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At least 163 records · Page 9Linked to original sources
PubMed2006

cDNA cloning, expression and homology modeling of a luciferase from the firefly Lampyroidea maculata.

The cDNA of a firefly luciferase from lantern mRNA of Lampyroidea maculata has been cloned, sequenced and functionally expressed. The cDNA has an open reading frame of 1647 bp and codes for a 548-residue-long polypeptide. Noteworthy, sequence comparison as well as homology modeling showed the highest degree of similarity with H. unmunsana and L. mingrelica luciferases, suggesting a close phylogenetic relationship despite the geographical distance separation. The deduced amino acid sequence of the luciferase gene of firefly L. maculata showed 93% identity to H. unmunsana. Superposition of the three-dimensional model of L. maculata luciferase (generated by homology modeling) and three dimensional structure of Photinus pyralis luciferase revealed that the spatial arrangements of Luciferin and ATP-binding residues are very similar. Putative signature of AMPbinding domain among the various firefly species and Lampyroidea maculata was compared and a striking similarity was found. Different motifs and sites have been identified in Lampyroidea maculata by sequence analysis. Expression and purification of luciferase from Lampyroidea maculata was carried out using Ni-NTA Sepharose. Bioluminescence emission spectrum was similar to Photinus pyralis luciferase.

Amino Acid Sequence↗
PubMed2003

[Construction of an expression vector with a bicistron of green fluorescent protein and HCV 5'NCR controlled luciferase gene and its expression in hepatocytes].

OBJECTIVE: To construct an expression vector with a bicistron of green fluorescent protein (GFP) and HCV 5'NCR controlled luciferase gene and to explore its expression in hepatocytes. METHODS: With molecular cloning techniques, the DNA fragment of HCV 5'NCR and its controlled luciferase gene was obtained from plasmid pCMVNCRLuc and subcloned into pEGFP-C1, an expression plasmid of GFP. A recombinant plasmid pGFPNCRLuc with a bicistron of GFP and HCV 5'NCR controlled luciferase gene was constructed, and liposome-mediated transient expression was detected in hepatocyte QSG7701 cells. RESULTS: The recombinant plasmid expressed both GFP and luciferase at a high level. The expression activity of GFP showed no significant difference compared to that of pEGFP-C1 (P > 0.05), nor did that of luciferase to pCMVNCRLuc (P > 0.05). CONCLUSION: An expression vector with a bicistron of GFP and HCV 5'NCR controlled luciferase gene was successfully constructed. GFP and luciferase could express independently. This study can promote the establishment of an anti-HCV drug screen system.

Genes, Reporter↗
PubMed2006

[Comparison of the relative luciferase activity in secondary CEF by different heterogenous strong promoters, MDV gB promoter and the composed promoters].

To improve the protection efficiency of the recombinant Marek' s disease viruses (MDV) in chickens with or without maternal antibodies,the work of selecting the optimal promoters for the construction of recombinant MDV was carried out. Combined with the efficient genetic manipulation, the composed promoters was constructed by use of the MDV gB core promoter with the regulatory elements from the early immediately promoter and enhancer of hCMV, the promoter and enhancer of SV40 or the partial enhancer of hCMV. And these composed promoters were ligased to the luciferase to construct the eukaryotic expressing vectors and named PhCMV-gB, Psv-gB and Pen-gB, respectively. In vitro, these vectors and internal standard plasmid (pSV-beta-LacZ) were transiently co-transfected into secondary CEF by FuGene 6 Transfection Reagent. Furthermore, cells were harvested 48 hours after transfection. Then the luciferase activity was detected by a luciferase assay kit, at the same time, the beta-galactosidase enzyme activity was detected by a beta-galactosidase enzyme assay kit, and the luciferase activity was corrected by the beta-galactosidase enzyme activity to get the relative luciferase activity. The relative luciferase activity was used as the transcriptional activity. By comparison of the relative luciferase activity of every promoter, it was found that these composed promoters could more effectively drive the reporter gene expression than the full legth of gB promoter did. Among them, PhCMV-gB robustly drove the reporter gene expression. On the other hand, PSV-gB and Pen-gB appeared to have the same strength; But compared with the commercial strong promoters, the transcriptional activity of the composed promoter were less than as or the same as that of the strong promoters. Therefore, at a sense, it can be proposed that these composed promoters have not only the characteristic of MDV gB promoter, but also that of the commercial strong promoters. These provide the choices for further developing the new-type recombinant MDV vaccine.

