Purification of Coronavirus Nucleocapsid Proteins

The nucleocapsid proteins of SARS-CoV (betacoronavirus lineage B), Pi-Bat CoV HKU5 (betacoronavirus lineage C), and rousettus bat coronavirus (Ro-BatCov HKU9; betacoronavirus lineage D) were obtained as described (2 (link),16 (link)). To produce a plasmid for DcCoV UAE-HKU23 nucleocapsid protein purification, primers 5�?�-CATGCCATGGGCATGTCTTTTACTCCTGGTAAGC-3�?� and 5�?�- CCGCTCGAGTATTTCTGAGGTGTTTTCAG-3�?� were used to amplify the gene encoding the nucleocapsid protein of DcCoV UAE-HKU23. The sequence encoding aa 1�?"672 of the nucleocapsid protein was amplified and cloned into the NcoI and XhoI sites of expression vector pET-28b(+) (Merck KGaA, Darmstadt, Germany). Recombinant nucleocapsid protein was expressed and purified by using the Ni²⁺-loaded HiTrap Chelating System (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer�?(tm)s instructions. The nucleocapsid protein of MERS-CoV was cloned and purified by using the method described above with primers 5�?�-GGAATTCCATATGATGGCATCCCCTGCTGCACCTC-3�?� and 5�?�- ATAAGAATGCGGCCGCATCAGTGTTAACATCAATCATT-3�?�.