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Information on EC 1.13.12.6 - Cypridina-luciferin 2-monooxygenase
for references in articles please use BRENDA:EC1.13.12.6
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EC Tree 1 Oxidoreductases
1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)
1.13.12 With incorporation of one atom of oxygen (internal monooxygenases or internal mixed-function oxidases)
1.13.12.6 Cypridina-luciferin 2-monooxygenase
IUBMB Comments
Cypridina is a bioluminescent crustacea. The luciferins (and presumably the luciferases, since they cross-react) of some luminous fish (e.g. Apogon, Parapriacanthus, Porichthys) are apparently similar. The enzyme may be assayed by measurement of light emission.
The enzyme appears in viruses and cellular organisms
- 1.13.12.6
-
bioluminescence
-
luminescence
- hilgendorfii
- luciferases
-
ostracod
- firefly
- analysis
- gaussia
- diagnostics
- molecular biology
- biotechnology
showSynonyms
clase, cypridina luciferase, vargula luciferase, cypridina noctiluca luciferase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CLuc
Cypridina luciferase
Cypridina luciferin 2-monooxygenase
-
-
-
-
Cypridina noctiluca luciferase
Cypridina-type luciferase
-
-
-
-
Fbp
-
biotinylated Cypridina luciferase conjugated to far-red fluorescent indocyanine derivative
FBP-IgG
-
conjugate of reduced monoclonal antibody to conjugated enzyme FBP
luciferase (Cypridina luciferin)
-
-
-
-
PGE2-luciferase
-
recombinant luciferase conjugated with N-hydroxysuccinimidyl ester of prostaglandin E2
Cypridina luciferase
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cypridina luciferin + O2 = oxidized Cypridina luciferin + CO2 + hnu
light, mechanism
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Cypridina luciferin + O2 = oxidized Cypridina luciferin + CO2 + hnu
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
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reduction
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
Cypridina-luciferin:oxygen 2-oxidoreductase (decarboxylating)
Cypridina is a bioluminescent crustacea. The luciferins (and presumably the luciferases, since they cross-react) of some luminous fish (e.g. Apogon, Parapriacanthus, Porichthys) are apparently similar. The enzyme may be assayed by measurement of light emission.
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CAS REGISTRY NUMBER
COMMENTARY
61969-99-1
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(S)-Cypridina luciferin I + O2
oxidized S-Cypridina luciferin I + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
1-{3-[6-(1H-indol-3-yl)-2-(1-methylpropyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl]propyl}guanidine + O2
N-[3-(3-carbamimidamidopropyl)-5-(1H-indol-3-yl)-1,4-dihydropyrazin-2-yl]-2-methylbutanamide + CO2 + hn
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
?
-
Substrates: fusion enzyme + 10 mM luciferin (dissolved in ethanol in 30 mM Tris-HCl (pH 7.6) with 10 mM EDTA) with ATP (250 mM), CoA (250 microM), and MgCl2 (5 mM) in 100 mM Tris-HCl, pH 7.8
Products: Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
Products: Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
?
DL-Cypridina luciferin + O2
oxidized DL-Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
luciferin + O2
oxidized luciferin + CO2 + hnu
-
Substrates: -
Products: -
Products: -
r
Vargula hilgendorfii luciferin + O2
oxidized Vargula hilgendorfii luciferin + CO2 + hv
Substrates: native or recombinant substrate protein
Products: -
Products: -
?
additional information
?
-
Cypridina sp.
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Substrates: evaluation of a method of measuring activity of Cypridina luciferase under conditions which minimize complicating with a relatively simple photoelectric light integrator, detailed overview
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: bioluminescence
Products: bioluminescence
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: bioluminescence, maximum wavelength emission at 465 nm
Products: bioluminescence, maximum wavelength emission at 465 nm
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
Products: conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: luciferase reaction catalyzed by CLuc is first order with respect to their luciferin. The rate of light generation by CLuc is linear with respect to the concentration of its substrate, Cypridina luciferin
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Cypridina sp.
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: very low bioluminescence rate with biluciferyl
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: Cypridina luciferin is [3-[3,7-dihydro-6-(1H-indol-3-yl)-2-[(S)-1-methyl-6-propyl]-3-oxoimidazo[1,2-a]pyrazin-8-yl]propyl]guanidine, equimolar complex of oxyluciferin with luciferase in an excited state is the light-emitter of Cypridina bioluminescence
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Cypridina luciferin + O2
?
-
Substrates: fusion enzyme + 10 mM luciferin (dissolved in ethanol in 30 mM Tris-HCl (pH 7.6) with 10 mM EDTA) with ATP (250 mM), CoA (250 microM), and MgCl2 (5 mM) in 100 mM Tris-HCl, pH 7.8
Products: Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
Products: Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: bioluminescence
Products: bioluminescence
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: bioluminescence, maximum wavelength emission at 465 nm
Products: bioluminescence, maximum wavelength emission at 465 nm
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
Products: conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Cypridina sp.
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
-
Substrates: -
Products: -
Products: -
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
Substrates: -
Products: -
Products: -
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
evidence that luciferase requires a divalent metal ion, possibly calcium for activity
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
chlorpromazine
which binds to human plasma alpha 1-acid glycoprotein, shows an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of human plasma alpha 1-acid glycoprotein and a recombinant Cypridina luciferase
Cypndina gamma-globulin
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Cypridina luciferase is completely inhibited by a rabbit antibody to Cypridina luciferase
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Urea
Cypridina sp.
