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

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.

Automated summary

A novel purification protocol was introduced to prepare nucleocapsid protein without any bound nucleic acids, revealing significant differences in the structural properties between the contaminated and uncontaminated protein. The study suggests that nucleic acid contamination may severely affect the molecular properties of the purified nucleocapsid protein, and describes the notable ability of the protein to form condensates resembling gel-like or solid structures.