β-1,3-木聚糖是紫菜等海藻中的重要结构多糖。多糖结合蛋白是多糖研究的关键工具,常被用作探针服务于多糖的原位分析;其中,碳水化合物结合结构域(carbohydrate-binding module,CBM)是一类被广泛应用的多糖结合蛋白。该研究从公共基因数据库中挖掘得到了一条潜在具有β-1,3-木聚糖结合能力的CBM6家族序列,并对该序列进行了异源表达及亲和纯化,得到电泳纯蛋白,命名为VaCBM6。该蛋白表现出对β-1,3-木聚糖的特异性结合能力,结合常数为3.0×105 (mol/L)-1。将该蛋白与绿色荧光蛋白EmGFP融合表达构建出首个β-1,3-木聚糖的特异性荧光探针,以该探针为工具实现了紫菜中β-1,3-木聚糖的荧光显微观察,证实了VaCBM6在β-1,3-木聚糖原位分析中的良好应用潜力。VaCBM6的挖掘与表征为β-1,3-木聚糖的研究与开发提供了良好工具。
β-1,3-xylan is an important structural polysaccharide distributed in seaweeds including Porphyra.Polysaccharide-binding proteins are critical tools in polysaccharide research, serving as probes for the in situ analysis of polysaccharides.Carbohydrate-binding module (CBM) is a class of widely used polysaccharide-binding proteins.In the present study, a potential β-1,3-xylan binding sequence from the CBM6 family was discovered from the public gene database.The pure protein, named VaCBM6, was obtained by gene cloning, heterologous expression, and affinity purification.VaCBM6 exhibited a specific binding capacity to β-1,3-xylan, with an association constant of 3.0×105 (mol/L)-1.Furthermore, the first specific fluorescent probe for β-1,3-xylan was constructed by fusing VaCBM6 with the green fluorescent protein EmGFP, based on which the fluorescence microscopic observation of β-1,3-xylan in Porphyra was realized.The potential of VaCBM6 in the in situ analysis of β-1,3-xylan was confirmed.The discovery and characterization of VaCBM6 provided a promising tool for the future investigation and application of β-1,3-xylan.
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