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

Characteristic Scales of Baryon Acoustic Oscillations from Perturbation Theory: Non-linearity and Redshift-Space Distortion Effects

An acoustic oscillation of the primeval photon-baryon fluid around the decoupling time imprints a characteristic scale in the galaxy distribution today, known as the baryon acoustic oscillation (BAO) scale. Several on-going and/or future galaxy surveys aim at detecting and precisely determining the BAO scale so as to trace the expansion history of the universe. We consider nonlinear and redshift-space distortion effects on the shifts of the BAO scale in $k$-space using perturbation theory. The resulting shifts are indeed sensitive to different choices of the definition of the BAO scale, which needs to be kept in mind in the data analysis. We present a toy model to explain the physical behavior of the shifts. We find that the BAO scale defined as in Percival et al. (2007) indeed shows very small shifts ($\lesssim$ 1%) relative to the prediction in {\it linear theory} in real space. The shifts can be predicted accurately for scales where the perturbation theory is reliable.Comment: 21 pages, 9 figures, references and supplementary sections added, accepted for publication in PAS

Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation

The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) <i>in vitro</i>. Since the detection of antigen-induced approximation of the two antibody variable region fragments V_H and V_L by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which V_H and V_L were each fused and expressed in <i>Escherichia coli</i> cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems

Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants

Firefly luciferase has been widely used in biotechnology and biophotonics due to photon emission during enzymatic activity. In the past, the effect of amino acid substitutions (mutants) on the enzymatic activity of firefly luciferase has been characterized by the Michaelis constant, KM. The KM is obtained by plotting the maximum relative luminescence units (RLU) detected for several concentrations of the substrate (luciferin or luciferyl-adenylate). The maximum RLU is used because the assay begins to violate the quasi-steady state approximation when RLU decays as a function of time. However, mutations also affect the time to reach and decay from the maximum RLU. These effects are not captured when calculating the KM. To understand changes in the RLU kinetics of firefly luciferase mutants, we used a Michaelis–Menten model with the non-steady state approximation. In this model, we do not assume that the amount of enzyme–substrate complex is at equilibrium throughout the course of the experiment. We found that one of the two mutants analyzed in this study decreases not only the dissociation rate (koff) but also the association rate (kon) of luciferyl-adenylate, suggesting the narrowing of the structural pocket containing the catalytic amino acids. Furthermore, comparative analysis of the nearly complete oxidation of luciferyl-adenylate with wild-type and mutant firefly luciferase reveals that the total amount of photons emitted with the mutant is 50-fold larger than that with the wild type, on average. These two results together indicate that the slow supply of luciferyl-adenylate to the enzyme increases the total number of photons emitted from the substrate, luciferyl-adenylate. Analysis with the non-steady state approximation model is generally applicable when enzymatic production kinetics are monitored in real time