Prset b his vector

Recombinant TRAIL proteins were purified as described previously.^{38 (link)} In brief, the coding portion of the extracellular portion of the TRAIL molecule (amino acids 95–281) was subcloned into a pRSET(B)-His vector (Invitrogen, Groningen, the Netherlands) and expressed in an Escherichia coli expression system. The His-TRAIL fusion protein was purified by metal chelate column chromatography using Ni-NTA resin, according to the manufacturer’s recommendations (Qiagen, Hilden, Germany) and dialyzed. Lipopolysaccharide endotoxin was further removed from the purified TRAIL using an Acrodisc syringe filter (Pall, Port Washington, NY) and reached the targeted endotoxin level of <0.1 EU/ml as determined by a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA).

Recombinant TRAIL proteins were purified as described previously (27 (link)) and used at 100 μg/mouse i.p. for EAE treatment. For self-purified TRAIL, in brief, the coding portion of the extracellular portion of the TRAIL molecule (amino acids 95–281) was subcloned into a pRSET(B)-His vector (Invitrogen, Groningen, the Netherlands) and expressed in an Escherichia coli expression system. The His-TRAIL fusion protein was purified by metal chelate column chromatography using Ni-NTA resin, according to the manufacturer’s recommendations (Qiagen, Hilden, Germany) and dialyzed. Lipopolysaccharide endotoxin was further removed from the purified TRAIL using an Acrodisc syringe filter (Pall, NY, USA) and reached the targeted endotoxin level of < 0.1 EU/ml as determined by a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific). For in vitro assays, TRAIL was purchased from Genescript (Piscataway, NJ, USA) used at a concentration of 10 µg/ml.