Fig. 5

Confirmation of interactions between truncated N and Nsp3 proteins using purified proteins. The aa 2–111 (A) and aa 2–180 of Nsp3.1 (B) and truncated N proteins aa 2–419 (A), aa 2–365 (D), aa 2–254 (E), aa 43–419 (G) and aa 43–365 (H) were fused with glutathione S-transferase (GST) and 6xHis tag at the N-terminus, respectively. The aa 2–111 and aa 2–180 of Nsp3.1 and various truncated N proteins were co-expressed in Escherichia coli BL21 (DE3). The cell lysates were mixed with glutathione Sepharose-4B beads, washed and eluted with lysis buffer containing 15 mM reduced glutathione. The elutes were digested with PreScission protease, and undigested proteins were cleaned with glutathione Sepharose-4B beads. A The interaction between GST-Nsp3.1 (aa 2–111) and His-N (aa 2–419) was verified using GST-pull down assay. B The interaction affinity of aa 2–180 of Nsp3.1 and aa 2–419 of N protein was quantified using MST. The proteins without tags were analysed by gel filtration, and the fractions around the peak related to the protein complex were examined using SDS-PAGE and coomassie blue staining (C–E, G and H). F Schematic diagram of PI values of various N regions generated using Expasy ProtParam tool