蛋白体外结合实验联合质谱分析鉴定Num1的互作蛋白
Identification of Num1-interacting Proteins by Pull-down Experiments and Mass Spectrometry
投稿时间:2018-03-22  修订日期:2018-05-04
DOI:
中文关键词: 芽殖酵母  动力蛋白  Num1  PA结构域  纺锤体定位
英文关键词: Budding yeast  Dynein  Num1  PA domain  Spindle orientation
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
唐仙英 生命科学学院 xytangscuec@163.com 
肖瑶 中南民族大学  
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中文摘要:
      为了解析芽殖酵母Num1蛋白在纺锤体定位和线粒体中的调节机制,在大肠杆菌中表达并纯化了在Num1的功能中起核心作用的PA(patch assembly)结构域的重组蛋白,通过体外蛋白结合实验(Pull-down)分离了酵母细胞中与PA结构域相结合的蛋白复合物,并对其进行了质谱分析。鉴定出一系列新的Num1互作蛋白,包括许多核糖体蛋白,参与蛋白折叠、分配及转运的内质网和高尔基复合体蛋白,参与基因转录和翻译的核酸酶和蛋白酶,以及其他一些功能各异的蛋白。研究结果为进一步研究Num1的作用机制奠定了基础。
英文摘要:
      To explore the regulatory mechanisms of Num1 during spindle orientation and in mitochondria in budding yeast, recombinant proteins of the patch assemble (PA) domain, which plays central roles in Num1’s functions, were expressed and purified from E. Coli cells. Protein complexes that bound to the PA domain were isolated from yeast cells through pull-down experiments and analyzed by mass spectrometry. A series of novel Num1-interacting proteins were identified. These include many ribosomal proteins, proteins involved in protein folding, sorting and transportation in the ER and Golgi complexes, some nucleases and protein enzymes involved in gene transcription and translation, and other proteins with various functions. These results lay a foundation for further study of the mechanism by which Num1 works.
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