Human recombinant Hsp70 was purified from Escherichia coli cells transformed with pMSHsp70 plasmid and detoxified with the use of polymyxin B–agarose gel (Sigma, St.Loise, MO, USA) as described elsewhere [10 (link)].
Recombinant human HMGB1 was isolated from E. coli BL21 (DE3) Star™, transformed with pET24a(+)-T7-HMGB1-His tag plasmid. The induction of HMGB1 expression was performed by the addition of 0.1mM IPTG to LB growth media followed by agitation at 200 rpm at 25 °C for 4 h. The cells were collected by centrifugation at 3000 rpm for 20 min at 4 °C and then lysed using an ultrasonic system in lysozyme-containing buffer. HMGB1 was then purified using HisPur ™ Ni-NTA Resin (Thermo Scientific) according to the manufacturer`s protocol.
For PPI and pull-down assay Hsp70 and HMGB1 were biotinylated with EZ—Link® Sulfo-LC-Biotin (Thermo Scientific, USA) in accordance with the manufacturer’s instruction.
HBHP and HBHP-scr peptides were synthesized with biotin on N-terminus by NPF Verta (Saint Petersburg, Russia).
Recombinant human HMGB1 was isolated from E. coli BL21 (DE3) Star™, transformed with pET24a(+)-T7-HMGB1-His tag plasmid. The induction of HMGB1 expression was performed by the addition of 0.1mM IPTG to LB growth media followed by agitation at 200 rpm at 25 °C for 4 h. The cells were collected by centrifugation at 3000 rpm for 20 min at 4 °C and then lysed using an ultrasonic system in lysozyme-containing buffer. HMGB1 was then purified using HisPur ™ Ni-NTA Resin (Thermo Scientific) according to the manufacturer`s protocol.
For PPI and pull-down assay Hsp70 and HMGB1 were biotinylated with EZ—Link® Sulfo-LC-Biotin (Thermo Scientific, USA) in accordance with the manufacturer’s instruction.
HBHP and HBHP-scr peptides were synthesized with biotin on N-terminus by NPF Verta (Saint Petersburg, Russia).
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