Animals↗
PubMed1980

[Kinetics and mechanism of inactivation and reactivation of immobilized luciferase from fireflies Luciola mingrelica and the role of sulfhydryl in these processes].

The role of SH-groups of the enzyme in the catalytic and inactivation processes of soluble and immobilized on Sepharose 4B luciferase from the fireflies Luciola mingrelica was studied. A significant role of SH-groups of the soluble and immobilized enzyme was established by titrating the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid. It was shown that the rate of inactivation of immobilized luciferase is limited by oxidation of the SH-groups of the enzyme; therefore dithiothreitol effectively protects the immobilized enzyme against inactivation. The kinetics of inactivation and reactivation of immobilized luciferase in the presence of dithiothreitol were studied. Dithiothreitol had no protective effect on soluble luciferase, which is indicative of another limiting step of inactivation of soluble luciferase. A mechanism of inactivation of Luciola mingrelica luciferase is proposed. The kinetic pattern of these processes is postulated and the rate constants of individual steps of inactivation of luciferase immobilized on Sepharose 4B were determined.

Animals↗
PubMed1996

[Participation of DnaK chaperone and ATP in in vivo folding of firefly luciferase, synthesized by E. coli cells].

The time-courses of protein accumulation and luciferase activity of Luciola mingrelica firefly luciferase synthesized on plasmid pJG lambda in E. coli strains omega 238 and B178groE7 (DnaK- and GroEL-deficient, correspondingly) and omega 237 and W3110 that are control to them, have been studied. It was shown that the amounts of the luciferase protein synthesized by all strains was approximately equal to 3.0-3.9% of the total cell protein. Luciferase was synthesized in a catalytically inactive form and transformed into an active enzyme during incubation at 21 degrees C. In the DnaK-deficient E. coli strain omega 238, the enzyme transformation into a catalytically active form did not occur which provides evidence for participation of the DnaK chaperone in transformation of newly synthesized luciferase into catalytically active form. Luciferase folding was accelerated with an increase in ATP concentration inside the cell. It is possible to increase the ATP concentration inside the cell by treatment with polymyxin and addition of ATP to the culture medium, which significantly accelerates the luciferase folding.

Adenosine Triphosphate↗
PubMed1996

Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia.

Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.

Animals↗
PubMed2005

Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo.

Photoproteins have played a major role in advancing our understanding of biological processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. hGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under standard imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.

Animals↗
PubMed2001

Single cell reporter assay using cell surface displayed Vargula luciferase.

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.

Journal Article↗
PubMedDate not supplied

Immobilization of luciferase from a firefly lantern extract on glass strips as an alternative strategy for luminescent detection of ATP.

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at -80 degrees C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research.

Adenosine Triphosphate↗
PubMedDate not supplied

Detection of luciferase having two kinds of luminescent colour based on optical filter procedure: application to an enzyme immunoassay.

This paper reports on our study using several optical filters known to be efficient in separating compounds having various levels of maximum luminescence, to separate information from three kinds of Luciola lateralis luciferase with a maximum luminescence of 559 nm, 604 nm and 607 nm. Simultaneous luminescence of Luciola lateralis luciferase was determined by measuring the luminescence through a band pass filter or sharp cut filter (BPB50, 53, 58, No.58, SC58, 60, 62, 64). It was possible to determine luciferase with a maximum luminescence lambda(max) of 559 nm (yellow-green) utilizing the band pass filter (BPB 50), described here. Meanwhile, luciferase with a lambda(max) of 607 nm (red) could be determined by calculations based on the bioluminescent intensity through the band pass filter and sharp cut filter (SC58). In addition, we also applied a simultaneous bioluminescent enzyme immunoassay of pepsinogen I (PGI) and pepsinogen II (PGII) in which two kinds of biotinylated luciferase (Luciola lateralis) labelled as an enzyme producing yellow-green light (lambda(max) = 559 nm) and red light (lambda(max) = 607 nm) were used. In the proposed method, PGI and PGII in serum were simultaneously captured in a sandwich-type immune reaction between anti-PGI and anti-PGII monoclonal antibody-coated magnetic particles, and streptavidin-biotinylated luciferase biotinylated anti-PGI and anti-PGII monoclonal antibodies triplexes, respectively. The result was a calibration range for PGI of 2-200 ng/mL, and for PGII of 1-100 ng/mL. In conclusion, the correlation of PG values in serum between the proposed method (simultaneous assay) and an individual specific bioluminescent immunoassay (specific assay) were satisfactory.

