Expression and production of recombinant SARS-CoV-2 RBD protein in E. coli system: comparison of performance,...

The worldwide COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). The receptor binding domain (RBD) of SARS-Cov-2 is located on the spike protein, which binds and interacts with ACE2. The RBD serve as a crucial target for the development of virus-neutralizing antibodies, vaccinations, and inhibiting agents. The goal of this study was to synthesis recombinant RBD (rRBD) protein and evaluate its expression, function, and stability in different strains of Escherichia coli (E. coli). The rRBD domain gene sequence was engineered utilizing in silico methodologies. The optimized sequence was inserted into pET-28a vector using XhoI and EcoRI restriction sites. Three E. coli expression strains BL21 (DE3), Rosetta-gami, and Shuffle T7 were chosen for protein synthesis. Cloning of the target gene fragment was confirmed using PCR. The validated recombinant vector was cloned into three E. coli strains. SDS-PAGE and Western blot analysis were used to examine the expression of the rRBD. Far-UV circular dichroism (CD) spectroscopy was used to examine secondary structure. ELISA experiments quantified the interaction of rRBD protein with HRP-conjugated anti-His antibody and serum from COVID-19 patients detected with anti-human immunoglobulin-HRP labeled. Protein stability was assessed under different temperature conditions. The rRBD protein was best expressed in all three bacterial strains at 1 mM concentration of IPTG. A 14 kDa band corresponding to the rRBD protein was identified in all three bacterial strains via Western blot. BL21 had the greatest amount of rRBD expression. The rRBD generated from Rosetta-gami exhibited the most robust affinity for anti-His antibodies. All three expressed proteins exhibited similar secondary structure, characterized by a predominance of random coil and β-sheet content. They also exhibited similar affinity to antibodies against SARS-Cov-2 generated from patients. Storage at -20 °C and 4 °C with suitable glycerol concentrations ensured optimal long-term protein stability. This study reveals that E. coli is still a good and flexible host system to produce functional SARS-Cov-2 RBD protein. Even though the strains had different levels of expression and binding efficacy, all of the proteins they made had comparable structures and antigenic properties. The findings validate the feasibility of using bacterial expression systems for the cost-effective production of RBD-based diagnostic reagents.