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Co-expression and Purification of Recombinant Ego1 in Complex with Ego3 from Saccharomyces cerevisiae
Ma Rui1, 2, Li Ang2, Wang Yujiong1, Zhang Yong2*
1School of Life Sciences, Ningxia University, Yinchuan 750021, China; 2School of Life Sciences and Biotechnology,Shanghai Jiao Tong University, Shanghai 200240, China
Abstract: Scaffold and adaptor proteins play crucial roles in many key signaling cascades. Ego1 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 Ego1 and Ego3 gene to co-express recombinant Ego1 and Ego3 protein. Co-expression of Ego1 and Ego3 in E.coli led to a soluble protein complex. By coexpression after co-transformation with different antibiotic-resistant duet plasmids into cells， the protein ratio problem could be improved in the complex. Furthermore， we subcloned several truncated Ego1 and full-length Ego1 into binary vectors and characterized the binding between the truncated Ego1 and Ego3 by co-purification. Finally，we obtained a stable truncated Ego1 (35-184) fragment in complex with Ego3 via co-transformation of pRSFDuet-Ego1 (35-184) and pACYDuet-Ego3.
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