Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts
Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts
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DOI：
10.1111/j.1365-313x.2007.03214.x
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发表时间：
2007-10-01
期刊：
影响因子：
7.2
通讯作者：
Kato, Naohiro
中科院分区：
文献类型：
--
作者：
Fujikawa, Yukichi;Kato, Naohiro
我们开发了一种分裂荧光素酶互补测定法来研究拟南芥原生质体中的蛋白质-蛋白质相互作用。在此测定中，海肾荧光素酶的 N 端和 C 端片段分别翻译融合至诱饵蛋白和猎物蛋白。当蛋白质相互作用时，分裂荧光素酶被激活并发出可以通过微孔板发光计测量的冷光。通过首先在 96 孔板中用 DNA 载体转化原生质体来测量裂解荧光素酶活性。通过两个独立的体外 DNA 重组反应 Gateway 和 Cre-loxP 构建表达诱饵和猎物基因的 DNA 载体。作为概念证明，我们检测了拟南芥原生质体中核组蛋白 2A 和 2B 之间的蛋白质-蛋白质相互作用，以及膜蛋白 SYP（植物突触蛋白）51 和 SYP61 之间的相互作用。
We developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.