The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Sanjiv Sam Gambhir - One of the best experts on this subject based on the ideXlab platform.
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BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals
The FASEB Journal, 2009Co-Authors: Abhijit De, Andreas Markus Loening, Sanjiv Sam GambhirAbstract:Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (∼85 nm), BRET3 shows improved...
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crystal structures of the luciferase and green fluorescent protein from Renilla reniformis
Journal of Molecular Biology, 2007Co-Authors: Andreas Markus Loening, Timothy D Fenn, Sanjiv Sam GambhirAbstract:Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.
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red shifted Renilla reniformis luciferase variants for imaging in living subjects
Nature Methods, 2007Co-Authors: Andreas Markus Loening, Sanjiv Sam Gambhir, Anna M WuAbstract:The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.
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Self-illuminating quantum dot conjugates for in vivo imaging
Nature Biotechnology, 2006Co-Authors: Min Kyung So, Andreas Markus Loening, Sanjiv Sam Gambhir, Chenjie Xu, Jianghong RaoAbstract:Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.
Milton J Cormier - One of the best experts on this subject based on the ideXlab platform.
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[29] Protein-protein interactions as measured by bioluminescence energy transfer in Renilla
Methods in Enzymology, 2004Co-Authors: William W. Ward, Milton J CormierAbstract:Publisher Summary This chapter discusses protein–protein interactions that occur in a variety of biochemical systems. Classic examples include (1) proteins containing multiple subunits, (2) self-associating oligomeric proteins, (3) protein self-assembly systems, (4) antigen–antibody systems, and (5) multienzyme complexes. Firefly luciferase is active as a 50,000-molecular weight monomer at low-protein concentrations, but at higher-protein concentrations, in low-ionic strength buffer, the monomers associate to form fully active dimers. The active form of bacterial luciferase is a dimer of nonidentical subunits. In the sea pansy ( Renilla reniformis) , luciferase 12 can also be shown to associate with an accessory protein known as the “green-fluorescent protein (GFP).” The chapter presents experimental evidence for protein–protein interaction between luciferase and GFP in the Renilla system and describes how this interaction can be measured by the assay of in vitro energy transfer.
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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, Dennis J Okane, A A Escher, 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.
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isolation and expression of a cdna encoding Renilla reniformis luciferase
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Walter W Lorenz, Richard O Mccann, Mathew Longiaru, Milton J CormierAbstract:Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.
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amino acid sequence of the ca2 triggered luciferin binding protein of Renilla reniformis
FEBS Letters, 1990Co-Authors: Santosh Kumar, Milton J Cormier, Maria Harrylock, Kenneth A Walsh, Harry CharbonneauAbstract:Abstract The complete amino acid sequence of the Ca2+-triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated Mr of 20541. LBP is a member of the EF-hand superfamily of Ca2+-binding proteins and bears three predicted EF-hand domains. The sequence and organization of EF-hand domains are similar to those of the Ca2+-dependent photoprotein, aequorin.
Andreas Markus Loening - One of the best experts on this subject based on the ideXlab platform.
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BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals
The FASEB Journal, 2009Co-Authors: Abhijit De, Andreas Markus Loening, Sanjiv Sam GambhirAbstract:Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (∼85 nm), BRET3 shows improved...
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crystal structures of the luciferase and green fluorescent protein from Renilla reniformis
Journal of Molecular Biology, 2007Co-Authors: Andreas Markus Loening, Timothy D Fenn, Sanjiv Sam GambhirAbstract:Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.
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red shifted Renilla reniformis luciferase variants for imaging in living subjects
Nature Methods, 2007Co-Authors: Andreas Markus Loening, Sanjiv Sam Gambhir, Anna M WuAbstract:The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.
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Self-illuminating quantum dot conjugates for in vivo imaging
Nature Biotechnology, 2006Co-Authors: Min Kyung So, Andreas Markus Loening, Sanjiv Sam Gambhir, Chenjie Xu, Jianghong RaoAbstract:Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.
Harry Charbonneau - One of the best experts on this subject based on the ideXlab platform.
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amino acid sequence of the ca2 triggered luciferin binding protein of Renilla reniformis
FEBS Letters, 1990Co-Authors: Santosh Kumar, Milton J Cormier, Maria Harrylock, Kenneth A Walsh, Harry CharbonneauAbstract:Abstract The complete amino acid sequence of the Ca2+-triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated Mr of 20541. LBP is a member of the EF-hand superfamily of Ca2+-binding proteins and bears three predicted EF-hand domains. The sequence and organization of EF-hand domains are similar to those of the Ca2+-dependent photoprotein, aequorin.
Walter W Lorenz - One of the best experts on this subject based on the ideXlab platform.
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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, Dennis J Okane, A A Escher, 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.
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the sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in candida albicans
Journal of Bacteriology, 1996Co-Authors: Thyagarajan Srikantha, Walter W Lorenz, Luong K. Tsai, A Klapach, L A Laughlin, J A Gorman, David R. SollAbstract:The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.
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isolation and expression of a cdna encoding Renilla reniformis luciferase
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Walter W Lorenz, Richard O Mccann, Mathew Longiaru, Milton J CormierAbstract:Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.