Purification of human TBC1D7 protein and its functional mechanism in mTOR signaling
GAI Zhongchao1， WANG Bing2
1.School of Food and Biological engineering， Shaanxi University of Science and Technology， Xi'an 710021， China；2.Key Laboratory of Biology and Medical Engineering， Guizhou Medical University， Guiyang 550025， China
Abstract：mTOR signaling plays crucial roles in various biological processes of eukaryotes， the key component of mTOR signaling is the mTORC1 complex， which is mainly regulated by TSC complex. TSC complex is composed by TSC1， TSC2 and TBC1D7， and the TBC1D7 subunit is taken as the research object in this study. First， sequence alignments showed that TBC1D7 was highly conserved between different species， and 3D structural analysis suggested that there was a conserved surface on TBC1D7 protein. Then， TBC1D7 was cloned into pET28a vector， and transformed into E.coli competent cells for protein expression. After Ni2＋ affinity chromatography， His tag was removed by thrombin digestion， and protein minture was subject into SEC column for future purification. Functional studies suggested that the up-regulation of TBC1D7 in HCM cells would inhibit the activity of mTORC1， while on the contrary， down-regulation of TBC1D7 would stimulate the activity of mTORC1. This present study provides an effective method for TBC1D7 expression and addressed the specific function of TBC1D7 in mTOR signaling.
盖中朝,王 兵. 人类TBC1D7蛋白的表达纯化及其在mTOR通路中的作用机制研究[J]. 华中师范大学学报(自然科学版), 2020, 54(1): 79-88.
GAI Zhongchao,WANG Bing. Purification of human TBC1D7 protein and its functional mechanism in mTOR signaling. journal1, 2020, 54(1): 79-88.
2020, Vol. 54 
