Protein purification

Small scale purification

30 ml of cells after induction were collected by centrifugation and resuspended in 400 µl buffer (25 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 7.5). The suspension was sonicated for 2 minutes (45% ampl, 6s On, 3s Off) and the lysate was centrifuged for 30 minutes in a table centrifuge at 13.6 rpm. Proteins were purified using Thermo Scientific™ HisPur™ Ni-NTA Spin Columns according to manufacturer's instructions (wash buffer: 25 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH 7.5; elution buffer: 25 mM HEPES, 300 mM NaCl, 300 mM I imidazole, pH 7.5). Eluted protein solutions were dialyzed using Thermo Scientific™ Slide-A-Lyzer™ MINI Dialysis Device, 7K and 10K MWCO, 0.1 mL (25 mM HEPES, 100 mM NaCl, 50% glycerol, 2 mM dithiothreitol, pH 7.5). Protein concentrations were measured spectrophotometrically (NanoDrop 1000) and target protein fractions in the samples were evaluated densitometrically from the gel using ImageJ program.

Large scale purification

Cells after induction were collected by centrifugation and resuspended in 5 ml of buffer per 1g of dry cells (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, pH 8). The suspension was sonicated for 1 minute for every 1 g oc cells (45% ampl, 6s On, 3s Off) and lysate was centrifuged for 60 minutes at 13.6 rpm. Proteins were subjected for Ni ions affinity chromatography on Cytiva 1 ml Histrap FF column. The column was washed with 15 ml of wash buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 5 mM 2-Mercaptoethanol, pH 8) and eluted with buffer gradient 3%-100% over 10 ml (elution buffer: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, 5 mM 2-Mercaptoethanol, pH 8). Eluted protein solutions were dialyzed using 10k MWCO dialysis membrane against dialysis buffer with of without glycerol (25 mM HEPES, 100 mM NaCl, 50% glycerol, 2 mM dithiothreitol, pH 7.5). Protein concentrations were measured spectrophotometrically (NanoDrop 1000) and target protein fractions in the samples were evaluated densitometrically from the gel using ImageJ program.