酿酒酵母全蛋白制备方法多样,破碎细胞和制备温度等操作条件差异很大,有必要进行综合比较和量化分析,从而有利于选择适于双向电泳的最优制备方法。文中以蛋白释放率、全蛋白浓度为指标,对制备酿酒酵母全蛋白的常用方法进行了比较分析。在文献报道的玻璃珠破碎制备全蛋白方法的基础上,采用单因素和正交试验设计对破碎次数、玻璃珠用量、装液比等进行了优化,得到了制备酿酒酵母提取全蛋白的最佳条件,蛋白释放率达到了9.23%,蛋白质量浓度为1.21 g/L,较优化前的方法分别提高了0.46倍和0.32倍。通过蛋白质双向电泳进行验证,优化后的电泳图像低丰度蛋白分辨率高,总蛋白点个数由124个增加到181个,35 kDa以下的低丰度蛋白的数量提高了1倍。结果表明,该制备方法可以很好地满足酿酒酵母蛋白质组学研究要求。

Several methods for the preparation of holoprotein from Saccharomyces cerevisiae for dimensional electrophoresis were compared and optimized in the present paper due to the great differences existed in the operating conditions.The comprehensive comparison and quantitative analysis were made according to the yield and concentration of total proteins.Based on the glass beads breaking method reported previously,breaking times,dosage of glass beads and loading ratio of liquid were further optimized by single factor and orthogonal test.The optimal yield and concentration of proteins were 9.23% and 1.21 g /L,which were increased 0.46 and 0.32 fold respectively.Through the twodimensional electrophoresis,low abundance proteins under 35 kDa whose number increased one fold in the optimized electrophoresis image was in high resolution and the number of total protein points increased from 124 to 181.The results showed that the protein sample deriving from the optimized method was proved to be fit for the proteomic analysis of Saccharomyces cerevisiae.