Purification and Loading of CD1a Glycoprotein

The glycoprotein CD1a was expressed in a mammalian expression system and purified as previously described^{9 (link)}. Following an endoglycosidase H (New England BioLabs) treatment, the purified CD1a was first loaded with the ganglioside GD₃ (GD₃) (Matreya LLC) that was dissolved in a solution containing 0.5% tyloxapol (Sigma) and 10 mM Tris buffer at pH 8.0. CD1a was first incubated with GD₃ overnight at room temperature at a molar ratio of 1:15. The CD1a sample loaded with GD₃ was further purified using ion exchange chromatography (MonoQ 10/100 GL-GE Healthcare). Urushiol C15:2 (Chemos) was dissolved in a solution containing 10 mM Tris buffer at pH 8.0 / 0.5% tyloxapol / 50% acetone (Sigma). The GD₃–CD1a sample was then incubated overnight with urushiol at a 1:15 molar ratio and at room temperature in order to achieve the GD₃ displacement by urushiol. A subsequent purification step involving ion exchange chromatography (MonoQ 10/100 GL) was performed to remove the excess of urushiol C15:2, GD₃ –CD1a, and detergent.