Ni sepharose high performance affinity media

ThDP, NAD⁺, CoA, DTT, isopropyl 1-thio-β-D-galactopyranoside, imidazole, thiamin-HCl, glycerol, DNase I, and Micrococcal nuclease were from Affymetrix (Thermo Fisher Scientific, Waltham, MA, USA); 2-oxoglutaric acid, 2-oxoadipic acid, iodoacetamide, benzamidine HCl, disuccinimidyl dibutyric urea (DSBU or BuUrBu), 1.1′-carbonyl-diimidazole (CDI), and dimethyl sulfoxide were from Sigma-Aldrich (St. Louis, MO, USA). Formic acid solution was obtained from Fluka Analytical (Buchs, Switzerland); MS grade trypsin protease was from Pierce; Ni Sepharose^® high-performance affinity media was from GE Healthcare (Chicago, IL, USA).

All purification steps were performed at room temperature as described previously [17 (link)]. His-tag affinity spin columns (His SpinTrap; GE Healthcare BioSciences, Pittsburgh, PA, USA) were used to purify the protein. The column was first equilibrated with binding buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.5). Cell extracts (in 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.5) were applied to the column, and then the column was washed twice with wash buffer (50 mM sodium phosphate, 500 mM NaCl, 50 mM imidazole, 20% ethanol, pH 7.5). The His-tagged protein was eluted with elution buffer (50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5).
For large volume purification, Ni Sepharose high performance affinity media (GE Healthcare BioSciences, Pittsburgh, PA, USA) and Glass Econo-Column^® Columns 2.5 × 10 cm (BioRad, Hercules, CA, USA) were used.

Recombinant rat histidine-tagged HMGB1 cDNA was cloned into pET28a vector (Clontech, Mountain View, CA, USA) as previously published [38 (link)] and transformed into BL21 (DE3) strain (Stratagene, Santa Clara, CA, USA). The protein, with 99 % homology to human HMGB1, was purified with Ni Sepharose High Performance affinity media (GE Healthcare, Chalfont St. Giles, UK) according to the protocol supplied by the manufacturer, followed by gel filtration on a Superdex 75 column (GE Healthcare) with phosphate-buffered saline (PBS), pH 7.4, as running buffer. Endotoxin was removed by addition of 1 % Triton X-114 and incubation at 4 °C for 30 minutes, followed by incubation in a 37 °C water bath for 10 minutes, and subsequently centrifugation at 18,300 g/25 °C for 10 minutes. This procedure was repeated once and yielded endotoxin levels below 0.003 EU/μg protein, according to the Limulus amoebocyte lysate assay (analyzed by the clinical laboratory at Karolinska University Hospital, Stockholm, Sweden). The protein preparation was also free from DNA as evaluated by agarose gel electrophoresis and staining for DNA with GelRed (Biotium, Hayward, CA, USA).