1a.Objectives (from AD-416):
To clone and express Ara h 2 and Ara h 6 in soluble form, and to subject these clones to various chemical treatments known to occur during processing, in order to identify specific modifications.
1b.Approach (from AD-416):
a.) Clone Ara h 2/Ara h 6 into various types of DNA vectors towards making soluble protein. b.) Purify this allergen and subject the purified allergen to processing induced modification towards identification of specific amino acids involved in enhanced IgE binding following roasting.
Tulane, ARS, and a mutual student have worked on cloning Ara h 2, a major peanut allergen, into various DNA vectors in order to obtain soluble recombinant Ara h 2. This has been successful, and we are in the process of subjecting Ara h 2 to a simulated roasting system (Maillard or browning reaction) followed by digestion and mass spectroscopy, which will allow us to identify the specific chemical modifications that occur due to the roasting process. This will also allow us to identify specific-processing induced modifications that contribute to alterations in allergenic properties of roasted peanuts.