The novel SARS-CoV-2 betacoronavirus SARS-CoV-2 causes coronavirus disease 2019, which turned into a global pandemic in March 2020. The spike protein of the SARS-CoV-2 virus is a structural transmembrane glycoprotein that is crucial for viral entry into the host cell. The virus attaches to the host cell receptor with the C-terminal domain of the spike protein called the receptor-binding domain (RBD). The binding of the RBD causes a conformational change in the spike protein, allowing fusion of the viral envelope and the host cell membrane. In the master’s thesis, we expressed genes for recombinant RBD proteins of the original strain of the SARS CoV-2 virus and the SARS-CoV-2 delta plus strain in Escherichia coli bacteria. Mutations specific to the RBD protein of the delta plus strain (L452R, T478K, and K417N) were successfully introduced into the RBD transcript using a simple asymmetric overlap extension polymerase chain reaction. Both versions of the RBD protein were synthesised in E. coli in the form of insoluble inclusion bodies, which were dissolved in a buffer with a high concentration of urea. We tested several methods of refolding denatured RBD proteins from inclusion bodies with step-wise dialysis proving to be the most successful method. We also checked the conformational stability of the RBD of the wild-type virus and the delta plus mutant virus. The presence of the secondary structure of proteins was determined by recording the far UV circular dichroism spectrum, and the conformation of proteins was additionally evaluated by measuring the fluorescence of tryptophan residues. We found that the RBD protein of the original strain maintains higher stability during freezing and thawing than the RBD version from the delta plus strain.
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