S100 gel filtration column

All samples were overexpressed using Escherichia coli BL21(DE3^*) cells grown in either Luria-Bertani media (EMD Millipore, Billerica, MA, USA) or M9 minimal media for NMR experiments. Samples for backbone relaxation dispersion were grown in ²H₂O-based media with ¹⁵N-labeled ammonium chloride (Cambridge Isotopes, Tewksbury, MA, USA).
All samples were purified using an anion-exchange column Q-sepharose (GE Healthcare, Pittsburgh, PA, USA) with buffers A (25 mM HEPES, pH 7.5, 1 mM Na₂EDTA) and B (buffer A with 1 M NaCl) using a 0–50% gradient of buffer B. Samples were concentrated with a Corning Spin-X UF centrifugal concentrator (Sigma Aldrich, St. Louis, MO, USA) to a volume of ~1 mL then purified on a S100 gel filtration column (GE Healthcare) in buffer A with 200 mM NaCl.

The expression vector was transformed into E. coli BL21 (DE3) cells (Novagen). For large scaled protein expression, cultures were grown in LB medium of 0.8 liter at 37°C for 4 h, induced with 0.4 mM isopropyl-β-_D-thiogalactopyranoside, and incubated overnight at 20°C. After centrifuging at 6,000 x g at 4°C for 15 min, the cell pellets were resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, and 2 mM β-mercaptoethanol) and then lysed by sonication. The crude extract was then centrifuged at 12,000 x g at 4°C for 25 min to remove the insoluble pellet. The supernatant was incubated with 1-ml Ni-NTA beads at 4°C for 1 h and then loaded into an empty column. After allowing the supernatant to flow through, the beads were washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 mM imidazole, and 2 mM β-mercaptoethanol), and the protein was eluted with elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, and 2 mM β-mercaptoethanol). The protein was then loaded onto a S-100 gel-filtration column (GE Healthcare) equilibrated with running buffer (20 mM Tris, pH 8.5, 100 mM NaCl, and 2 mM dithiothreitol). The purity of the fractions collected was analyzed by SDS-PAGE and the protein was concentrated to 30 mg/ml by Amicon Ultra-4 10-kDa centrifugal filter (Millipore).