Simplified Methods for Purification of Peanut Allergenic Proteins: Ara h 1, Arah 2, and Ara h 3 (1)
Ara h 1 is the most abundant of the peanut seed proteins, accounting for 12 _ 16% of the total protein content in peanuts. Ara h 1 is recognized by more than 90% of serum IgE from peanut allergic patients, indicating its importance in the etiology of peanut allergy. Ara h 1 exists as a stable trimer of monomers (~ 64 kDa) bound to each other. The stable structure of the trimer contributes to its resistance to protease digestion.
Ara h 2 is a seed storage protein classified as a 2S albumin, and accounts for 5.9 _ 9.3% of the total protein content in peanuts. It comprises two isoforms (~ 16 kDa and ~ 18 kDa).
Ara h3 is a single chain protein (~ 58 kDa) which is cut into 13 _ 38 kDa acidic subunits and a 21 kDa basic subunit by protein processing, and the processed subunits are linked by disulfide bonds. The bound subunits as monomers form a hexamer.
It was found that fractionation at 70 _ 75% AS and 75 _ 80% AS following 0 _ 70% ASF eliminated the 37 kDa and 21 kDa contaminating proteins from the fraction at 80 _ 90% AS. On the other hand, 70 _ 80% ASF following 0 _ 70% ASF did not eliminate the 37 kDa and 21 kDa proteins from the fraction at 80 – 90% AS. Since the elimination of Ara h 3 is key in the purification of Ara h 1, the 70 _ 75% and 75 _ 80% ASFs are indispensable procedures. The low molecular mass fraction was eliminated by repeated ultrafiltration (200,000 MWCO) and concentration, and the protein was observed as a single 64-kDa band on SDS-PAGE. The degree of purification for Ara h 1 was 91.3%.
The 40 _ 50% ASF precipitate was dialyzed and Ara h 2 was highly concentrated, and contamination with proteins of smaller molecular mass than Ara h 2 was minimized. The supernatant (0.8 mL) was subjected to ultrafiltration (30,000 MWCO) and concentration. The filtrate was further subjected to ultrafiltration (3,000 MWCO) and the concentrate was recovered. The degree of purification was 86.4%. When TB was used for the ultrafiltration with the 30,000 unit, proteins of ~ 23 kDa and ~ 30 kDa, including Ara h 2, passed through the membrane unit and the purification of Ara h 2 was not successful. However, by using water instead of TB the purification was successfully carried out as mentioned above. It was speculated that the contaminating proteins might be less solubilized in water than in TB or they might form large molecules in distilled water, preventing passage through the membrane. Notably, an ~ 15 kDa protein was an occasional contaminant of Ara h 2. The protein is suspected to be Ara h 6, a 2S albumin and a homologue of Ara h 2. The amino acid sequence of Ara h 6 shares 59% identity with Ara h 2, and its resistance to protease digestion and allergenic potency are comparable to those of Ara h 2.
Structural analysis of the purified Ara h 3 by MALDI-TOF-MS revealed varied structures composed of both acidic subunits (~ 13 _ 38 kDa), which are processed at specific sites, and basic subunits (~ 21 kDa). The total band intensity of the bands at ~ 37kDa, ~ 34kDa, ~25kDa, and ~ 21kDa on the gel under reducing condition was designated as Ara h 3 and used for the purity evaluation. Ara h 3 exists as a relatively large 350 kDa hexamer in its native state.
The purification protocol for peanut allergens Ara h1, Ara h2, and Ara h3 in this study.
