The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties

Tarczewska, Aneta; Kolonko-Adamska, Marta; Zarębski, Mirosław; Dobrucki, Jurek; Ożyhar, Andrzej; Greb-Markiewicz, Beata

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.