DEPTO R蛋白的表达纯化条件及与mTOR-TRD2亚结构域的相互作用

Expression and Purification of DEPTOR and Its Interaction with mTOR-TRD2

测试并优化了哺乳动物雷帕霉素靶蛋白(m TOR)的FAT结构域及其亚结构域TRD2和DEPTOR蛋白在大肠杆菌中的表达条件,获得了TRD2和DEPTOR蛋白成功表达并大量纯化的条件.结果表明,麦芽糖结合蛋白(MBP)融合m TOR的TRD2亚结构域可以在菌株BL21(DE3)中大量表达.DEPTOR-G270P突变比野生型DEPTOR在BL21(DE3)中的表达量提高约5倍,且该突变并不影响DEPTOR自身的二级结构.通过凝胶过滤实验发现,m TOR的TRD2亚结构域和DEPTOR之间可能存在相互作用.

The inhibition of mammalian target of rapamycin（ m TOR） is important to cancer treatment.DEPTOR is currently known as an endogenous m TOR inhibitor and could interact with m TOR FAT domain to suppress m TOR activity in vivo. It showed that MBP fusion m TOR TRD2 subdomain could be expressed in BL21（ DE3） with conversional protocol and DEPTOR-G270 P mutation could be expressed 5-fold more than wild-type DEPTOR in BL21（ DE3）. Adding glycerol（ volume fraction 10%） in purification buffer would improve DEPTOR-G270 P purity. CD spectra assay showed that this purification protocol could produce DEPTOR in large quantity without affecting its secondary structure. Gel-filtration assay indicated possible interactions between m TOR-TRD2 subdomain and DEPTOR,which may provide insights into m TOR-DEPTOR interaction and give new information about m TOR inhibition.