RESUMO
At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices. © 2022 Walter de Gruyter GmbH, Berlin/Boston.
RESUMO
At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices.
RESUMO
From the receptor-binding domain (RBD) of the SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19), a RBD-hFc fusion protein was obtained at the Center of Molecular Immunology (Havana, Cuba). This fusion protein was used in the construction of a diagnostic device for COVID-19 called Ultramicroenzyme-Linked Immunosorbent Assay (UMELISA)-SARS-CoV-2-IgG and it is currently been used in the studies of biological activity of the Cuban vaccine Abdala (CIGB-66). In this work, Circular Dichroism (CD) is used to characterize this protein. Using Far Ultraviolet Circular Dichroism (FAR-UV CD), it was determined that the protein has a secondary structure in the form of a sheet-beta fundamentally. Using this technique, a thermodynamic study was carried out and it was determined that the melting temperature (Tm) of the protein is 71.5 degrees C. Information about the tertiary structure of the protein was obtained using Near Ultraviolet Circular Dichroism (NEAR-UV CD) and Molecular Fluorescence;they indicates that the protein has a three-dimensional folding associated with the aromatic amino acids in its structure, where tryptophan (Trp) is located inside the folded structure of the protein while tyrosine (Tyr) is exposed to the solvent.