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 [56 (link)] The concentration of bacterial lipopolysaccharide in the final preparation was lower than 0.1 MU/mL, according to LAL test (E-toxate, Sigma, St. Louis, USA); this value is much lower than one that can have any endotoxic effect.
Polymyxin b agarose gel
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Polymyxin B–agarose gel is a laboratory product used for the purification and isolation of specific biomolecules. It consists of agarose beads covalently linked to the antimicrobial compound polymyxin B. This product can be used in affinity chromatography techniques to capture and separate target analytes from complex mixtures.
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4 protocols using polymyxin b agarose gel
1
Recombinant Hsp70 Purification and Endotoxin Removal
2
Preparation and Purification of Recombinant Hsp70 and HMGB1
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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).
3
Recombinant Human Hsp70 Purification
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Recombinant human Hsp70 was obtained from E. coli transformed with a pMSHSP plasmid75 (link). Briefly, chaperone was purified with anion exchange chromatography employing DEAE-Sepharose (GE Healthcare, USA) with subsequent ATP-affinity chromatography using ATP-Agarose (Sigma-Aldrich, USA). Bacterial lipopolysaccharides (LPS) were depleted employing Polymyxin B-Agarose gel (Sigma) with the endotoxin content below 0.1 EU/mg Hsp70.
4
Purification and Labeling of Recombinant Hsp70
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Recombinant human Hsp70 was purified from bacteria transformed with a pMSHsp70 plasmid, as described elsewhere [43 (link)]. Hsp70 solution was further cleaned by incubation with Polymyxin B-agarose gel (Sigma-Aldrich, USA) and sterilized by filtration through a 0.2 µm filter (Millipore). According to the E-Toxate assay (Sigma-Aldrich, USA), the level of lipopolysaccharide in the final Hsp70 preparation was lower than 0.1 MU/ml. For microscopy Hsp70 was conjugated to Alexa555 dye (Invitrogen) according to the manufacturer's protocol. For biochemical experiments, Hsp70 was biotinylated using succinimide-NHS-biotin (Sigma-Aldrich, USA). The control protein, bovine serum albumin (BSA), was labelled as above. In some experiments Hsp70 was incubated with a specific RSIII polyclonal antibody generated in our laboratory. To understand which domain of Hsp70 is responsible for its intracellular transport we incubated Hsp70 before introducing with C-terminal binder PES (25mM, Sigma) or N-terminal binder MKT077 (25 mM, Sigma).
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