title: 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.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
authors.forename: Sung Bae
authors.initials: SB
authors.lastname: Kim
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Otani
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D001517:Coleoptera / Q000201:enzymology*; D008024:Ligands; D008156:Luciferases / Q000032:analysis* / Q000378:metabolism*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011944:Receptors, Androgen / Q000032:analysis / Q000378:metabolism
chemical_list: D000596:Amino Acids; D008024:Ligands; D010446:Peptide Fragments; D011944:Receptors, Androgen; D008156:Luciferases