Mbr 032p

The 2SY medium was supplemented with 10 g/L proline and 200 mM ArgHCl, and 1 mL of the glycerol stock of the transformed B. choshinesis was added to each well of a 96-well deep-well plate. The plate was covered by a gas permeable seal and incubated at 1000 rpm at 30 °C for 60 h in a plate incubator (MBR-032P, TAITEC). Supernatants were collected after performing a round of centrifugation at 40,000 g for 20 min and then sanitized by filtering. The supernatants were mixed at a 1:1 ratio with 200 mM Tris-HCl and 500 mM NaCl (pH 7.4) and then loaded onto a Ni Sepharose Excel (Cytiva) column. The column was washed with wash buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole (pH 7.4). His₆-tagged RBD was eluted using an elution buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 200 mM imidazole (pH 7.4). The His₆-tag of RBD was cleaved using TEV protease. RBD was further purified using SEC with a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) or HiLoad 16/60 Superdex 75 pg column (GE Healthcare) that was equilibrated with phosphate-buffered saline (PBS). The monomer fraction was then collected. The elution profile of the proteins was monitored at 280 nm.

TM medium that was supplemented with 10 g/L of proline, 200 mM ArgHCl, and 1 mL of glycerol stock of the transformed B. choshinesis was added to each well of a 96-well deep-well plate. The plate covered by a gas permeable seal and incubated at 1000 rpm at 30 °C for 60 h in a plate incubator (MBR-032P, TAITEC). Supernatants were collected after performing a round of centrifugation at 40,000 g for 20 min and then sanitized by filtering. The supernatants were mixed at a 1:1 ratio with 200 mM Tris-HCl and 500 mM NaCl (pH 7.4) and then loaded onto a Ni Sepharose Excel (Cytiva) column. The column was washed with wash buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole (pH 7.4). His₆-tagged C121 Fab was eluted using an elution buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 200 mM imidazole (pH 7.4). His₆-tagged C121 Fab was further purified using SEC with a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) that was equilibrated with PBS. The monomer fraction was then collected. The elution profile of the proteins was monitored at 280 nm.