Characterization of the Dog Lipocalin Allergen Can f 6

Abstract

Allergies to dogs are a critically important clinical problem that affect between 10%-30% of humans worldwide and continue to increase. Four of the seven currently named dog component allergens (Can f 1, Can f 2, Can f 4, and Can f 6) are lipocalins. Sensitization to Can f 6 is linked to the highest likelihood of allergic symptoms. Accurate biochemical and biophysical data for each is limited. The cDNA encoding 6xHis-tagged Can f 6 was cloned, expressed in bacteria, and purified using a HisPur Ni-NTA Superflow Agarose nitrilotriacetic acid (NTA) column, followed by fast protein liquid chromatography (FPLC) size exclusion column, and then pooling fractions of the appropriate molecular weight. Initial crystallization trials were performed at room temperature by hanging drop vapor diffusion. Data were collected on a cryoprotected crystal on the 24-ID-C Synchrotron at the Advanced Photon Source at Argonne National Laboratory, processed, solved, and refined using Phaser and the CCP4 program suite. The purified recombinant Can f 6 protein has been crystalized. The resulting high-resolution x-ray diffraction data was used to solve the tertiary structure of the protein. This is the third dog lipocalin allergen whose structure has been characterized. This Can f 6 structure can now be used to identify potential sites of cross reactivity with other lipocalins and define allergenic epitopes that can be therapeutic targets.