The Experts below are selected from a list of 9153 Experts worldwide ranked by ideXlab platform
Aladar A Szalay - One of the best experts on this subject based on the ideXlab platform.
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Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.
Molecular genetics and genomics : MGG, 2002Co-Authors: Yubao Wang, Gefu Wang, Shahrokh Shabahang, Aladar A SzalayAbstract:Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the 'humanized' GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.
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A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.
Molecular & general genetics : MGG, 2001Co-Authors: Yubao Wang, Gefu Wang, D. J. O'kane, Aladar A SzalayAbstract:We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.
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visualizing and quantifying protein secretion using a Renilla luciferase gfp fusion protein
Luminescence, 2000Co-Authors: Jingxue Liu, Aladar A Szalay, Yubao Wang, Alan P EscherAbstract: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.
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The Renilla Luciferase-Modified GFP Fusion Protein is Functional in Transformed Cells
BioHydrogen, 1998Co-Authors: Yubao Wang, Dennis J. O'kane, Gefu Wang, Aladar A SzalayAbstract:The cDNA of Renilla reniformis luciferase (ruc) has been cloned and used successfully as a marker gene in a variety of transgenic species. Similarly, the transfer and expression of green fluorescent protein (GFP) cDNA (gfp) and its mutants from Aequorea victoria resulted in high levels of GFP in transformed cells, allowing convenient visualization of gene expression under the microscope.
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expression of the Renilla reniformis luciferase gene in mammalian cells
Journal of Bioluminescence and Chemiluminescence, 1996Co-Authors: Walter W Lorenz, Milton J Cormier, A A Escher, Dennis J Okane, Aladar A SzalayAbstract:: A cDNA encoding the Renilla reniformis luciferase was expressed in similan and murine cells in a transient and stable manner, respectively. Light emission catalyzed by luciferase was detected from transfected cells both in vitro and in vivo. This work establishes the Renilla luciferase gene as a new efficient marker of gene expression in mammalian cells.
Ludmila A Frank - One of the best experts on this subject based on the ideXlab platform.
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ca 2 triggered coelenterazine binding protein from Renilla as an enzyme dependent label for binding assay
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Vasilisa V Krasitskaya, Ludmila A Frank, Svetlana V Markova, Galina A Stepanyuk, S I Korneeva, A N KudryavtsevAbstract:The recombinant Ca2+-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.
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Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2008Co-Authors: Galina A Stepanyuk, Ludmila A Frank, Zhijie Liu, John Lee, Svetlana Markova, Bicheng WangAbstract:Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three “EF-hand” Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 A, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.
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coelenterazine binding protein of Renilla muelleri cdna cloning overexpression and characterization as a substrate of luciferase
Photochemical and Photobiological Sciences, 2008Co-Authors: Maxim S Titushin, Ludmila A Frank, Svetlana V Markova, Galina A Stepanyuk, Natalia P Malikova, John J LeeAbstract:The Renilla bioluminescent system in vivo is comprised of three proteins—the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.
John J Lee - One of the best experts on this subject based on the ideXlab platform.
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coelenterazine v ligated to ca2 triggered coelenterazine binding protein is a stable and efficient substrate of the red shifted mutant of Renilla muelleri luciferase
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Galina A Stepanyuk, Svetlana V Markova, James Unch, Natalia P Malikova, John J LeeAbstract:It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.
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structure based mechanism of the ca2 induced release of coelenterazine from the Renilla binding protein
Proteins, 2009Co-Authors: John P Rose, Zhijie Liu, Galina A Stepanyuk, John J Lee, Bicheng WangAbstract:The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex.
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coelenterazine binding protein of Renilla muelleri cdna cloning overexpression and characterization as a substrate of luciferase
Photochemical and Photobiological Sciences, 2008Co-Authors: Maxim S Titushin, Ludmila A Frank, Svetlana V Markova, Galina A Stepanyuk, Natalia P Malikova, John J LeeAbstract:The Renilla bioluminescent system in vivo is comprised of three proteins—the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.
Jeremy W Peck - One of the best experts on this subject based on the ideXlab platform.
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detecting protein protein interactions using Renilla luciferase fusion proteins
BioTechniques, 2002Co-Authors: Peter D Burbelo, Adam E Kisailus, Jeremy W PeckAbstract:We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.
Galina A Stepanyuk - One of the best experts on this subject based on the ideXlab platform.
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ca 2 triggered coelenterazine binding protein from Renilla as an enzyme dependent label for binding assay
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Vasilisa V Krasitskaya, Ludmila A Frank, Svetlana V Markova, Galina A Stepanyuk, S I Korneeva, A N KudryavtsevAbstract:The recombinant Ca2+-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.
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coelenterazine v ligated to ca2 triggered coelenterazine binding protein is a stable and efficient substrate of the red shifted mutant of Renilla muelleri luciferase
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Galina A Stepanyuk, Svetlana V Markova, James Unch, Natalia P Malikova, John J LeeAbstract:It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.
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structure based mechanism of the ca2 induced release of coelenterazine from the Renilla binding protein
Proteins, 2009Co-Authors: John P Rose, Zhijie Liu, Galina A Stepanyuk, John J Lee, Bicheng WangAbstract:The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex.
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Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2008Co-Authors: Galina A Stepanyuk, Ludmila A Frank, Zhijie Liu, John Lee, Svetlana Markova, Bicheng WangAbstract:Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three “EF-hand” Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 A, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.
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coelenterazine binding protein of Renilla muelleri cdna cloning overexpression and characterization as a substrate of luciferase
Photochemical and Photobiological Sciences, 2008Co-Authors: Maxim S Titushin, Ludmila A Frank, Svetlana V Markova, Galina A Stepanyuk, Natalia P Malikova, John J LeeAbstract:The Renilla bioluminescent system in vivo is comprised of three proteins—the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.