scholarly journals An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

2015 ◽  
Vol 484 ◽  
pp. 148-153 ◽  
Author(s):  
Bruce R. Branchini ◽  
Tara L. Southworth ◽  
Danielle M. Fontaine ◽  
Dawn Kohrt ◽  
Munya Talukder ◽  
...  
Keyword(s):  
Firefly Luciferase ◽  
Atp Detection

Ultrasensitive ATP Detection Using Firefly Luciferase Entrapped in Sugar-Modified Sol−Gel-Derived Silica

2004 ◽  
Vol 126 (22) ◽  
pp. 6878-6879 ◽  
Author(s):  
Jorge A. Cruz-Aguado ◽  
Yang Chen ◽  
Zheng Zhang ◽  
Nadine H. Elowe ◽  
Michael A. Brook ◽  
...  
Keyword(s):  
Sol Gel ◽  
Firefly Luciferase ◽  
Atp Detection

scholarly journals 5,5-Dialkylluciferins are thermal stable substrates for bioluminescence-based detection systems

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243747
Author(s):  
Ce Shi ◽  
Michael P. Killoran ◽  
Mary P. Hall ◽  
Paul Otto ◽  
Monika G. Wood ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Firefly Luciferase ◽  
Breakdown Product ◽  
Detection Systems ◽  
Oxidative Breakdown ◽  
Luciferase Enzyme ◽  
Cost Efficient ◽  
The Individual ◽  
Detection Reagent ◽  
Atp Detection

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.

Cell-surface-localized ATP detection with immobilized firefly luciferase

2006 ◽  
Vol 352 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Makiko Nakamura ◽  
Masayasu Mie ◽  
Hisakage Funabashi ◽  
Kimiko Yamamoto ◽  
Joji Ando ◽  
...  
Keyword(s):  
Cell Surface ◽  
Firefly Luciferase ◽  
Atp Detection

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Sulfonamides Are an Overlooked Class of Electron Donors in Luminogenic Luciferins and Fluorescent Dyes

2018 ◽  
Author(s):  
Deepak K. Sharma ◽  
Spencer T. Adams ◽  
Kate L. Liebmann ◽  
Adam Choi ◽  
Stephen Miller
Keyword(s):  
Fluorescent Dyes ◽  
Bright Light ◽  
Firefly Luciferase ◽  
Electron Donors ◽  
Light Emitting

Many fluorophores, and all bright light-emitting substrates for firefly luciferase, contain hydroxyl or amine electron donors. Here we show that sulfonamides can serve as replacements for these canonical groups. Unlike “caged” carboxamides, sulfonamide analogues enable bioluminescence, and sulfonamidyl luciferins, coumarins, rhodols, and rhodamines are fluorescent in water. Sulfonamide donors thus have previously unappreciated potential to expand the functional repertoire of luminescent molecules.

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

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