硫化叶菌超表达载体构建及Mre11的表达

Construction of Sulfolobus over-expression vector and expression of Mre11

利用araS启动子并在穿梭载体pZC1及pEXA的基础上构建硫化叶菌超表达载体pZC2,成功超表达了带有C端6×His标签序列的DNA双链断裂修复蛋白Mre11,进一步采用共纯化法鉴定到Mre11蛋白的作用配体Rad50,确定上述蛋白在高浓度盐(500mmol/L NaCl)中仍能形成MR复合体,表明该复合物在逆性条件下可能仍保持DNA断链修复的功能。进一步共纯化分析表明,基因组DNA片段是MR复合体形成的必需条件,在缺乏基因组DNA片段的情况下古菌分子伴侣蛋白结合并可能保护Mre11,该表达系统能够有效地应用于鉴定蛋白质的体内作用网络。

It is important in expressing,purifying and analyzing crucial protein in vivo of solfataricus for the study of important life activities of extremely thermophilic archaea.The over-expression vector is constructed on the basis of pZC1 and pEXA vector inserted by araS promoter.Mre11 with 6×His-tag in C-terminal are expressed successfully using it.Rad50 which interplays with Mre11 is detected by co-purification.And it is ensured that the frame of the complex is still unchanged in high concentration of salt（500 mmol/L NaCl）.It indicates that the complex still keeps the function of DNA repair in adverse situation.Further analysis of co-purification makes it clear that genome DNA fragments are the essential condition of foming the MR complex.In the absence of genome DNA fragments,the molecular chaperone of archaea may bind and protect Mre11.So this expression system possesses important sense in identifying the interact network of protein in vivo.