小麦面粉Puroindoline蛋白的提取与纯化

Puroindoline蛋白是小麦面粉中一种非常重要的蛋白质，不仅影响和决定了籽粒的硬度，而且有抗G^＋、G^-菌以及抗真菌的作用。用含4％TritonX-114、100mmol／L pH7．8Tris-HCl缓冲液处理小麦面粉来分离Puroindoline蛋白。经处理后得到的蛋白质混合溶液首先用分子筛葡聚糖G-75纯化，每个收集管内的组分经SDS-PAGE分析，分子量小于31kD的蛋白质组分被回收和集中，回收的蛋白质组分经PEG20000浓缩后，再用离子交换柱羧甲基纤维素（CM-23）进行纯化。其洗脱液分别是双蒸水和NaCl，梯度为0．05～0．7mol／L、8mmol／L pH5．5的MES缓冲液，回收只含15kD的蛋白质的组分，接着用PEG20000浓缩。最后冷冻干燥得到Puroindoline蛋白。

Extraction and Purification of Puroindoline from Wheat Flour

Puroindoline,influenced and decided the wheat hardness and exhibiting activity against gram-positive bacteria,gram-negative bacteria and fungi,is a very important protein in wheat flour.In this studies,the puroindoline was extracted from wheat flour in the 100 mmol/L Tris-HCl pH7.8 buffer,containing 4% Triton X-114.Then the protein mixture including puroindoline was successively purified by Sephadex G-75,and each fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fractions containing polypeptides with apparent molecular weights of less than 31 kD were pooled and loading on Carboxymethyl-cellulose(CM-23) after concentrated by PEG20000,and each fraction was analyzed as preceding.In those ways,they were eluted by distilled water and applying a gradient from 0.05 mol/L to(0.7 mol/L) NaCl in 8 mmol/L MES pH5.5 buffer respectively.The fractions containing polypeptides apparent molecular weight 15 kD were pooled and concentrated by PEG20000,at last the puroindoline was obtained by freeze-dried.