Eves Robert, Davies Peter L
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.
Methods Mol Biol. 2025;2942:81-91. doi: 10.1007/978-1-0716-4627-4_7.
In this method, a bacterial lectin is allowed to bind to its target polysaccharide in the form of a matrix suspension that can be recovered by centrifugation or filtration. After an initial test to show that the lectin is fully bound to the matrix, a measured amount of competing free sugar is added to the matrix resuspended in buffer. After mixing, the suspension is centrifuged, and the protein content of the supernatant fraction is measured before being returned to the matrix with the addition of another aliquot of sugar. The cycle of mixing, centrifugation, and quantification of protein (lectin) in the supernatant fraction is continued until the released protein reaches plateau levels. The procedure is repeated with a variety of different sugars to compare their efficiencies at binding and displacing the lectin from its polysaccharide ligand. This procedure is illustrated here using a PA14-type bacterial lectin that has affinity for fucosylated glycans and will, as a result, bind to dextran-based resins like Superdex 200 (S200). A second example features the release of Escherichia coli maltose-binding protein from amylose resin by mono- and disaccharides. The purpose of this assay is to determine the relative binding affinity of different sugars for a bacterial lectin.
在这种方法中，使一种细菌凝集素以基质悬浮液的形式与它的目标多糖结合，该悬浮液可通过离心或过滤回收。在进行初步测试以表明凝集素已完全结合到基质上之后，将一定量的竞争性游离糖添加到重悬于缓冲液中的基质中。混合后，将悬浮液离心，并在添加另一等份糖后将上清液部分的蛋白质含量测量出来再返回至基质中。重复进行混合、离心以及对上清液部分中的蛋白质（凝集素）进行定量的循环，直到释放的蛋白质达到稳定水平。用多种不同的糖重复该过程，以比较它们在从多糖配体上结合和取代凝集素方面的效率。这里以对岩藻糖基化聚糖具有亲和力的PA14型细菌凝集素为例进行说明，该凝集素因此会与基于葡聚糖的树脂如Superdex 200（S200）结合。第二个例子是单糖和双糖从直链淀粉树脂上释放大肠杆菌麦芽糖结合蛋白。该测定的目的是确定不同糖对细菌凝集素的相对结合亲和力。
