Bioluminescent Indicator for Determining Protein−Protein Interactions Using Intramolecular Complementation of Split Click Beetle Luciferase

Abstract

Click beetle luciferase (CBLuc) is insensitive to pH, temperature, and heavy metals, and emits a stable, highly tissue-transparent red light with luciferin in physiological circumstances. Thus, the luminescence signal is optimal for a bioanalytical index reporting the magnitude of a signal transduction of interest. Here, we validated a single-molecule-format complementation system of split CBLuc to study signal-controlled protein-protein (peptide) interactions. First, we generated 10 pairs of N- and C-terminal fragments of CBLuc to examine respectively whether a significant recovery of the activity occurs through the intramolecular complementation. The ligand binding domain of androgen receptor (AR LBD) was connected to a functional peptide sequence through a flexible linker. The fusion protein was then sandwiched between the dissected N- and C-terminal fragments of CBLuc. Androgen induces the association between AR LBD and a functional peptide and the subsequent complementation of N- and C-terminal fragments of split CBLuc inside the single-molecule-format probe, which restores the activities of CBLuc. The examination about the dissection sites of CBLuc revealed that the dissection positions next to the amino acids D412 and I439 admit a stable recovery of CBLuc activity through an intramolecular complementation. The ligand sensitivity and kinetics of the single molecular probe with split CBLuc were discussed in various cell lines and in different protein-peptide binding models. The probe is applicable to developing biotherapeutic agents on the AR signaling and for screening adverse chemicals that possibly influence the signal transduction of proteins in living cells or animals.

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

“…In this system, the amino-and carboxy-terminal domains of luciferase are fused to strongly interacting peptides that are linked by an enzyme-specific sequence. [27][28][29][30][31][32][33][34] Cleavage of the linker permits the interacting peptides to associate, allowing reconstitution of luciferase activity. Coppola et al 15 described development of a split firefly luciferase reporter strategy in which DEVD is used as the intervening cleavage sequence and also reported the ability to detect activation of apoptosis in vivo after treatment with chemotherapeutic agents.…”

Techniques for the analysis of protein-protein interactions in vivo

“…Table IV provides an overview of the different systems used in planta, where the luciferases from the North American firefly (Photinus pyralis) and the sea pansy (Renilla reniformis) are utilized exclusively, while in mammalian cells, other luciferases derived from click beetle, Gaussia spp. and nanoLUC also have been used (Remy and Michnick, 2006;Kim et al, 2007;Dixon et al, 2016).…”

Determination of the Androgenicity of Ligands Using a Single-chain Probe Carrying Androgen Receptor N-Terminal Peptides

“…We previously introduced an integrated-molecule-format (IMF) bioluminescent indicator for determining ligand-activated protein-protein (peptide) interactions. [8][9][10] This IMF probe provides an advanced methodology for determining weak protein-protein (peptide) interactions of interest. Here, the methodological advantage of IMF probes was demonstrated through an exploration of the binding chemistry between AR LBD and the functional peptides in AR NTD.…”