Ni sepharose hp

The truncated M protein (NCBI registration number PP378623) and recombinant N protein (NCBI registration number PP378624) were produced according to the M and N genes of the PRRSV CH-1R strain (NCBI registration numbers AY032626: ORF6 and ORF7) in GenBank. The truncated and recombinant gene sequences were sent to Qingke Biological Co., Ltd. (Xi’an, China) for synthesis (Figure S1). Recombinant plasmids pET28a-SUMO-M and pET28a-N were transformed into competent Escherichia coli BL21 cells to obtain recombinant Escherichia coli. Transformed cells were plated on Luria–Bertani (LB) plates supplemented with 100 mg/L kanamycin. After obtaining single colonies, positive clones were grown in LB broth supplemented with 100 mg/L kanamycin and 1 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG; TakaraBio, Shiga, Japan) at 16 °C with strong shaking for 8 h to induce recombinant protein expression. Cells were harvested and sonicated after the induction period. The truncated M protein and recombinant N protein were purified with a commercial protein purification product (Ni Sepharose HP, GE) and assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using PRRSV-positive serum. The proteins were then divided into 1 mg/mL aliquots-and stored at − 70 °C.

Human sAC protein used in all assays was purified from Sf9 cells via baculovirus expression. Biochemical potency and jump dilution assays used N-terminal tagged GST-sAC_t (Litvin et al., 2003 (link)); SPR used highly pure C-terminal tagged sAC_t-His6 that was prepared via sequential chromatography that included, in this order, affinity (Ni-Sepharose HP from GE Healthcare), ion exchange (Mono Q from GE Healthcare), and size exclusion (Superdex 200 from GE Healthcare) chromatographies. The synthesis of TDI-10229 is described in Fushimi et al., 2021 (link) and the synthesis of other compounds is described in Miller et al., 2022 .