Recombinant HMGB1 Protein Expression and Purification

Human HMGB1 cDNA was cloned into the pETM-11 vector and expressed in Escherichia coli strain BL21 (DE3) cells. The protein expressed a 6-residue N-terminal histidine tag with a tobacco etch virus (TEV) cleavable linker and was purified by FPLC using Ni-sepharose affinity chromatography (HisTrap HP, GE Healthcare, Uppsala, Sweden) in an ÄKTA explorer (GE Healthcare). The histidine tag was cleaved using TEV protease (Sigma-Aldrich, Stockholm, Sweden) at a ratio of 1:20. Proteolytic TEV cleavage leaves a GA scar at the N-terminal. Endotoxins were removed using Triton-X114 two phase extraction. Protein purity was confirmed using SDS-PAGE gel electrophoresis analysis (21 (link)).