应用于二维电泳细胞蛋白质样品提取的复合蛋白酶抑制剂的优化

Optimization of Protease Inhibitor Mixture for Protein Extraction from Cells in Proteomics Study of Streptomyces avermitilis

阿维链霉菌在复合培养基中生长时合成大量蛋白酶以满足菌体分解有机氮源进行生长代谢的需要。而大量蛋白酶的存在对二维电泳的蛋白质组分析细胞蛋白质样品的提取带来了很大的困难。根据阿维链霉菌胞内蛋白酶的组成,以EDTA、PMSF、Bestatin、Pepstatin和E-64等5种蛋白酶抑制剂为基础,通过单因子实验和正交实验优化得到了高效的蛋白酶抑制剂复合配方。验证实验表明,该复合蛋白酶抑制剂在阿维链霉菌细胞蛋白质样品提取中,具有良好的蛋白酶活性抑制效果。

A lot of proteases are synthesized for cell growth and metabolism by Streptomyces avermitilis during growing in the complex medium.However,the 2-DE protein sample preparation for intracellular proteomics analysis of S.avermitilis became difficult because of the hydrolysis of protein by massive proteases when cells were disrupted.An effective protease inhibitor mixture was obtained by single factor and orthogonal optimum design based on the knowledge of intracellular protease composition of S.avermitilis.By verifying expriments,it was validated that the protease inhibitor mixture can effectively protect large molecular weight proteins from enzymolysis during sample preparation.