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).