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PubMed1996

[Physicochemical properties of recombinant luciferase from the firefly Luciola mingrelica and its mutant forms].

Physico-chemical properties of the recombinant L. mingrelica luciferase synthesized by E. coli cells have been studied. The catalytic and spectral properties of recombinant luciferase were similar to those of the native enzyme but the former was less stable in the presence of the additional Cys residue. The mutant forms of L. mingrelica firefly luciferase with point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala and Thr-204-->Asp, have been constructed using the method of site-specific mutagenesis. Mutations Cys-82,260,393-->Ala changed slightly the Km values for ATP and luciferin but did not influence kcat. The Cys-393-->Ala mutant appeared to be more stable in comparison with the native enzyme. Mutation Thr-204-->Asp resulted in a 8-fold increase in the ATP binding constant and in a 2-fold increase in the kcat, thus indicating that Thr-204 may be located in the ATP-binding region of luciferase. Dithiothreitol, ethylene glycol, bovine serum albumin and trehalose had a stabilizing effect on the native, recombinant and mutant luciferases.

Adenosine Triphosphate↗
PubMed1998

Immobilization of recombinant firefly luciferase. Physicochemical properties and application.

Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated Sepharose was investigated. The catalytic properties and analytical characteristics of the immobilized recombinant and native luciferases were comparatively studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase are close to those of the native luciferase, but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within the 0.01-10000 nM range.

Animals↗
PubMed2025

Functional Characterization of Luciferase in a Brittle Star Indicates Parallel Evolution Influenced by Genomic Availability of Haloalkane Dehalogenase.

Determining why convergent traits use distinct versus shared genetic components is crucial for understanding how evolutionary processes generate and sustain biodiversity. However, the factors dictating the genetic underpinnings of convergent traits remain incompletely understood. Here, we use heterologous protein expression, biochemical assays, and phylogenetic analyses to confirm the origin of a luciferase gene from haloalkane dehalogenases in the brittle star Amphiura filiformis. Through database searches and gene tree analyses, we also show a complex pattern of the presence and absence of haloalkane dehalogenases across organismal genomes. These results first confirm parallel evolution across a vast phylogenetic distance, because octocorals like Renilla also use luciferase derived from haloalkane dehalogenases. This parallel evolution is surprising, even though previously hypothesized, because many organisms that also use coelenterazine as the bioluminescence substrate evolved completely distinct luciferases. The inability to detect haloalkane dehalogenases in the genomes of several bioluminescent groups suggests that the distribution of this gene family influences its recruitment as a luciferase. Together, our findings highlight how biochemical function and genomic availability help determine whether distinct or shared genetic components are used during the convergent evolution of traits like bioluminescence.

Echinodermata↗
PubMedDate not supplied

Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.

Adenosine Triphosphate↗
PubMedDate not supplied

Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli.

We have cloned the cDNA for luciferase from lantern poly(A)+ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.

Amino Acid Sequence↗
PubMedDate not supplied

Stable luciferase transfected cells for studying steroid receptor biological activity.

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.

Animals↗
PubMedDate not supplied

Expression of firefly luciferase gene in Erwinia amylovora.

In this study we describe an efficient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the firefly luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our findings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.

Animals↗
PubMed2001

Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter.

A sequence of 1624 bp 5' to the apurinic/apyrimidinic (AP) endonuclease structural gene of Dictyostelium discoideum (APEA) has been inserted upstream of the luciferase reporter gene in pVTL2, an autonomously replicating nuclear plasmid in this organism. Cells transformed with this plasmid, designated pVTL-AL, displayed strong luciferase induction during treatment with the DNA-damaging agent bleomycin. For example, a luciferase activity of 45-fold above the constitutive level was observed for 20 hours of growth in axenic medium with 0.002 U/mL of bleomycin. The response was bleomycin concentration-dependent. Cell survival was greater than 90% for all treatments. The level of luciferase expression was highly dependent on the cell growth conditions, with the greatest induction observed for stationary phase axenically-grown cells. This effect may be related to a variation of plasmid copy number with growth conditions.

