Chelating sepharose fast flow column

The E. coli W3110 TatExpress cell line (37 (link)) was transformed with the pEXT22 vector containing TorA–BT6, TorA–BT6M1, or TorA–BT6M0 (Table S1). 500-ml cultures were grown in 2-liter Erlenmeyer flasks at 30 °C with 220 rpm agitation. At an A₆₀₀ of ∼0.6, protein production was induced with 0.5 mm IPTG and cultures were incubated for 24 h, after which cells were harvested by centrifugation (3,900 × g, 30 min, 4 °C). To obtain periplasmic fractions, cells were resuspended in 10 ml of chilled buffer F. 10 ml of chilled milliQH₂O was added followed by 800 μl of 1 mg ml⁻¹ of lysozyme and samples were incubated on ice for 10 min. 800 μl of 1 m MgSO₄ was added and the solution was centrifuged (16,600 × g, 30 min, 4 °C) with the supernatant collected as the periplasm.
Periplasmic fractions were applied to a Chelating Sepharose Fast Flow column (GE Healthcare) pre-equilibrated with 10 mg ml⁻¹ of nickel sulfate. The column was washed with 20 ml of buffer A and 20 ml of buffer G (50 mm HEPES, pH 7.4, 500 mm NaCl, 50 mm imidazole). Protein was eluted with 10 ml of buffer H (50 mm HEPES, pH 7.4, 100 mm NaCl, 400 mm imidazole) and the elution fractions were collected.

Over expression of CbiX and its variants was performed in the E. coli strain BL21 codonplus (RIPL) (Stratagene). All cells expressing protein were grown at 37°C in 500 ml volumes of Luria–Bertani (LB) broth in 2 l flasks from overnight starter cultures to an OD₆₀₀ of 0.8–1.0 and transferred to 18°C before induction with 0.1 mM IPTG. After further incubation for 16 h the cells from 2 l culture were harvested and re-suspended in 25 ml of Buffer A (50 mM Tris-HCl, 200 mM NaCl, pH 7.5), containing a trace amount of DNaseI and EDTA-free protease inhibitor tablet (Roche). The total cell lysate were obtained by sonication on ice for 5 min and removing the insoluble material by centrifuging at 15 000 g for 40 min. The total cell lysate was applied to 5 ml of chelating sepharose fast flow column (5 ml) (GE, Healthcare), previously charged with NiCl₂ and equilibrated with Buffer A. The column was first washed with 3 column volumes of Buffer A followed by washes with 10 column volumes of 50 mM Tris-HCl, 200 mM NaCl and 25 mM imidazole (pH 7.5), and the protein was eluted with 50 mM Tris-HCl (pH 8.0), 400 mM imidazole, according to the manufacturer's instructions. All the CbiX variants were also produced in the same manner. Purity of the samples was checked by running SDS-PAGE 10% Bis-Tris NuPAGE gels (Invitrogen).