U.S. Department of Agriculture, Agricultural Research Service, Pacific West Area, Western Regional Research Center, 800 Buchanan Street, Albany, California 94710, United States.
J Agric Food Chem. 2014 Dec 31;62(52):12695-700. doi: 10.1021/jf5045102. Epub 2014 Dec 15.
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. 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.
为了理解食物过敏原的致敏性，需要对其进行生化特性分析。此类研究需要相对大量的高度纯化的过敏原。杏仁中 Pru du 4 的含量较低，要想以可溶性形式在大肠杆菌中表达，就需要一个表达标签。使用 MBP 标签来增强其表达和可溶性。首次将 Sumo 用作肽酶识别位点。用 Sumo 蛋白酶去除表达标签，然后通过色谱法纯化得到野生型 Pru du 4。用化学变性研究过敏原的稳定性。确定 Pru du 4 折叠-展开转变的吉布斯自由能为 5.4 ± 0.7 kcal/mol。
