none

47 kDa (Pastorello et al. 2002) [671]
48 kDa (Lauer et al. 2004) [1117]

Freeze-dried hazelnut extract was dissolved to a protein concentration of 4 mg/ml and dialysed against starting buffer (50 mM Tris/HCl, pH 8). After filtration through a 0.45 µm filter (Sartorius, Göttingen, Germany) the protein solution was applied to a 20 ml HiPrep 16/10 DEAE FF column (Amersham Pharmacia Biotech, Uppsala, Sweden). Bound proteins were eluted with a linear salt gradient (50 mM Tris/HCl, pH 8, 1 M NaCl) at a flow rate of 5 ml/min. Further purification of the eluted fractions was performed by concanavalin A affinity chromatography (Amersham Pharmacia Biotech) in 20 mM Tris/HCl (pH 7.3)/0.5 M NaCl. Elution was carried out with alpha-D-methyl-mannoside (10, 25, 50 and 100 mM) (SigmaAldrich, Steinheim, Germany). Fractions (0.5 ml) were analysed by SDS/PAGE and immunoblotting and the protein was found to be approximately 98% pure (Lauer et al. 2004) [1117].

Recombinant Cor a 11 was expressed using a pET100D construct in E. coli BL21 Star cells (Invitrogen). Protein synthesis was induced with 1 mM isopropyl beta-D-thiogalactoside for 5 h at 37 °C. Bacteria were harvested by centrifugation (3000xg, 20 min, 4°C) and stored at -80°C. The pellet from a 1-litre bacterial culture was resuspended in lysis buffer (50 mM NaH₂PO₄, 500 mM NaCl and 2 mM imidazole, pH 8) and subjected to three freezethaw cycles using liquid nitrogen (frozen 3 times in liquid nitrogen). rCor a 11 was purified by metal-chelate affinity chromatography using a commercial kit (Qiagen) (Lauer et al. 2004) [1117].

Pastorello et al. (2002) [671] define the 47 kDa allergen, which has the Cor a 11 N-terminal sequence, as major whilst Lauer et al. (2004) [1117] found that less than 50% of their sera contained reactive IgE to Cor a 11.

A partial N-terminal sequence of a 48 kDa hazelnut allergen with high similarity to vicilin was also found by Müller et al. (2000) [1116]