Animals↗
PubMedDate not supplied

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.

Animals↗
PubMed1989

Site-directed mutagenesis of bacterial luciferase: analysis of the 'essential' thiol.

It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the alpha subunit, has been replaced with a seryl residue. The resulting alpha 106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH2 dissociation constant. The alpha 106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (alpha 106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position alpha 106. An alpha 106Ala variant luciferase was also active, but the alpha 106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the alpha 106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at alpha 106.

Aldehydes↗
PubMed1989

Introduction to beetle luciferases and their applications.

All beetle luciferases have evolved from a common ancestor: they all use ATP, O2, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.

Amino Acid Sequence↗
PubMed1989

New approaches to the preparation and application of firefly luciferase.

Allowing for the lipid nature of firefly luciferase we have developed a new method for obtaining high-activity and high-stability enzyme preparations for bioluminescent microassay. The method includes the step of differential centrifugation in presence of stabilizing additives which entails a partial purification of the enzyme and its essential stabilization likely due to the fact that luciferase retains its lipid environment which plays an important role in catalysis. The resultant luciferase preparation is stable in solution at 4 degrees C for 2-3 months and allows the detection of down to 10(-11) M ATP. A new method has been offered for luciferase immobilization on film carriers precoated with a phospholipid layer. By sorption of the enzyme on such carriers, the samples of immobilized luciferase have been obtained suitable for constructing chemiluminescent biosensors, in the form of luciferase-containing films. There are many-fold applications for detection of ATP micro-quantities.

Animals↗
PubMedDate not supplied

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase.

The interaction of triazine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methylthio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 x 10(-30) m3. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 x 10(-30) m3 or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.

Aldehydes↗
PubMed2004

Identification of luciferyl adenylate and luciferyl coenzyme a synthesized by firefly luciferase.

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.

Adenosine Triphosphate↗
PubMed2001

Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase.

The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.

Animals↗
PubMed2003

Characterization of transcriptional activity of taurine transporter using luciferase reporter constructs.

Although taurine transporter (TAUT) activity has been known to be regulated by diverse intracellular and extracellular factors involved in the signal transduction pathway, such as protein kinase C, intracellular Ca concentration, and glucocorticoids, little is known concerning the underlying mechanisms. Evidence suggests that such stimulation-mediated changes in TAUT activity in mammalian cells are partly achieved through the modulation of TAUT transcription activity. In order to better understand the regulation of TAUT transcription activity and subsequently the role of taurine in the signal transduction pathway, we have cloned and sequenced the 5' flanking region of the human TAUT gene, and characterized the TAUT promoter region in human cells. For these reasons, the TAUT luciferase reporter vector was constructed using the 5' flanking region of the TAUT gene (1800 bp). The TAUT luciferase reporter vector was then transfected into SiHa cells, and luciferase activity was measured. The construct containing its own promoter of TAUT (pGL3 b TAU31) showed a 10 fold higher luciferase activity compared to the value found in the empty vector (pGL3 b). This implies a functional transcription of the homologous TAUT promoter. Similar results were obtained with the exon deleted construct [pGL3 b TAU31(-e)]. We also constructed the TAUT luciferase reporter gene (pGL3 pro TAU13) using a heterologous promoter. About 2.5 fold higher luciferase activity was observed in cells transfected with this construct containing the heterologous promoter compared to the value found in the control vector (PGL3 pro).

Base Sequence↗