Expression and purification of recombinant HMGB1 and HMGB1-M

The pET28a-HMGB1 and pET28a-HMGB1-M plasmids (50 ng) were transformed into 100 µl protease-deficient Escherichia coli, strain BL21 (Beijing Solarbio Science & Technology Co., Ltd.) at 42 °C for 90 sec, according to the DH5α transformation protocol, The bacteria were incubated in ZYP-5052 medium (Amresco, LLC, Solon, OH, USA) containing kanamycin (50 µg/ml) overnight at 37°C with vigorous agitation. The bacterial solution was centrifuged at 4°C, 8,000 × g for 10 min. Then, cell lysis and purification of the recombinant protein were performed with 1 ml HisTrap FF Crude columns (GE Healthcare Life Sciences, Little Chalfont, UK), according to the manufacturer's protocols. The affinity-purified recombinant proteins were further desalted using Zeba Spin Desalting Columns (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The recombinant proteins were further purified by filtration and stored in aliquots at −80°C until use. Contaminating lipolysaccharide (LPS from protein preparations) was detected by a Pierce LAL Chromogenic Endotoxin Quantitation kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The LPS content in purified HMGB1 and HMGB1-M samples was 1.31 and 1.11 pg/µl, respectively. For all DC or RAW264.7 stimulation experiments with the recombinant proteins, polymyxin B (Sigma-Aldrich; Merck KGaA) was added to the cell culture medium at 6 units of polymyxin B per picogram of LPS. Proteins were detected by Coomassie blue staining at room temperature overnight following SDS-PAGE gel and protein purity was determined using BandScan software version 5.0 (Glyko, Novato, CA, USA).