酿酒酵母TORC1信号通路重要调控元件Ego1／Ego3

支架和连接蛋白在调节信号级联传导中起着重要作用。最近，Ego1／Ego3蛋白复合物被证实作为定位在细胞内体膜表面的支架蛋白在TORCl信号通路感知氨基酸丰度方面发挥重要调控作用。研究中通过克隆酵母Ego1与Ego3基因，构建了双T7启动子的双元原核表达载体。通过在大肠杆菌中共表达、共破菌以及带有不同抗性的两个质粒共转化的方法来重组表达Ego1/Ego3复合物。结果发现，Ego1可与Ego3蛋白形成可溶性的复合物，但纯化后蛋白比例存在明显差异，且Ego1有降解现象。通过构建一系列带组氨酸标签截短的Ego1与全长Ego3共表达，依靠pulldown纯化来鉴定Ego1与Ego3相互作用的区段。最终发现，共转化pRSFDuet-mcs1 Ego1（35-184）和pACYDuet-mcsl Ego3共表达、纯化到较稳定的比例较一致的复合物，从而为进一步对Ego1／Ego3蛋白复合物进行生化鉴定和结构生物学研究奠定了基础。

Co-expression and Purification of Recombinant Ego1 in Complex with Ego3 from Saccharomyces cerevisiae

Scaffold and adaptor proteins play crucial roles in many key signaling cascades. Egol is a scaffold protein and together with Ego3 forms a subunit of EGO complex that is shown to be responsible for localization of TORC1 to the endosomal membrane surface. To biochemically and biophysically characterize the Ego1/ Ego3 complex, we constructed various binary expression vectors with Egol and Ego3 gene to co-express recombinant Egol and Ego3 protein. Co-expression of Ego1 and Ego3 in E.coli led to a soluble protein complex. By co- expression after co-transformation with different antibiotic-resistant duet plasmids into cells, the protein ratio prob- lem could be improved in the complex. Furthermore, we subcloned several truncated Ego 1 and full-length Ego 1 into binary vectors and characterized the binding between the truncated Ego1 and Ego3 by co-purification. Finally, we obtained a stable truncated Ego 1 （35-184） fragment in complex with Ego3 via co-transformation of pRSFDuet- Ego 1 （35-184） and pACYDuet-Ego3.