Development of rapid immunoasssay using nanoluc-derived peptide tags

Introduction Conventional immunoassays require multiple steps, and are labor and time-consuming. Hence, rapid and handy immunoassays with high sensitivity are desired. Protein-fragment complementation assay (PCA) of an enzyme such as luciferase will be a promising approach to realize such homogeneous immunoassay. Previously, a luciferase from the deep sea shrimp Oplophorus gracilirostris (NanoLuc, Nluc) was divided into two fragments, SmBit (11 aa) and LgBit (18 kDa) [Fig. 1 left], and each fused with interacting partners [1]. When expressed in mammalian cells, upon interaction, increased luminescence was observed. However, detection of PCA using purified proteins in vitro has been elusive. The affinity between the two antibody variable region fragments, VL and VH, remarkably increases when antigen binds to them (Open-sandwich principle) [2]. In this study, first, LgBit and SmBit were fused with VH and VL to detect antigen peptide [Fig. 1, left]. Next, LgBit was divided into MidBit and SmBit2 (11 aa), and SmBit and SmBit2 were each fused with VL and VH, respectively, to obtain antigen-dependent luminescent signal upon reconstitution with MidBit [Fig. 1, right]. Methods cDNA for SmBit, SmBit2 and MidBit were synthesized by Eurofin Genomics (Tokyo, Japan). VL and VH fragments of anti-BGP antibody [3] were each fused with LgBit and SmBit, or fused with SmBit and SmBit2, respectively. They were expressed as a fusion protein with thioredoxin and His6 tag in E. coli SHuffle Express T7 LysY, and purified by TALON immobilized metal affinity resin. Please click Additional Files below to see the full abstract

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction

Protein-fragment complementation assays (PCAs) are commonly used to assay protein–protein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we developed a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more stable probes, and higher signal readout in a shorter time period, and it also worked in cellulo

Protein-Protein Interaction Assays Using Split-NanoLuc

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

Mathematical model of the firefly luciferase complementation assay reveals a non-linear relationship between the detected luminescence and the affinity of the protein pair being analyzed

© 2016 Dale et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively