Vivaspin turbo 10 kda centrifugal concentrator

The protocol was adapted from previous reports^{40 ,41} . SurA was overproduced in E. coli BL21(DE3) by growing cells in LB (10 g l⁻¹ NaCl) containing 50 µg ml⁻¹ kanamycin up to an OD₆₀₀ of ~1.0. The temperature was shifted to 16 °C and 0.1 mM (final concentration) IPTG was added and the cells incubated at 16 °C for ~16–18 h. Cells were collected (6,200g, 4 °C, 15 min), resuspended in buffer A (20 mM Tris/HCl, pH 8.0) and disrupted by sonication. The soluble fraction was incubated with 2 ml per l of culture volume of Ni-NTA agarose beads (Qiagen) and rotated overnight at 4 °C on a tube roller. Beads were washed in buffer B (20 mM Tris/HCl, 50 mM imidazole, pH 8.0) and the protein was eluted in buffer C (20 mM Tris/HCl, 500 mM imidazole, pH 8.0). Eluted fractions were dialysed against buffer D (20 mM Tris/HCl, 10% glycerol) overnight at 4 °C, then concentrated to ~5 ml and applied to a Superdex 75 (16/600) column (GE Healthcare), in filtered and degassed buffer D at 1 ml min⁻¹. Eluted fractions were analysed by SDS–PAGE to assess protein purity and yield. Fractions containing SurA were combined and concentrated to 250–300 µM in a Vivaspin Turbo 10 kDa centrifugal concentrator (Sartorius), and stored in aliquots at −80 °C.

An adapted protocol was used^{42 (link)}. OmpT was overproduced as cytoplasmic inclusion bodies in E. coli BL21(DE3) by growing cells in LB (10 g l⁻¹ NaCl) containing 50 µg ml⁻¹ kanamycin up to OD₆₀₀ ~0.5-0.6, adding 1 mM IPTG and incubating for 4 h at 37 °C. Cells were collected (6,200g, 4 °C, 15 min), resuspended in buffer A (50 mM Tris/HCl, 5 mM EDTA, pH 8.0) and disrupted by sonication. The insoluble fraction was collected by centrifugation (4,500g, 4 °C, 15 min) and resuspended in buffer B (50 mM Tris/HCl, 2% Triton X-100, pH 8.0), then incubated for 1 h at room temperature with gentle shaking. Inclusion bodies were pelleted (4,500g, 4 °C, 15 min) and washed twice in buffer C (50 mM Tris/HCl, pH 8.0) by incubating for 1 h at room temperature, then solubilized in buffer D (25 mM Tris/HCl, 6 M guanidine-HCl, pH 8.0). The supernatant was filtered, concentrated to ~5 ml in a Vivaspin Turbo 10 kDa centrifugal concentrator (Sartorius), and applied to a Superdex 75 (26/600) column (GE Healthcare) with filtered and degassed buffer D at 1 ml min⁻¹. Eluted fractions were analysed by SDS–PAGE to assess protein purity and yield. Fractions containing OmpT were combined and stored in aliquots at −80 °C.