Recombinant Firefly Luciferase Research Articles

A micro luminescence-based assay to measure serum susceptibility in Escherichia coli

Neonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.

Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays

Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.

Direct reagentless detection of the affinity binding of recombinant His-tagged firefly luciferase with a nickel-modified gold electrode

The direct reagentless electrochemical detection of recombinant firefly luciferase binding with a gold electrode modified with nickel complex of 1,16-di[4-(2,6-dihydroxycarbonyl)pyridyl]-1,16-dioxa-8,9-dithiahexadecane has been carried out.

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

A Real-Time Method for Measuring cAMP Production Modulated by Gαi/o-Coupled Metabotropic Glutamate Receptors

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Firefly Luciferase Inhibitor-Conjugated Peptide Quenches Bioluminescence: A Versatile Tool for Real Time Monitoring Cellular Uptake of Biomolecules

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

RNA-directed packaging of enzymes within virus-like particles.

RNA-directed packaging of enzymes within virus-like particles.

Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-alpha. Exposure of primary murine neurons to tumor necrosis factor-alpha resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease.

Protein transduction by lipidic peptide dendrimers

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology.

Firefly luciferase terminally degraded by mild heat exposure: Implications for reporter assays

Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and β-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with β-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.

Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction.

The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.

Glucose-stimulated oscillations in free cytosolic ATP concentration imaged in single islet beta-cells: evidence for a Ca2+-dependent mechanism.

Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K(+) channel activity and hence oscillatory Ca(2+) influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet beta-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose. In human beta-cells, oscillations were observed at both 3 and 15 mmol/l glucose, but the oscillations were of a longer wavelength at the higher glucose concentration (167 vs. 66 s). Mouse beta-cells displayed oscillations in both cytosolic free [Ca(2+)] and [ATP] only at elevated glucose concentrations, both with a period of 120 s. To explore the causal relationship between [Ca(2+)] and [ATP] oscillations, the regulation of each was further investigated in populations of MIN6 beta-cells. Incubation in Ca(2+)-free medium lowered cytosolic [Ca(2+)] but increased [ATP] in MIN6 cells at both 3 and 30 mmol/l glucose. Removal of external Ca(2+) increased [ATP], possibly by decreasing ATP consumption by endoplasmic reticulum Ca(2+)-ATPases. These results allow a model to be constructed of the beta-cell metabolic oscillator that drives nutrient-induced insulin secretion.

A Metal-Chelating Pluronic for Immobilization of Histidine-Tagged Proteins at Interfaces: Immobilization of Firefly Luciferase on Polystyrene Beads

Pluronic is a surface-active poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO−PEO) triblock copolymer. The PEO chains of the triblock form a hydrophilic, protein-repelling interface at hydrophobic surfaces. The protein-repelling property of Pluronic surfactants was used as an activity-preserving foundation on which to develop a scheme for specific protein immobilization at hydrophobic interfaces through immobilized metal affinity. A nitrilotriacetic acid (NTA) group was coupled to the terminal hydroxyl groups of the PEO chains of Pluronic F108 to create a metal-chelating Pluronic (F108-NTA). Recombinant firefly luciferase (FFL) with C-terminal histidine tags was used as a test protein. Histidine-tagged FFL adsorbed strongly to untreated polystyrene beads but retained much less than 1% of its bioluminescence activity. An equivalent amount of histidine-tagged FFL bound to polystyrene beads treated with the chelating Pluronic, but only in the presence of Ni2+ ions. The firefly luciferase immobilized on the chelating Pluronic retained at least 93% of its bioluminescence activity. The results demonstrate that the chelating Pluronic is a simple and versatile reagent for specific, oriented immobilization of histidine-tagged proteins on hydrophobic interfaces.

Streptavidin-Biotin Based Bioluminescent Enzyme Immunoassay Using Biotinylated Acetate Kinase and Recombinant Firefly Luciferase

Abstract Streptavidin-biotin system has been introduced to a bioluminescent enzyme immunoassay using biotinylated acetate kinase and recombinant firefly luciferase. The streptavidin-biotin based bioluminescent enzyme immunoassays (BLEIAs) for thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) were developed in which the complexes of biotinylated acetate kinase, streptavidin, and biotinylated antibody were used. In BLEIA for TSH, acetate kinase activity was determined by endpoint assay and for hCG the enzyme activity was measured by rate assay. The measurable ranges of TSH and hCG were 0.02 μIU/ml to 50 μIU/ml and 0.003 to 2 μIU/ml, respectively. The proposed streptavidin-biotinylated acetate kinase system was shown to be applicable to several sandwich type enzyme immunoassays employing biotinylated antibody with high sensitivity.

Bioluminescent Homogeneous Enzyme Binding Assay for Biotin Using Luciferase as a Label

Abstract Bioluminescent homogeneous enzyme binding assay was developed for biotin using recombinant firefly luciferase as a label. Biotinylated luciferase was prepared by the reaction of luciferase with biotinyl-e-aminocaproic acid N-hydroxyl succinimide ester. The biotinylated luciferase was inactivated by adding avidin. However, the inactivation was prevented in the presence of an excess amount of biotin. The measurable range of biotin was 15 pg/assay tube to 500 pg/assay tube. The detection limit of biotin was 15 pg/assay tube. The coefficient of variation (CV, n=5) for each point ranged from 1.4% to 11.9% (mean 7.9%). The proposed method is very convenient, rapid and sensitive by using homogeneous enzyme binding assay system and bioluminescent reaction as detection system.

Expression of recombinant firefly luciferase in prokaryotic and eukaryotic cells.

Expression of recombinant firefly luciferase in prokaryotic and eukaryotic cells.