摘要
泛素结合酶是一类在泛素化修饰过程中起重要生物化学功能的蛋白。对于被预测为泛素结合酶的蛋白进行体外泛素化活性检测,是确定其泛素结合酶功能必不可少的试验步骤,试验结果可以为后续研究提供重要的线索。目前体外泛素化反应存在体外表达纯化蛋白工作量大、反应复杂、假阴性率高等问题。本研究针对以上问题进行了改良,开发出一种新的植物泛素结合酶活性体外检测方法,该方法无需裂解细胞提取、纯化蛋白,也不需要做体外反应,简便易行,结果更稳定可靠。应用该方法对花生泛素结合酶Ah UBC34和盐芥泛素结合酶Ts UBC33进行了检测,结果证明这两个蛋白都具有泛素结合酶活性。
The ubiquitin-conjugating enzymes play key biochemical roles in the ubiquitination modification. The in vitro ubiquitination assay is essential to the study of the predicted ubiquitin-conjugating enzyme,which supplies important clues for the following study. So far,the in vitro ubiquitin-conjugating enzyme activity analysis needs huge amount of works such as protein expression and purification. The process is complex,and the ratio of false negative is high. In this study,we developed a new system to detect the plant ubiquitin-conjugating enzyme activity which omits the protein purification and in vitro reaction steps in the old method. This new system is more convenient and stable. We applied this system to detect Arachis hypogaea UBC34 and Eutrema salsugineum( Thellungiella salsuginea) UBC33,and proved the ubiquitin-conjugating enzyme activity of these two proteins.
出处
《山东农业科学》
2018年第3期120-124,共5页
Shandong Agricultural Sciences
基金
山东省自然科学基金项目(ZR2014YL043
ZR2014CQ046)
国家自然科学基金项目(31401034)
山东省农业科学院农业科技创新工程项目(CXGC2016C03)
关键词
泛素结合酶
体外泛素化检测
检测方法
花生
盐芥
Ubiquitin-conjugating enzyme
In vitro ubiquitination assay
Assay method
Peanut
Eutrema salsugineum
