Protein-Protein Interaction Assays Using Split-NanoLuc

Abstract

Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further developments are anticipated.Bioluminescence -Analytical Applications and Basic Biology 2 very bright luminescence. Based on this attractive enzyme, PCA systems were developed [13,14]. This innovation on the NanoLuc PCA improves the luminescent signal, which is markedly better than the conventional PCA signal obtained using other luciferases. Herein, we will focus on the new PCA technology and its application, and further discuss potential improvements in the system. PCA using NanoLucVerhoef et al. constructed a PCA system using NanoLuc [14]. They made several pairs of NanoLuc fragments by cutting at several loop regions, and selected a pair comprised of the N-terminal 52-amino acid (aa) fragment and the C-terminal 119-aa fragment (Figure 2A). These fragments were used to successfully detect the interaction between the transactivation domain fragment of p53 and Mdm2.At almost the same time, Dixon et al. developed another NanoLuc-based PCA system designated NanoLuc Binary Technology (NanoBiT) [13]. This was devised by first identifying a dissection site from 90 candidate sites. An 18-kDa N-terminal fragment and 13-aa C-terminal fragment were selected. The K D value between these fragments was 6 μM. This low affinity was suitable for PCA, but their use was hampered by the very low stability of the N-terminal fragment. The sequence of the N-terminal fragment was optimized from an N-terminal library containing 15,000 variants. The optimization increased the luminescent signal by 300-fold when the two fragments were interacting, which was 37% that of the wild-type NanoLuc. However, the affinity between the N-and C-terminal fragments became too strong for PCA (K D = 900 nM). As a next step, the sequence of the C-terminal peptide was optimized from 350 variants. Finally, two fragments were obtained. They were designated LgBiT (18 kDa) and SmBiT (11 aa). These exhibited significantly low

Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent

“…Ternary Nluc was previously engineered − as an extension of NanoBiT, a binary Nluc-based complementation technology . NanoBiT was generated by splitting Nluc, a 10-strand β-barrel luciferase, between C-terminal strands 9 and 10.…”

“…Ternary Nluc is an extension of NanoBiT, where additional dissection was carried out to remove and isolate β-strand 9 from LgBiT − (Figure a). The result is a ternary reporter complementation system consisting of two small β-strand peptide reporters that can be attached to the interacting target proteins and a 17 kDa polypeptide (referred to as LgTrip) that can be part of a detection reagent.…”

A Homogeneous Split-Luciferase Assay for Rapid and Sensitive Detection of Anti-SARS CoV-2 Antibodies

“…Different sensors such as fluorescent proteins, transcription factors, proteases and more have been successfully used in various designs. Among them, split luciferases have been shown to have the advantages of high signal/noise ratio and rapid reconstitution, making them widely used [17][18][19] . However, the conventional strategy of splitting luciferase into two fragments has limitations.…”

Luciferase-Based Biosensors in the Era of the COVID-19 Pandemic

“… 19 − 21 In contrast to PCAs with green fluorescent protein, luciferase-based PCAs allow for a reversible approach for the detection of temporal dynamics of protein–protein interactions. 22 In their application to virology, split luciferase assays have proven incredibly useful in studying protein–protein interactions between viral and host cell proteins. They have also been used to quantify viral protease activity and have further been applied successfully in examining viral cell entry by enveloped viruses.…”

“…The luciferase enzymatic activity is restored upon assembly of the N-terminal fragment with the C-terminal fragment . In general, the N-terminus of a luciferase enzyme determines its spectral characteristics, whereas the C-terminus contributes to its enzymatic function. − In contrast to PCAs with green fluorescent protein, luciferase-based PCAs allow for a reversible approach for the detection of temporal dynamics of protein–protein interactions . In their application to virology, split luciferase assays have proven incredibly useful in studying protein–protein interactions between viral and host cell proteins.…”