2.2. Expression and Purification of Recombinant 3AB Protein

The FMDV 3AB gene (O/Boeun/SKR/2017) was synthesized by Bioneer Corp. (Daejeon, Korea). The 3AB DNA fragments, (i) full-length (672 bp) and (ii) truncated (432 bp) were amplified and cloned into the pET28a vector. The recombinant plasmids were transformed into Escherichia coli (E. coli) BL21 (DE3) strain, and cells were cultured in Luria-Bertani medium containing 50 μg/mL kanamycin at 37 °C until the optical density at 600 nm reached 0.6. The expression of recombinant proteins was induced by adding 1 mM isopropyl-β-d-1-thiogalactopyranoside at 16 °C for 4 h. Pellets were harvested from the culture by centrifugation at 3000× g for 10 min, resuspended in lysis buffer [50 mM Na₂HPO₄, 300 mM NaCl, pH 8.0], and lysozyme was added to a final concentration of 1 mg/mL. After incubation on ice for 30 min, the cells were lysed by sonication (20 cycles of pulsed on-time 5 s and off-time 10 s at 20% amplitude). Cell lysates were centrifuged at 10,000× g, 4 °C for 30 min, and supernatants were mixed with nickel-nitrilotriacetic acid resin (Qiagen, Germantown, MD, USA) by inverting at 4 °C for 1 h. The mixture was loaded onto an Econo-Pac column (Bio-Rad, Hercules, CA, USA) and passed through gravity flow. The column was washed twice with wash buffer [50 mM Na₂HPO₄, 300 mM NaCl, and 10 mM imidazole, pH 8.0], and the recombinant 3AB protein was then eluted with elution buffer [50 mM Na₂HPO₄, 300 mM NaCl, and 250 mM imidazole, pH 8.0]. The eluted fractions were pooled and dialyzed against dialysis buffer [50 mM Tris-HCl and 150 mM NaCl, pH 7.6] using a Slide-A-Lyzer membrane cassette (Thermo Fisher Scientific) with a molecular weight cut-off of 10 kDa.