One-step purification of lectins from banana pulp using sugar-immobilized gold nano-particles
One-step purification of lectins from banana pulp using sugar-immobilized gold nano-particles
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DOI：
10.1093/jb/mvn038
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发表时间：
2008-06-01
影响因子：
2.7
通讯作者：
Suda, Yasuo
中科院分区：
文献类型：
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作者：
Nakamura-Tsuruta, Sachiko;Kishimoto, Yuko;Suda, Yasuo
为了获得凝集素，而无需繁琐的纯化步骤，我们开发了一种方便的方法，一步纯化凝集素使用糖固定化金纳米粒子（SGNP）。用60%硫酸铵沉淀来自植物材料的粗提取物中的蛋白质，并将沉淀物重新溶解在小体积的磷酸盐缓冲液中。然后在优化的条件下将所得溶液与适当的SGNP混合。在4 ° C下孵育过夜后，混合物中的凝集素与SGNP形成聚集体，其通过视觉检测并且容易通过离心沉淀。通过添加抑制性糖溶解聚集体，所述抑制性糖与SGNP上的非还原糖部分相同。根据由此获得的蛋白质的SDS-PAGE和MS，发现SGNP分离了高纯度的凝集素。例如，使用Glc alpha-GNP（α-葡萄糖固定化金纳米颗粒）从香蕉中分离的蛋白质通过胰蛋白酶消化肽-MS指纹法鉴定为香蕉凝集素。
To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nanoparticles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulphate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4 degrees C, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glc alpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.