A Protein鈥揚rotein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases

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• 作者：Yuki Ohmuro-Matsuyama ; Kota Nakano ; Aoi Kimura ; Keiichi Ayabe ; Masaki Ihara ; Takeshi Wada ; Hiroshi Ueda
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：August 20, 2013
• 年：2013
• 卷：85
• 期：16
• 页码：7935-7940
• 全文大小：322K
• 年卷期：v.85,no.16(August 20, 2013)
• ISSN：1520-6882

文摘

We report a novel bioluminescent protein鈥損rotein interaction (PPI) assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH₂-AMP) intermediate is produced from LH₂ and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH₂-AMP. We created and optimized two Fluc mutants; one is named 鈥淒onor鈥? which virtually lacks oxidative activity, while the other, named 鈥淎cceptor鈥? is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure proteins. When the interaction between the partners is induced, higher efficiency of LH₂-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.