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.