E. coli Tuner (DE3)/pET25b_phl cells were grown in standard LB broth low-salt medium (ForMedium, UK) containing 100 μM ampicillin at 37°C until the OD600 reached ~ 0.5. After induction with 0.2 mM isopropyl ß-d -1-thiogalactopyranoside (ForMedium, UK), cells were cultured for an additional 20 hours at 18°C, harvested by centrifugation at 12,000 g for 10 min and resuspended in buffer A (300 mM NaCl, 20 mM Tris/HCl, pH 7.5). Harvested cells were stored at -20°C prior to protein purification.
Cells were disrupted by sonication (VCX 500, Sonics & Materials, Inc., USA) and the soluble fraction was collected by centrifugation at 21,000 g at 4°C for 1 hour and filtrated through a 0.45 μm pore size filter (Carl Roth, Germany). Recombinant protein PHL was purified by affinity chromatography on mannose-agarose resin (Sigma-Aldrich, USA) equilibrated with buffer A using an ÄKTA FPLC system (GE Healthcare, UK). The protein was eluted isocratically. Protein purity was assessed by SDS-PAGE (12% gel) stained with Coomassie Brilliant Blue R-250/G-250 (Sigma-Aldrich, USA). The fractions containing pure PHL were extensively dialyzed against an appropriate buffer and used for further studies. If desired, PHL was concentrated using an ultrafiltration unit with a 10-kDa cut-off membrane (Vivaspin 20, Sartorius, Germany).
Cells were disrupted by sonication (VCX 500, Sonics & Materials, Inc., USA) and the soluble fraction was collected by centrifugation at 21,000 g at 4°C for 1 hour and filtrated through a 0.45 μm pore size filter (Carl Roth, Germany). Recombinant protein PHL was purified by affinity chromatography on mannose-agarose resin (Sigma-Aldrich, USA) equilibrated with buffer A using an ÄKTA FPLC system (GE Healthcare, UK). The protein was eluted isocratically. Protein purity was assessed by SDS-PAGE (12% gel) stained with Coomassie Brilliant Blue R-250/G-250 (Sigma-Aldrich, USA). The fractions containing pure PHL were extensively dialyzed against an appropriate buffer and used for further studies. If desired, PHL was concentrated using an ultrafiltration unit with a 10-kDa cut-off membrane (Vivaspin 20, Sartorius, Germany).