Animals↗
PubMed1994

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell-free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from its mRNA. It is shown that ribosome-bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half-time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.

Animals↗
PubMed2026

Screening of Estrogenic and Antiestrogenic Effects of Estradiol, Bisphenol A, and Fulvestrant Using 2D and 3D Breast Cancer Cell Systems With a Luciferase Reporter Gene Assay.

Endocrine-disrupting chemicals (EDCs) like bisphenol A (BPA) pose health risks by interfering with hormones. This study develops and utilizes in vitro 2D and 3D cell models to evaluate the estrogenic and antiestrogenic properties of compounds. Human breast cancer cell lines T47D and MCF7, stably transfected with a luciferase reporter gene (ERE-LUC), were first compared in 2D. Due to the significantly higher sensitivity and responsiveness observed in the T47D line during preliminary 2D screenings, this cell line was exclusively selected for the development of the 3D spheroid model. Cells were treated with 17β-estradiol (E2), BPA, and Fulvestrant (FUL) to assess cell viability and luciferase activity. In 2D models, T47D ERE-LUC cells showed higher responsiveness than MCF7 ERE-LUC, which failed to show significant luciferase induction with E2. In the 3D T47D model, cells exhibited significant and robust changes in luciferase activity in response to E2 and BPA, highlighting the enhanced fidelity of 3D cultures in replicating tissue conditions compared to their 2D counterparts. The study highlights the effectiveness of 3D models over 2D in evaluating estrogenic activity. Specifically, the 3D T47D ERE-LUC system serves as a superior, sensitive, and reliable platform for screening EDCs, offering benefits in cost, data speed, and reduced in vivo reliance.

Humans↗
PubMed1997

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

Biopolymers↗
PubMed2004

Androgen responsiveness of Renilla luciferase reporter vectors is promoter, transgene, and cell line dependent.

Renilla based reporters are frequently used as transfection controls for luciferase transcriptional reporter assays. However, recent evidence suggests that a commonly used reporter (HSV-thymidine kinase driven Renilla) is responsive to androgen receptor (AR) and glucocorticoid receptors in the presence of the cognate ligands, dihydrotestosterone (DHT) and dexamethasone (DEX), respectively [1]. We further validate this important technical difficulty by illustrating that in LNCaP prostate cancer cells, spurious Renilla luciferase activity is a function of (a) the promoter driving Renilla expression, (b) the presence of co-transfected transgenes, and (c) the androgen responsiveness of the cell line used. Using inhibitors of transcription and translation we showed that transcript interference or translational modulation is not a major means by which androgens affect Renilla luciferase activity. As luciferase reporter assays are a frequent means of studying transcriptional co-regulation in the highly androgen dependent LNCaP cell line, our data serves as a cautionary note that alternative normalization techniques should be employed to avoid misinterpretation of data.

Androgens↗
PubMed1999

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

Horseradish peroxidase (HRP) catalyzes the oxidative chemiluminescent reaction of luminol, and firefly luciferase catalyzes the oxidation of firefly D-luciferin. Here we report a novel substrate, 5-(5'-azoluciferinyl)-2,3-dihydro-1,4-phthalazinedione (ALPDO), that can trigger the activity of HRP and firefly luciferase in solution because it contains both luminol and luciferin functionalities. It is synthesized by diazotization of luminol and its subsequent azo coupling with firefly luciferin. NMR spectral data show that the C5' of benzothiazole in luciferin connects the diazophthalahydrazide. The electronic absorption and fluorescence properties of ALPDO are different from those of its precursor molecules. The chemiluminescence emission spectra of the conjugate substrate display biphotonic emission characteristic of azophthalatedianion and oxyluciferin. It has an optimum pH of 8.0 for maximum activity with respect to HRP as well as luciferase. At pH 8.0 the bifunctional substrate has 12 times the activity of luminol but has 7 times less activity than the firefly luciferin-luciferase system. The specific enhancement of light emission from the cyclic hydrazide part of ALPDO helped in the sensitive assay of HRP down to 2.0 x 10(-13) M and of ATP to 1.0 x 10(-14) mol. Addition of enhancers such as firefly luciferin and p-iodophenol (PIP) to the HRP-ALPDO-H2O2 system enhanced the light output.

