Bl21 de3 plyse

For the recombinant HMGB1 protein, six-His-tagged recombinant human WT HMGB1, HMGB1 boxes A (aa 1-79) and B (aa 88-162), and acidic tail-deleted HMGB1 (ΔC-HMGB1, aa 1-185) were subcloned into pRSET B plasmid and produced in Escherichia coli BL21 (DE3) pLysE (Invitrogen) (4 (link), 16 (link)). A six-His-tagged HMGB1 (ΔN-HMGB1, aa 11-215), a form of HMGB1 with pro-inflammatory potential (29 (link)), was subcloned into pRSET B plasmid and produced in E. coli BL21 (DE3) pLysE. One mM DTT was added during protein purification and preservation. Endotoxin was removed using an LPS-binding column (Thermo Fisher Scientific, Inc.) or detergent-phase separation using Triton X-114 (30 (link)). LPS concentrations were less than 0.1 EU/μg protein, as determined using the limulus amebocyte lysate assay (Sigma). In addition, HMGB1 produced in NS0 mouse myeloma cell line (Euk-HMGB1, R&D Systems) was used to confirm the study.

EndoE, EndoE(E186Q) and EndoE(E662Q) were recombinantly expressed in E. coli as previously described using the GST gene fusion system from GE Healthcare [22] . Recombinant expression of the enolase (EF1961) was performed using the same protocol. Briefly, a 1299 bp fragment of EF1961 was amplified using the following primers: 5′-CGGGATCCACATGTCAATTATTACTGATA-3′ with a BamHI site (underlined) and 5′-CCCTCGAGTTATTTGTTTTTTAAGTTG-3′ with an XhoI site (underlined). The PCR fragment was cloned into the pGEX-5x-3 vector (GE Healthcare) and transformed into the E. coli BL21 (DE3) pLysE (Invitrogen) strain for expression of EF1961.