Protein – protein interaction (PPI) assay is a key technology for various fields from basic molecular biology to diagnosis and drug discovery. Previously, PPI assays based on fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA in short) are generally used. FRET- and BRET-based assays are established methods and their reproducibility is high. To conduct these assays, fluorescent proteins and luciferases are generally used, however, the interaction between large proteins is difficult to detect, because the signal intensity decreases inversely correlated with the 6^th power of the distance between the probes. In addition, the attainable signal/background (S/B) ratios are rather low. On the other hand, PCA is a user-friendly assay and shows high S/B ratio, and many enzymes and fluorescent proteins can be used. However, the signal intensity and the stability of the probes are low due to the split form of the probes.

Fig. 1.

To overcome the problems of these conventional methods, we developed a new PPI assay utilizing a unique property of firefly luciferase (Fluc) reaction. Namely, the light-emitting reactions of Fluc can be divided into two half-reactions, the adenylation step and the following oxidative luminescent steps. In the adenylation step, D-luciferin is reacted with ATP to produce a reaction intermediate luciferyl-adenylate (LH₂-AMP), while in the following steps LH₂-AMP is added with oxygen, decarboxylated, and converted to light-emitting oxyluciferin (OxL). To detect PPI, we made two mutant Flucs without luminescent activity on its own. One mutant called Donor is defective in converting LH₂-AMP to OxL. On the other hand, another mutant called Acceptor is defective in converting D-luciferin and ATP to LH₂-AMP. When the Donor and the Acceptor are each fused with an interacting protein, and are brought into close proximity due to the interaction, the Donor provides LH₂-AMP to the Acceptor, and the Acceptor produces OxL to emit light. We named this PPI assay as firefly luminescent intermediate-based protein interaction assay, FlimPIA.

FlimPIA already manifests several merits as an in vitro PPI assay. First, the interaction between larger proteins can be detected compared with FP-FRET assay and Fluc PCA, because the signal intensity of FlimPIA decreases more slowly according to the distance between the probes. Second, the S/B ratio attained was higher than that of those other assays. Third, the detection limit of PPI was the lowest in these assays. Fourth, the stability of probes is higher than that of Fluc PCA, probably due to the use of full-length enzyme as a probe. Fifth, the signal intensity was higher than that of Fluc PCA. Although FlimPIA performed in cellulo provides fewer benefits at this moment, we believe it will be improved soon.

FlimPIA is based on the unique principle of compensating the catalytic steps of an enzyme. Fluc belongs to an acyl/adenylate-forming enzyme superfamily. The chemical reactions catalyzed by the enzyme in this superfamily all consist of two half-reactions including an adenylation step, to which the principle of FlimPIA will be applied. Hopefully, by employing other enzymes in this family of enzymes, more useful PPI assays and other applications will be developed.

Yuki Ohmuro-Matsuyama, Hiroshi Ueda
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Japan
Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan

Publication

Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.
Kurihara M, Ohmuro-Matsuyama Y, Ayabe K, Yamashita T, Yamaji H, Ueda H
Biotechnol J. 2016 Jan

Read offline:     

Related Articles:

	Non-destructive inspection of metabolic status in… Bacteria are able to utilize various carbon sources, and change the intracellular metabolic fluxes depending on the availability. Whereas much effort has been taken to understand the underlying mechanisms, most…
	Cyagen Biosciences – Helping you choose the right… While many animal models are available “ off the shelf ” through various repositories and collaborations, generation of novel animal models has allowed for more effective studies, not limited by…
	Novel biophysical assay reveals that bacterial cell… Bacterial infections are increasingly difficult to treat with antibiotics due to antibiotic resistance. Knowledge on how of bacteria multiply needs to be profoundly expanded as a requirement for the development…
	Do you want a specific signal? Go local! Imaging our… Cyclic adenosine monophosphate (cAMP) is a ubiquitous intracellular second messenger, a small molecule that conveys the information carried by hormones, neurotransmitters and other extracellular stimuli to the intracellular environment. cAMP…
	Unregulated oxygen levels in mammalian cell culture… As aerobic organisms we continuously utilize oxygen from the surrounding environment which ultimately participates in the formation of useful bioenergetic intermediates. Approximately 21% of the air we breathe is composed…
	Mechanistic details of a copper-catalyzed C-H… Exploiting chemical or biological reactions by different spectroscopic techniques has become a powerful method for mechanistic analysis. Such studies enable chemists to understand several reaction features, optimize reaction conditions and…