Periplasmic Protein Extraction from E. coli

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.