A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction
A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction
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DOI：
10.5772/intechopen.75644
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发表时间：
2018-04
期刊：
影响因子：
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通讯作者：
Y. Ohmuro-Matsuyama;H. Ueda
中科院分区：
文献类型：
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作者：
Y. Ohmuro-Matsuyama;H. Ueda
蛋白质片段互补试验（PCA）通常用于测定蛋白质-蛋白质相互作用（PPI）。虽然基于细胞或裂解物中的萤火虫荧光素酶（Fluc）的PCA是一种用户友好的方法，给出了高的信号/背景（S/B）比，但由于分离的Fluc片段的不稳定性，它们难以在体外使用。为了解决这一问题，我们开发了一种新的蛋白质-蛋白质相互作用检测方法，命名为FlimPIA，该方法使用两种突变的Flucs，每种突变的Flucs催化野生型酶催化的两个半反应之一。在通过拴系蛋白对近似后，由于反应中间体腺苷酸的更有效的转移，两个突变体产生更高的信号。FlimPIA与体外裂解Fluc检测法相比，具有检测距离长、探针稳定性好、检测时间短等优点，并可用于细胞内检测。
Protein-fragment complementation assays (PCAs) are commonly used to assay protein–pro- tein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we devel oped a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more sta - ble probes, and higher signal readout in a shorter time period, and it also worked in cellulo.