Johnsson N, Varshavsky A
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10340-4. doi: 10.1073/pnas.91.22.10340.
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 (Nub) 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 Nub. However, if C(ub) and the altered Nub 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.
我们描述了一种用于体内蛋白质相互作用的检测方法。包含泛素（一种由76个残基组成的单结构域蛋白质）的蛋白质融合体在体内会被泛素特异性蛋白酶迅速切割，这些蛋白酶能够识别泛素的折叠构象。当泛素的C末端片段（C(ub)）作为与报告蛋白的融合体表达时，只有在同一细胞中也表达了泛素的N末端片段（Nub）时，该融合体才会被切割。通过体内切割检测可检测到从其片段中重建天然泛素的情况，但在突变的Nub中则不会出现这种情况。然而，如果C(ub)和改变后的Nub分别与在体内相互作用的多肽相连，那么包含C(ub)的融合体的切割就会恢复，从而产生一种普遍适用的检测方法，用于检测体内蛋白质相互作用的动力学和平衡方面。这种方法被称为USPS（基于泛素的分裂蛋白传感器），它使得在活细胞中该相互作用的天然位点上，能够将蛋白质-蛋白质相互作用作为时间的函数进行监测。
