Barral Patricia, Tejera María Luisa, Treviño Miguel Angel, Batanero Eva, Villalba Mayte, Bruix Marta, Rodríguez Rosalía
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Protein Expr Purif. 2004 Oct;37(2):336-43. doi: 10.1016/j.pep.2004.06.012.
Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.
橄榄花粉是地中海国家过敏的主要原因之一。油橄榄6（Ole e 6）是一种橄榄花粉过敏原，是一种小的（5.8 kDa）酸性蛋白（pI 4.2），目前尚未分离或鉴定出同源蛋白。Ole e 6已在甲基营养型酵母毕赤酵母中高效表达。将编码Ole e 6的cDNA插入质粒载体pPIC9中，并在GS115酵母细胞中过表达。重组产物通过尺寸排阻色谱，然后进行反相高效液相色谱纯化。采用N端测序、氨基酸组成分析、圆二色光谱（CD）、核磁共振（NMR）和IgG结合实验对纯化后的蛋白进行表征。NMR数据显示，约50%的重组蛋白中第28位的甲硫氨酸发生了氧化，尽管这改变了其电泳行为，但并未影响重组Ole e 6的折叠或IgG结合特性。在毕赤酵母中表达的重组形式的Ole e 6可用于结构和生化研究。
