Purification of Fabs directed to BRIL

Generation and cloning of Fab fragments against the BRIL fusion protein were described in ref. ¹⁸. Fab fragments were expressed in E. coli BL21-CodonPlus (DE3)-RIPL. Cells were cultured in 2x YT media at 37 °C, and the expression was induced with 1 mM IPTG for 4 h at 37 °C when the OD600 reached 0.6–0.8. Cells were lysed by sonication, and the lysate was cleared by centrifugation at 50,000×g for 30 min. The supernatant was passed over 1–2 ml of Protein A Sepharose 4B resin (ThermoFisher), pre-equilibrated in 50 mM HEPES/KOH pH 7.5, 300 mM NaCl. The resin was washed with 10 column volumes of buffer. Fabs were eluted with 0.1 M acetic acid, and fractions were collected dropwise into 1.5-ml tubes containing buffer to bring the final concentration in each tube to 100 mM Tris/HCl pH 8.0, 100 mM NaCl. Fractions containing Fabs were combined, concentrated on Amicon 30 kDa molecular weight cut-off filters, and further purified by SEC on a Superdex 200 column equilibrated in 20 mM HEPES/KOH pH 7.5, 150 mM NaCl. All purification steps were carried out at 4 °C.

To confirm Yop1 is a dimer in detergent, SEC-purified Yop1-BRIL or Yop1(I145R)-BRIL in DDM were incubated with a 2.5-fold molar excess of BRIL Fabs for 1 h at 4 °C, followed by subsequent chromatography on a Superose 6 column equilibrated in 25 mM HEPES/KOH pH 7.4, 100 mM NaCl, 0.03% DDM. The peak fractions were collected and diluted to 10 µg/ml for negative-stain EM analysis.