli BL21(DE3).