Purification and characterization of 2S albumin from Nelumbo nucifera

The 2S albumins are a group of seed storage proteins that have recently attracted considerable attention in the field of allergen science due to their allergenic potential. A new 2S albumin from seeds of Nelumbo nucifera (Nn-2S alb) was purified to electrophoretic homogeneity by the combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The protein has a molecular mass of about 12 kDa estimated by SDS–PAGE, in good agreement with 12.5 ± 0.01 kDa determined by ESI–MS. Circular dichroism data showed that protein contained about 66% α-helices as estimated by K2D3, indicating that the protein was predominantly helical. The sedimentation coefficient (s°20,w) of the predicted model was 1.72 ± 0.21 S. The predicted 3-dimensional structure of the Nn-2S alb revealed that the protein has a region of 12 amino acids which largely corresponds to the conserved immuno-dominant epitope of 2S allergens. Surface and ribbon representations of Nn-2S albumin model, showing hydrophobic cavity in gray (A), and hypervariable region (yellow in B) and the amino acids residues (sticks) that constitute hydrophobic cavity (B).

2S清蛋白（2S albumins）是一类种子贮藏蛋白，因其具有致敏潜能，近年来在变应原学领域受到广泛关注。本研究从莲（Nelumbo nucifera）种子中纯化得到一种新型2S清蛋白（命名为Nn-2S alb），通过硫酸铵分级沉淀、凝胶过滤层析与离子交换层析联用的方法，将其纯化至电泳纯。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估算，该蛋白的相对分子质量约为12 kDa，与电喷雾电离质谱（ESI-MS）测得的12.5 ± 0.01 kDa结果吻合良好。圆二色谱数据显示，通过K2D3软件估算，该蛋白约含66%的α-螺旋，表明其主要以螺旋结构形式存在。该蛋白预测模型的标准沉降系数（s°20,w）为1.72 ± 0.21 S。对Nn-2S alb的三维结构预测结果表明，该蛋白存在一段由12个氨基酸残基组成的区域，该区域与2S变应原保守的免疫显性表位高度对应。本研究同时展示了Nn-2S清蛋白模型的表面视图与带状结构视图：其中图A以灰色标注疏水空腔，图B则以黄色标注高变区，并展示了构成该疏水空腔的氨基酸残基（棍状模型）。