Purification of Hexahistidine-Tagged Proteins

Purification of hexahistidine‐tagged proteins was performed by standard immobilized metal affinity chromatography using HisPur Cobalt resin (Thermo Scientific) under native conditions. For 3 mL cultures from 24 DWP, IMAC was performed using 0.2 mL resin in small gravity feed columns. The resin was washed with 2 × 2 mL of water and equilibrated with 2 × 2 mL of 50 mM phosphate buffer (pH 7.4). Cell lysates on 24 DWP were cleared by centrifugation (3220g, 20 min, 4°C) and loaded onto the columns. The columns were rinsed with 2 mL of 50 mM phosphate buffer (pH 7.4), washed with 4 × 2 mL of wash buffer (50 mM sodium phosphate, 10 mM imidazole, 300 mM sodium chloride; pH 7.4), and then rinsed with 2 mL of 50 mM sodium phosphate (pH 7.4) before elution with 3 × 0.2 mL of 50 mM sodium phosphate, 50 mM EDTA (pH 7.4). For 10 mL cultures, the same protocol was used with the following changes: medium samples were 1:2 diluted (total volume 10 mL), periplasmic and cytoplasmic fractions were diluted in 2.5 mL of 200 mM sodium phosphate buffer and made up to 10 ml with water to reduce the salt concentration. Samples were prepared for SDS‐PAGE analysis and 10 μL were loaded in 4–20% Criterion™ TGX™ Precast Midi Protein Gel, 26 well (BioRad).
For the detection of proteins by WB analysis the method as detailed in Guerrero‐Montero, Dolata, et al. (2019 (link)) was performed, with the exception that was transferred to the polyvinylidene fluoride‐membrane (GE Healthcare) by rapid semi‐dry transfer using the Invitrogen Power Blotter XL System according to the manufacturer's instructions.