Detoxi gel endotoxin removing column

The expression and purification of wild-type α-syn was performed as detailed previously [7 (link)]. Bacterial endotoxins were removed using a detoxi-gel endotoxin removing Columns (ThermoFisher scientific) according to manufacturer’s instruction. Levels in endotoxin-free α-syn were recorded as 0.006 ng/ml (stomacher 80 biomaster seward) in α-syn preparations (autoclaved endotoxin-free plastics and endotoxin-free water were used at all stages of preparations). Protein was then aliquoted into separate eppendorfs into filtered PBS (0.02 μM filter, Whatman) flash-frozen, stored at − 80 °C and thawed once before use. Clusterin was obtained as previously described [11 (link), 50 (link)].

Human Gal-1C2S, a Cys²-to-Ser-substituted form of the wild type, which is thought to be resistant to the oxidation-dependent inactivation of its carbohydrate affinity without affecting carbohydrate-binding ability [8 (link)] and mouse Gal-1C2S were expressed in Escherichia coli and affinity-purified using an asialofetuin-immobilized Sepharose column, as described previously [21 (link)]. For the mouse Gal-1 (mGal-1) wild-type-expressing plasmid, an artificial gene encoding the mGal-1 protein with optimized codon usage was synthesized by Fasmac (Kanagawa, Japan) and subcloned into the NdeI and BamHI sites of the pET21a vector. The resultant pET-mGal-1WT plasmid was used for protein expression as described above. Contaminated lipopolysaccharide was removed from the affinity-purified protein dissolved in phosphate-buffered saline (PBS; 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, and 2.68 mM KCl; pH 7.4) using Detoxi-Gel Endotoxin Removing Columns (Thermo Fisher Scientific, Waltham, MA, USA), and the removal was checked using Limulus ES-2 Single Test Wako 0.03 (Fujifilm Wako), as described previously [22 ]. Heat-inactivation of recombinant Gal-1 protein was performed by boiling it for 5 min.

Recombinant ClfA N123 (amino acids 40 to 559) (30 (link)) and ClfA N23 (amino acids 220 to 559) proteins (30 (link)), SdrC (amino acids 182 to 496) (31 (link)), and ClfB (amino acids 44 to 542) (32 (link)) were cloned and expressed from genomic DNA of S. aureus strain Newman and purified from Escherichia coli by Ni²⁺ affinity chromatography, as previously described (33 (link)). GST-tagged ClfA N1 (amino acids 40 to 228) was purified on a GSTrap FF purification column (GE Healthcare). Endotoxin was removed from all the proteins using Detoxi-Gel endotoxin-removing columns (Thermo Scientific).