2.3 Recombinant allergen production
For recombinant protein production, the protein sequences of Api m 1.0101, Lol p 1.0101 and Lol p 5.0101 were obtained from the Allergen Nomenclature website (allergen.org).32,33 All three constructs were generated with the Api m 1 N-terminal leader sequence for secretion, as well as a 6-His tag for purification and a BirA tag for biotinylation (Figure 1A ). To prevent unwanted effects of catalytic activity, mutations were introduced in Api m 1 (H67Q) and Lol p 1 (H104V), as published previously.34,35 All constructs were codon-optimized for Spodoptera frugiperda and cloned into the pFastBac vector (Thermo Fisher Scientific), prior to incorporation into a Bacmid for baculovirus production. Bacmids were transfected into Sf21 cells, which were subsequently cultured at 27oC. Supernatants from infected Sf21 cultures were clarified by centrifugation and the 6-His tagged proteins were purified through retention on a cobalt column. Supernatants were gravity-fed through a 25 ml column packed with 4 ml Talon NTA-cobalt-agarose beads (Clontech, Mountain View, CA, US). Beads were washed with PBS and allergens eluted with PBS, pH 8.5, containing 200 mM imidazole. Eluate was dialyzed against 10 mM TRIS, pH 7.5. The purified recombinant proteins were incubated overnight at RT with BirA enzyme for targeted biotinylation (2.5 µg/ml BirA in 10 mM TRIS containing 62.5 mM Bicine-HCl, 12.5 mM ATP, 12.5 mM MgOAc, 62.5 µM D-biotin). Following subsequent dialysis against PBS, tetramerization was performed with fluorochrome-conjugated streptavidin (PE, APC and BV711 conjugates; all from BD Biosciences, San Jose, CA, US) at a 4:1 molar ratio of allergen:streptavidin.