|FREGEVU, CECILE - School Of Engineers Of Purpan|
|KOTHARY, MAHENDRA - Food And Drug Administration(FDA)|
|Harden, Leslie - Les|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2014
Publication Date: 12/1/2014
Citation: Zhang, Y., Du, W., Fregevu, C., Kothary, M.H., Harden, L.A., Mchugh, T.H. 2014. Expression, purification, and characterization of almond (Prunus dulcis) allergen Pru du 4. Journal of Agricultural and Food Chemistry. 62(52):12695-12700. doi: 10.1021/jf50451021.
Interpretive Summary: Pru du 4 is an almond allergen. It belongs to the profilin protein family. Profilins are known to be involved in both food and pollen allergies. Characterization of allergens will allow a better understanding of the allergenicity of food allergens and their cross-reactivities. This study reported the production of recombinant Pru du 4 in its native form and the investigation of its stability. The results showed that using sumo as a peptidase recognition site, other allergens may also be produced in their native form in bacteria even if their native form in natural food are produced after post-translational signal peptide removal. This opens a new avenue for studying the importance of conformational epitopes.
Technical Abstract: Biochemical characterizations of food allergens are required for understanding the allergenicity of food allergens. Such studies require a relatively large amount of highly purified allergens. Profilins from numerous species are known to be allergens, including food allergens, such as almond (Prunus dulcis) allergen Pru du 4, and pollen allergens, such as birch allergen Bet v 2. The level of Pru du 4 in almond is low and its expression in a soluble form in Escherichia coli required an expression tag. An MBP tag was used to enhance its expression and solubility. Sumo was used for the first time as a peptidase recognition site. The expression tag was removed with a sumo protease and the resulting wild-type Pru du 4 was purified chromatographically. The stability of the allergen was investigated with chemical denaturation. The Gibbs free energy of Pru du 4 folding-unfolding transition was determined to be 5.4 ± 0.7 kcal/mol.