SPLIT UBIQUITIN AS A SENSOR OF PROTEIN INTERACTIONS IN-VIVO
SPLIT UBIQUITIN AS A SENSOR OF PROTEIN INTERACTIONS IN-VIVO
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DOI：
10.1073/pnas.91.22.10340
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发表时间：
1994-10-25
影响因子：
11.1
通讯作者：
VARSHAVSKY, A
中科院分区：
文献类型：
--
作者：
JOHNSSON, N;VARSHAVSKY, A
我们描述了体内蛋白质相互作用的测定。含有泛素（一种76个残基的单结构域蛋白）的蛋白融合物在体内被泛素特异性蛋白酶快速切割，所述蛋白酶识别泛素的折叠构象。当泛素的C-末端片段（C-ub）表达为与报告蛋白的融合物时，仅当泛素的N-末端片段（N-ub）也在同一细胞中表达时，融合物才被切割。通过体内切割测定可检测到的天然泛素从其片段的这种重建，在突变改变的N-ub中未观察到。然而，如果C-ub和改变的N-ub各自连接到在体内相互作用的多肽，则含有C-ub的融合物的切割被恢复，产生用于体内蛋白质相互作用的动力学和平衡方面的普遍适用的测定。这种方法，称为USPS（基于泛素的分裂蛋白传感器），使得有可能监测蛋白质-蛋白质相互作用作为时间的函数，在活细胞中这种相互作用的天然位点。
We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C-ub) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (N-ub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered N-ub. However, if C-ub and the altered N-ub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C-ub is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.