Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis (1)
Proteins were extracted by stirring 40 g of defatted peanut meal in 400 mL of 50 mM Tris/HCl, pH 8.2, at 4C, overnight. The resulting suspension was clarified by centrifugation (10000xg for 10 min, 4C) and the supernatant filtered through four layers of cheesecloth prior to ammonium sulphate fractionation. The fraction precipitated between 40 and 80% ammonium sulphate saturation at 4C, was collected by centrifugation (10 000xg for 20 min, 4C) and resuspended in 200 mL of 20 mM Tris/HCl, pH 8.0 containing 1 mM EDTA, prior to dialysis against 10 L of 20 mM Tris/HCl, pH 8.0 (overnight, 48C) using 3.5 kDa MW cut off (MWCO) dialysis tubing. The dialysate was clarified by centrifugation (30 000xg for 30 min, 4C) and the supernatant passed through a 0.2 lm filter prior to chromatography.
The crude extract was loaded onto a Superdex 75 column equilibrated and eluted with 20 mM Tris/HCl, pH 8, at a flow rate of 0.75 mL/min. Fractions from gel filtration were loaded onto an anion exchange column equilibrated with 20 mM Tris/HCl, pH 8. Bound proteins were eluted at a flow rate of 5 mL/min using a step gradient as follows: five column volumes (CV) of 20 mM Tris/HCl, pH 8; 15 CV of 20 mM Tris/HCl, pH 8, 62.5 mM NaCl; five CV of 20 mM Tris/HCl, pH 8, 125 mM NaCl and five CVof 20 mM Tris/ HCl, pH 8, 250 mM NaCl. The Fractions were then loaded onto a Vydac C18 preparative RP-HPLC column. The bound proteins were eluted with an ACN gradient from 15–40% using 1:2000 v/v, trifluoracetic acid (TFA), water as buffer A and 1:2000 v/v, TFA, ACN as buffer B. Peaks eluting at 25 and 30 min were shown to be enriched in Ara h 2 and Ara h 6 by SDS-PAGE.
SDS-PAGE of this fraction showed the presence of three major bands, two of approximately 17 kDa which were assumed to correspond to Ara h 2 and Ara h 2.02 and a third of about 15 kDa which was shown by MS to correspond to Ara h 6. The combined fractions from gel-filtration were therefore separated by anion exchange chromatography with elution with a stepped salt gradient. SDS-PAGE showed that the fractions from this separation were highly enriched in Ara h 6 (Peak 1) and in Ara h 2 and Ara h 2.02 (peaks 2 and 3). All fractions gave a peak at about 25 min which contained two proteins of Mr about 17 kDa corresponding to Ara h 2/2.02 and a peak at about 30 min which contained a major protein of about 15 kDa corresponding to Ara h 6.
Ara h 1 is glycosylated and was therefore readily purified from defatted peanut meal using a lectin affinity column (with concanavalin A which binds to mannose present in the glycan chain) followed by gel filtration chromatography. SDS-PAGE showed a major band of 67 kDa with a minor band of about 33 kDa. Analytical gel permeation chromatography showed that the native trimeric protein has a mass of about 235 kDa.
Peanut 11S globulins, Ara h 3/4, were purified from defat- ted peanut flour by anion exchange chromatography followed by gel filtration and analytical size exclusion chromatography. SDS-PAGE under reducing conditions of fractions containing Ara h 3/4 showed two groups of bands which corresponded to the N- terminal acidic (about 40–43 kDa) and C-terminal basic (about 20 kDa) subunits.
Recombinant Ara h 8 was synthesised in E. coli with a histidine tag to allow purification using IMAC. SDS- PAGE of the purified protein showed a single major band migrating at about 20 kDa.
The published sequences of Ara h 2 components; the longer sequence corresponding to Ara h 2.02 and the shorter sequence to Ara h 2. It was therefore concluded that the slow and fast 17 kDa bands in our preparation cor- responded to Ara h 2.02 and Ara h.2, respectively. However, the N-terminal sequences determined for our components start five (Ara h 2.02) and seven (Ara h 2) residues downstream of the N-termini reported in other studies, which correspond to the predicted sites of signal peptide cleavage.
Sequence databases show two sequences encoding forms of Ara h 6, which differ in the presence of aspartate (D) or asparagine (N) two residues upstream of the N-terminus of the large subunit of Ara h 6.2. The form with aspartate was the precursor of Ara h 6.2, and cleavage adjacent to aspartate residues cer- tainly occurs in the 2S albumins of Arabidopsis catalysed by an aspartyl proteinase.
Pools of sera with IgE reactivity to Ara h1,Ara h3/4, Ara h2/6(pool1) and to Ara h8 (pool 2) were used for inhibition assays. The reactivity to total peanut protein extract was then deter- mined after preincubation with individual protein preparations. Pre-incubation with Ara h 1 resulted in reduced binding of serum pool 1 to the 7S globulin (Ara h 1) bands at about 67 kDa and 34 kDa but also reduced binding to Ara h 3/4 components at 40–43 kDa, due to cross reactivity. Similarly, preincubation with Ara h 3/4 resulted in reduced binding of serum pool 1 to the 11S globulin bands at 40–43 kDa and also reduced binding to the 7S globulin band at 34 kDa. Pre incubation with Ara h 2 and Ara h 6 resulted in reduced binding of serum pool 1 to the corresponding 2S albumin bands in the range of 11–17 kDa. A band at 17 kDa was recognised by serum pool 2, but the binding to this was significantly reduced on pre-incubation with rAra h 8.
Recently, Ramos et al. have shown that A. hypogaea cv Georgia Green has two genes encoding Ara h 2 and Ara h 2.02 and three genes encoding Ara h 6. The gene encoding Ara h 2 was assigned to the A genome, while that encoding Ara h 2.02 was assigned to the B genome. Two of the Ara h 6 genes are closely linked to the Ara h 2.02 gene on the B genome. The third Ara h 6 gene is on the A genome. However, none of the Ara h 6 genes encodes an asparagine residue at position 66.