Azo Compounds↗
PubMed1993

Prior arterial injury enhances luciferase expression following in vivo gene transfer.

We determined the time course of gene expression following DNA/Lipofectin transfection of normal or previously injured arterial segments using direct intraluminal infusion following surgical exposure. Constructs possessing the firefly luciferase cDNA regulated by Simian virus 40, Rous sarcoma virus, or alpha-actin promoter were incubated together with Lipofectin for 30 minutes. Arterial segments were assayed for luciferase activity following harvest at 2-21 days. Without prior injury, luciferase activity was only 2.5-fold greater than background two days following gene transfer. Arterial injury three days before gene transfer resulted in luciferase activity 12.5-fold over background levels. This observation has clinical implications with regard to gene therapy following angioplasty, a procedure that is associated with endothelial cell denudation and smooth muscle cell proliferation. Maintenance of gene expression for several days could ameliorate the early smooth muscle migration and proliferation following arterial injury.

Actins↗
PubMed1999

The emitting species dissociated from the enzyme can emit the light in Photinus pyralis luciferase system.

By fluorescent results, it has been proposed that the product (excited oxyluciferin) bounded in the firefly luciferase emits the light. Also it is generally accepted that two forms (keto and enol) of the oxyluciferin emit red and yellow-green light, respectively. In order to demonstrate the existence of free emitting species using His-tagged luciferase (His-luc), N-terminal 6x His-tagged luciferase (Photinus pyralis) was prepared and expressed in E. coli. After immobilization of His-luc on the membrane by IDA-Ni(2+)-His complex, His-luc clearly showed spectral changes of the emission toward red light. The luciferase-free product obtained from enzymatic reaction mixture in the presence of ATP and dATP emits the light with maximal wavelengths of 575 and 620 nm, respectively. Based on these results, we suppose that two different emitting species or two different energy levels of the emitting species are responsible for two different color lights.

Animals↗
PubMed2001

Probing local conformational changes during equilibrium unfolding of firefly luciferase: fluorescence and circular dichroism studies of single tryptophan mutants.

Firefly luciferase is a monomeric protein composed of two globular domains. There is a wide cleft between the two domains. The N-terminal domain can be further divided into A-, B-, and C-subdomains. Previous studies showed that in vitro unfolding of firefly luciferase induced by guanidinium chloride can be described as a four-state equilibrium with two inactive intermediates (Herbst, R., et al. (1997) J. Biol. Chem. 272, 7099-7105). In order to monitor spectroscopically the conformational changes that occur in the different domains and subdomains during the multi-state unfolding process, we constructed a series of single-tryptophan mutants. These mutants were purified and characterized and shown to retain essentially all of the structural properties of the wild-type luciferase. Under equilibrium conditions, the unfolding of each mutant protein were studied by means of fluorescence and circular dichroism. The results show that different conformational changes occur in specific regions, suggesting a sequential unfolding process for firefly luciferase. Under 2.5 M GdmCl, whereas the N-domain unfolds partially holding half of the secondary structure content, the C-domain unfolds almost completely. In the equilibrium intermediate I(2), the secondary structure might stem mostly from the A- and B- subdomains.

Animals↗
PubMed1998

NF-AT-luciferase reporter T cell lines as tools to screen immunosuppressive drugs.

The development of safer analogues of immunosuppressants such as cyclosporin A and FK506 is an important goal for a number of clinical applications ranging from transplantation to the treatment of autoimmune diseases. Here we show the generation and the characterization of Jurkat T cell lines stably transfected with a reporter construct containing the firefly luciferase gene under the control of NF-AT. These lines specifically respond in a cyclosporin A-sensitive manner to T cell antigen receptor-derived signals. Due to the high levels of luciferase activity expression fewer than 1000 cells are required for detection of luciferase. In addition, a simplified luciferase assay allows to reduce both the manipulations and the time required for the assay, making these lines potentially useful models for the automated screening of cyclosporin A and FK506 analogues.

Cyclosporine↗
PubMed1995

Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters.

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.

Adenoviruses, Human↗