Purification and structural stability of the peach allergens Pru p 1 and Pru p 3
Purification and structural stability of the peach allergens Pru p 1 and Pru p 3
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DOI：
10.1002/mnfr.200700274
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发表时间：
2008-11-01
影响因子：
5.2
通讯作者：
Shewry, Peter R.
中科院分区：
文献类型：
--
作者：
Gaier, Sonja;Marsh, Justin;Shewry, Peter R.
Pru p1（一个Bet v 1同源物）和Pru p3（一个非特异性脂质转移蛋白；nsLTP）是桃果中主要的致敏蛋白，但它们的丰度和稳定性不同。Pru p1是一种低丰度、高不稳定性的重组蛋白，在大肠杆菌中表达后得到纯化。桃皮中含有丰富的Pru p3，采用常规方法纯化。通过序列分析和质谱分析确定了蛋白质的性质。纯化后的蛋白与来自其他物种的相关过敏原的抗血清反应：Pru p1与抗betv1的血清反应，Pru p3与抗Mal d3的血清反应（来自苹果）。通过圆二色性（CD）和高场核磁共振（NMR）谱分析证实了其二级和三级结构的存在。CD光谱还表明，这两种蛋白在pH值为3时的稳定性和加热至95℃后的再折叠能力不同。因此，Pru p1即使在25℃下也能在pH值为3时展开，但在pH值为7.5时加热至95℃后仍能再折叠。相比之下，Pru p3在中性条件下加热后不能再折叠，但在pH 3下加热后很容易再折叠。
Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia coli. Pru p 3 is highly abundant in peach peel and was purified by conventional methods. The identities of the proteins were confirmed by sequence analysis and their masses determined by MS analysis. The purified proteins reacted with antisera against related allergens from other species: Pru p 1 with antiserum to Bet v 1 and Pru p 3 with antiserum to Mal d 3 (from apple). The presence of secondary and tertiary structure was demonstrated by circular dichroism (CD) and high field NMR spectroscopy. CD spectroscopy also showed that the two proteins differed in their stability at pH 3 and in their ability to refold after heating to 95 degrees C. Thus, Pru p 1 was unfolded at pH 3 even at 25 degrees C but was able to refold after heating to 95 degrees C at pH 7.5. In contrast, Pru p 3 was unable to refold after heating under neutral conditions but readily refolded after heating at pH 3.