酿酒酵母FFC2146胞内蛋白及胞外蛋白双向电泳条件优化及图谱建立

Optimization and construction of the intracellular and extracellular proteomic map of FFC2146

通过对酿酒酵母(Saccharomyces cerevisiae)的培养基、培养条件及蛋白质提取方案的优化,建立了酿酒酵母胞外和胞内蛋白双向电泳图谱制作方法。在YNB培养基中培养20 h,经过离心取上清-超滤-冻干可得到酿酒酵母胞外蛋白质样品;用SDS缓冲液悬浮酵母细胞-煮沸-超声-增溶,得到了酿酒酵母胞内蛋白质样品。经过双向电泳分离、硝酸银染色和PDQuest图像分析可以检测到了200多种酿酒酵母胞外蛋白和500多种酿酒酵母胞内蛋白。

The aim of this study was to optimize a proper method to extract intracellular and extracellular protein from Saccharomyces cerevisiae to construct the proteomic maps.Methods of protein extraction and cultivation condition were optimized.The cells were cultured in YNB medium for 20 h and cells were separated by centrifugation.The extracellular proteins in supernatants were obtained through ultra filtration-freeze drying.Cell pellets were resuspended in SDS lysis buffer.The cell suspension were boiled for 5 min,after solubilized by sonication and stored until use.The intra-or extracellular protein from S.cerevisiae were separated by 2-DE and stained with silver nitrate.The separated protein spots in gels were analyzed by PDQuest.The results showed that over 200 spots of extracellular proteins and about 500 spots of intracellular proteins had been separated by 2-DE.