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Licensed Unlicensed Requires Authentication Published by De Gruyter March 24, 2022

Use of Circular Dichroism in the characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc

  • Ingrid Ruíz , Jose A. Gómez and Laura García EMAIL logo

Abstract

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-β 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 °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.


Article note:

A collection of invited papers based on presentations at the Virtual Conference on Chemistry and its Applications (VCCA-2021) held on-line, 9–13 August 2021.



Corresponding author: Laura García, Faculty of Chemistry, University of Havana, 10400, Havana, Cuba, e-mail:

Annex

Table 5:

Secondary structure and its content in % of the RBD-hFc protein spectra obtained at a temperature range of 20–95 °C, with intervals of 5 °C (Results of deconvolution analysis of the spectra in Figure 5 using the BeStSel).

Temperature α-helix β-sheet Random coil
20 5.1 49.9 45.0
25 7.4 50.1 42.5
30 5.8 49.9 44.3
35 5.7 49.1 45.2
40 8.3 48.6 43.1
45 6.4 51 42.6
50 7.5 52.3 40.2
55 4.2 51 44.8
60 5.6 51.5 42.9
65 8.9 47.6 43.5
70 10.4 43.7 45.9
75 5.1 45.4 49.5
80 5.9 45.7 48.4
85 11.3 44.2 44.5
90 7.1 46.5 46.4
95 6.8 45.6 47.6

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Published Online: 2022-03-24
Published in Print: 2022-07-26

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