Dermato-Allergological Dept. K, CUH-Gentofte, Rigshospitalet Dept 7551, Blegdamsvej 9, 2100 København Ø, Denmark.
Microb Cell Fact. 2010 Sep 24;9:74. doi: 10.1186/1475-2859-9-74.
Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model.
The proteins were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS) after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating allergen-specific histamine release from human basophils.
All the three major wasp venom allergens were expressed on the yeast surface. A high-level expression, which was observed only for antigen 5, was needed for detection of IgE binding by FACS and for induction of histamine release. The non-modified S. cerevisiae cells did not cause any unspecific reaction in FACS or histamine release assay despite the expression of high-mannose oligosaccharides.In perspective the yeast surface display may be used for allergen discovery from cDNA libraries and possibly for sublingual immunotherapy as the cells can serve as good adjuvant and can be produced in large amounts at a low price.
酵母表面展示技术是一种将感兴趣的蛋白质与酵母表面蛋白融合表达，表达后仍附着在酵母细胞壁上的技术。我们的目的是研究在酿酒酵母 Saccharomyces cerevisiae 细胞表面表达的变应原是否保持其天然的变应原特性，以及酵母天然表面糖蛋白是否会干扰 IgE 结合。我们选择使用常见胡蜂 Vespula vulgaris 毒液中的主要过敏原：磷脂酶 A1、透明质酸酶和抗原 5 作为模型。
这些蛋白通过与 a-凝集素复合物蛋白 AGA2 融合表达在表面上。通过用针对过敏原 C 端连接的 C 标签的抗体染色细胞后，用荧光细胞术（FACS）确认表达。磷脂酶 A1 和透明质酸酶保留了其酶活性。磷脂酶 A1 严重抑制了酵母细胞的生长。用 FACS 证实，表达抗原 5 的酵母细胞可与黄蜂毒液过敏患者血清中的 IgE 抗体结合，但不能与对照血清结合。此外，表达抗原 5 的酵母细胞能够介导人嗜碱性粒细胞的过敏原特异性组胺释放。
三种主要的黄蜂毒液过敏原均在酵母表面表达。仅观察到抗原 5 表达高水平，这是通过 FACS 检测 IgE 结合和诱导组胺释放所必需的。尽管表达了高甘露糖寡糖，但未经修饰的 S. cerevisiae 细胞在 FACS 或组胺释放试验中不会引起任何非特异性反应。从 cDNA 文库中发现过敏原，可能作为舌下免疫疗法，酵母表面展示可能是一种有用的方法，因为细胞可以作为良好的佐剂，并且可以以低价格大量生产。
