2.1∣Expression and purification of the recombinant For t 2 protein from E. coli for sensitization of mice
E.coli -expressed For t 2 recombinant protein (rFor t 2) was purified by rapid affinity column chromatography as previously described.22 After using a His-tag affinity column, rFor t 2 was further purified using Endotoxin Detoxi-Gel (Pierce, Rockford, IL, USA) and sterilized with a 0.22 μm syringe filter (Millipore, Billerica, MA, USA). The endotoxin content was determined using an E-TOXATE kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Finally, the protein concentration was determined by the Coomassie brilliant G-250 protein-dye binding method of Bradford with bovine serum albumin as a standard according to manufacturer’s procedures (Bio-Rad, Hercules, CA, USA).