Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, Wako, Saitama, Japan.
Carbohydr Res. 2013 Jun 28;375:112-7. doi: 10.1016/j.carres.2013.04.032. Epub 2013 May 7.
Using ultrafiltration membrane, a simple method for screening protein-ligand interaction was developed. The procedure comprises three steps: mixing ligand with protein, ultrafiltration of the solution, and quantification of unbound ligands by HPLC. By conducting analysis with variable protein concentrations, affinity constants were easily obtained. Multiple ligands can be analyzed simultaneously as a mixture, when concentration of ligands was controlled. Feasibility of this method for lectin-glycan interaction analysis was examined using fluorescently labeled high-mannose-type glycans and recombinant intracellular lectins or endo-α-mannosidase mutants. Estimated Ka values of malectin and VIP36 were in good agreement indeed with those evaluated by conventional methods such as isothermal titration calorimetry (ITC) or frontal affinity chromatography (FAC). Finally, several mutants of endo-α-mannosidase were produced and their affinities to monoglucosylated glycans were evaluated.
利用超滤膜，开发了一种筛选蛋白-配体相互作用的简单方法。该方法包括三个步骤：配体与蛋白混合、溶液超滤和 HPLC 定量分析未结合的配体。通过分析不同蛋白浓度，可以轻松获得亲和常数。当控制配体浓度时，可以同时分析多种配体作为混合物。使用荧光标记的高甘露糖型聚糖和重组细胞内凝集素或内切-α-甘露糖苷酶突变体，考察了该方法用于凝集素-聚糖相互作用分析的可行性。甘露糖结合凝集素和 VIP36 的估计 Ka 值与等温滴定量热法（ITC）或前沿亲和色谱法（FAC）等常规方法评估的值非常吻合。最后，制备了几种内切-α-甘露糖苷酶突变体，并评估了它们与单葡萄糖化聚糖的亲和力。
