Ohmuro-Matsuyama Yuki, Ueda Hiroshi
Laboratory for Chemistry and Life Science, Institute for Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.
Methods Mol Biol. 2017;1596:119-130. doi: 10.1007/978-1-4939-6940-1_8.
We recently developed a protein-protein interaction assay, FlimPIA (Firefly luminescent intermediate-based Protein-protein Interaction Assay) based on the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named "Donor" and "Acceptor" where the oxidative luminescent activity of the Donor is almost eliminated and the adenylation activity of the Acceptor is suppressed. When the Donor and the Acceptor are each fused to one of two interacting partners, and put together to interact, the Donor and the Acceptor come sufficiently close such that the Acceptor can react with the luciferyl-adenylate intermediate (LH-AMP) produced by the Donor, and thus emit luminescence.FlimPIA can be used in vitro and in cultured cells. Owing to recent improvements, it has several advantages in terms of signal/background ratio, detectable size of interacting partner, and sensitivity over conventional protein-protein interaction assays based on Förster/fluorescence resonance energy transfer and protein-fragment complementation performed in vitro. Here, we describe a protocol to make use of the latest version of FlimPIA which shows even lower background and higher signal than previously described ones.
我们最近开发了一种蛋白质-蛋白质相互作用检测方法，即基于萤火虫荧光素酶（Fluc）催化机制的FlimPIA（基于萤火虫发光中间体的蛋白质-蛋白质相互作用检测方法），该机制可分为两个半反应：腺苷化步骤和氧化发光步骤。我们设计了两种突变型Fluc酶，分别命名为“供体”和“受体”，其中供体的氧化发光活性几乎被消除，受体的腺苷化活性受到抑制。当供体和受体分别与两个相互作用的伙伴之一融合，并放在一起相互作用时，供体和受体足够靠近，使得受体能够与供体产生的荧光素-腺苷酸中间体（LH-AMP）反应，从而发出荧光。FlimPIA可用于体外和培养细胞。由于最近的改进，与基于Förster/荧光共振能量转移和体外进行的蛋白质片段互补的传统蛋白质-蛋白质相互作用检测方法相比，它在信号/背景比、可检测的相互作用伙伴大小和灵敏度方面具有几个优点。在这里，我们描述了一种使用最新版本FlimPIA的方案，该方案显示出比先前描述的方案更低的背景和更高的信号。
