Superdex s200 26 60 column

ETEC H10407 supernatants were fractionated using a AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Life Sciences, Marlborough, MA, USA) to facilitate molecular characterization of the ESF. Acetone-precipitated supernatants from M9-grown ETEC cultures were resuspended in 10 mL 0.15 M NaCl, clarified by 0.22 µm filtration, and applied at 4 mL/min to a Superdex S200 26/60 column (GE Life Sciences) that had previously been equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl. The ESF-containing eluent was collected from the column between 110–130 mL, and dialyzed against 4 L of 20 mM Tris (pH 8.0) in preparation for further chromatography. The ESF-containing sample was applied to a 1 mL Resource Q anion exchange column (GE Life Sciences). The column was washed with 20 mM Tris (pH 8.0) until the OD₂₈₀ value reach baseline, and then the bound proteins were eluted with a gradient to 1 M NaCl in the same buffer. Fractions of 1 mL were collected and then screened for their ability to prevent IκBα degradation in response to TNF, separated by SDS-PAGE, and detected using Silver Staining (Thermo Scientific, Waltham, MA, USA). Proteins from active fractions were excised and digested in-gel with trypsin (Promega, Madison, WI, USA). Proteins were identified using mass spectrometry at the Mass Spectrometry & Analytical Proteomics Laboratory, University of Kansas.

EP was isolated from Suparen (kindly provided by DSM Food Specialties, Heerlen, the Netherlands) in 0.1 M sodium acetate buffer pH 4.6 as described previously (Köster et al., 2011 ▸ ). The sample was then subjected to size-exclusion chromatography using a Superdex S200 26/60 column (GE) and the same batch of buffer as for isolation. Protein-containing fractions were pooled, concentrated and flash-cooled in liquid nitrogen. The protein was then crystallized in a vapor-diffusion experiment in 48-well format using 250 µl reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 0.1 M ammonium acetate pH 7.0, 24–33%(w/v) PEG 4000. 1.5 µl protein solution at a concentration of 5 mg ml⁻¹ was mixed with an equal amount of reservoir solution. Trays were incubated at 20°C. Crystals appeared after 5–6 days and were then crushed using a seed-bead kit (Douglas Instruments) to prepare crystal seeds, which were then used in a second crystallization experiment, here using 27%(w/v) PEG 4000 in the reservoir and adding 0.1 µl of seed dilutions of 1:15–1:45 (seed stock:reservoir) to the freshly mixed drop of protein and reservoir. The seeded crystals appeared after three days.