Nakamura-Tsuruta Sachiko, Kishimoto Yuko, Nishimura Tomoaki, Suda Yasuo
Venture Business Laboratory, Kagoshima University, 1-21-40, Kohrimoto, Kagoshima 890-0065, Japan.
J Biochem. 2008 Jun;143(6):833-9. doi: 10.1093/jb/mvn038. Epub 2008 Mar 15.
To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nano-particles (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 Glcalpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.
为了在不经过繁琐纯化步骤的情况下获得凝集素，我们开发了一种使用糖固定化金纳米颗粒（SGNPs）一步纯化凝集素的简便方法。植物材料粗提物中的蛋白质用60%硫酸铵沉淀，沉淀物再溶解于少量磷酸盐缓冲液中。然后将所得溶液在优化条件下与适当的SGNPs混合。在4℃孵育过夜后，混合物中的凝集素与SGNPs形成聚集体，通过肉眼观察即可检测到，并且通过离心很容易沉淀。通过加入与SGNPs上非还原糖部分相同的抑制糖来溶解聚集体。根据所得蛋白质的SDS-PAGE和质谱分析，发现SGNPs分离出的凝集素纯度很高。例如，使用Glcalpha-GNP（α-葡萄糖固定化金纳米颗粒）从香蕉中分离出的一种蛋白质，通过胰蛋白酶消化肽质谱指纹法被鉴定为香蕉凝集素。
