Codon-Optimized FCoV N Protein Expression

The gene encoding the FCoV N (GenBank: AY994055.1) was codon optimized for bacterial expression and synthesized by GenScript. The sequence was incorporated in pET-30a(+) backbone between NdeI and HindIII restriction sites for the expression of C-terminally His-tagged N. The Rosetta (DE3) E. coli cells transformed with the resulting plasmid were induced with 1 mM IPTG upon entering into exponential growth phase (A₆₀₀ 0.4). Cells were allowed to grow overnight at 16 °C and were harvested by centrifugation at 3000 rpm for 30 min at room temperature. Cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM dithiothreitol [DTT], 1% Triton-X100, 5 mM CHAPS, 0.1 mM phenyl methyl sulfonyl fluoride [PMSF]), followed by sonication on ice and clearance of the lysate by centrifugation at 4500g for 20 min. N was purified from clear lysates using AKTA pure protein purification system (GE Healthcare), as reported (30 (link)).