Fig. 3.

Expression of wild type and mutant N proteins. (A) Nucleotide (NT) sequencing of the mutant N proteins, including the T64P (ACA → CCA), T64N (ACA → AAT), and T123V (ACT → GTT) variants of TW IBV TP/64. The altered codons are underlined. (B and C) The full-length N gene in the pET-28 plasmid was transferred into E. coli BL21 (DE3). The N protein expression was induced by adding 1 mM IPTG for 4 h at 25 °C when the OD₆₀₀ value reached 0.4–0.6 (arrow head) (B). The cells were collected and suspended in the lysis buffer (described in Section 2). The crude cell lysates were applied to resin affinity columns and N protein fractions were eluted with a buffer containing 8 M urea. Concentrations of the purified N proteins were determined by the Bradford assay and by 10% SDS-PAGE stained with Coomassie brilliant blue (C). WT, wild type N protein from TW TP/64 strain.