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at 5.0 M urea, the half-time of the enzyme is 45 min, at 6.0 M urea 8 min, and at 7.5 M urea, the enzyme is inactivated within 1 min. r´Reversible, non-competitive inhibition of the luciferase activity occurs at concentrations up to 1.5 M
additional information
-
Apogon luciferase is not inhibited by a rabbit antibody to Cypridina luciferase
-
additional information
-
not inhibited by N-ethylmaleimide, N-methylmaleimide, iodoacetamide and p-chloromercuribenzoate
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
human plasma alpha 1-acid glycoprotein
a mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produces light. The total value of the luminescence intensity over 60 s is over 12.6fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Hemiplegia
The performance profile on the Wisconsin Card Sorting Test of a group of children with cerebral palsy aged between 9 and 12.
Neoplasms
Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.
Sepsis
Biologics and 30-Day Postoperative Complications After Abdominal Operations for Crohn's Disease: Are There Differences in the Safety Profiles?
Syphilis
Syphilis in the Americas: a protocol for a systematic review of syphilis prevalence and incidence in four high-risk groups, 1980-2016.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.03
1-{3-[6-(1H-indol-3-yl)-2-(1-methylpropyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl]propyl}guanidine
pH 7.4
0.00028
Cypridina luciferin
recombinant enzyme, pH and temperature not specified in the publication
0.0016
Cypridina luciferin
-
35 nM recombinant prostaglandin E2-luciferase diluted with 0.1 M Tris buffer (pH 7.4) containing 0.05% bovine serum albumin, 1 nM monoclonal anti-prostaglandin E2 antibody, incubation for 18 h, 4°C, in the dark, addition of 1 microM Cypridina luciferin in 0.1 M Tris-HCl buffer (pH 7.4) containing 0.3 M sodium ascorbate and 0.02 M Na2SO3 for bioluminescence reading
0.00045
Vargula hilgendorfii luciferin
recombinant enzyme, recombinant substrate, pH and temperature not specified in the publication
-
0.00052
Vargula hilgendorfii luciferin
recombinant enzyme, native substrate, pH and temperature not specified in the publication
-
additional information
additional information
-
Michaelis-Menten kinetics
-
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
-
Michaelis-Menten kinetics
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
recombinant luciferase exhibits 14% activity of wild-type Cypridina luciferase
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5 - 8
-
no difference in normalized bioluminescence spectra of conjugated enzyme in 100 mM sodium phosphate buffer (pH 6.5), 100 mM Tris-HCl buffer (pH 7.4) or 100 mM Tris-HCl buffer (pH 8) each containing 0.1 M NaCl
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
the enzyme Cluc contains a secretion signal peptide, which results in efficient secretion and folding of Cluc
-
brenda
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
Producibility and relative specific activity are apparently reduced in Cluc mutated in the phosphorylation sites, although the thermostability and secretion efficiency are not affected. Defects in the glycosylation modification are not related to secretion process and stability of the protein
physiological function
the enzyme enables Cypridina luciferin to emit light efficiently through an oxidation reaction
evolution
Parapriacanthus ransonneti, a bioluminescent fish, obtains not only its luciferin but also its luciferase enzyme from bioluminescent ostracod prey. Experiments where fish are fed with Vargula hilgendorfii, demonstrate the specific uptake of the luciferase to the fish's light organs. This kleptoprotein system allows an organism to use novel functional proteins that are not encoded in its genome and provides an evolutionary alternative to DNA-based molecular evolution
evolution
Parapriacanthus ransonneti, a bioluminescent fish, obtains not only its luciferin but also its luciferase enzyme from bioluminescent ostracod prey. The enzyme purified from the fish's light organs is identical to the luciferase of Cypridina noctiluca, a bioluminescent ostracod that they feed upon. This kleptoprotein system allows an organism to use novel functional proteins that are not encoded in its genome and provides an evolutionary alternative to DNA-based molecular evolution
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). LUCI_VARHI
555
0
61627
Swiss-Prot
Secretory Pathway (Reliability: 3)
B7PK58_IXOSC
329
0
37347
TrEMBL
other Location (Reliability: 2)
B7PQH0_IXOSC
1043
0
115877
TrEMBL
Mitochondrion (Reliability: 2)
G0R6M5_ICHMG
Ichthyophthirius multifiliis (strain G5)
658
2
74444
TrEMBL
other Location (Reliability: 1)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
200000
-
SDS-PAGE, reduced antibody-avidin conjugate of the conjugated enzyme (FBP-IgG)
52000 - 57000
-
determination of diffusion and sedimentation constant, gel filtration, sedimentation equilibrium
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
glycoprotein
glycosylation pattern analysis using LCMS/MS. In the recombinnat enzyme, two potential N-glycosylation sites are modified with plant-type oligosaccharide chains. Partial deglycosylation (trimming) of recombinant Cluc by beta-N-acetylhexosaminidase, HexNAc residues at the non-reducing termini of glycopeptides (Pronase E-digested) are completely eliminated at both glycosylation sites after beta-N-acetylhexosaminidase treatment (at Asn182 and Asn404). The glycan compositions after treatment displays trimannosyl cores (Hex3HexNAc2)+/-Fuc+/-Xyl. Specifically, because the trimannosyl core is fully occupied by Fuc and Xyl on Asn404, only a single signal corresponding to the trimannosyl core +Fuc +Xyl remains after treatment. This glycan-trimmed recombinant protein still exhibits nearly the same level of activity as the unmodified glycoprotein
glycoprotein
the enzyme has two N-glycosylation sites with the consensus sequence for N-glycosylation (Asn-X-Ser/Thr). The producibility and relative specific activity are apparently reduced in Cluc mutated in the phosphorylation sites, although the thermostability and secretion efficiency are not affected. N-glycosylation modifications and the proper amino acid sequence of the N-glycan binding sites of Cluc are required for the complete protein folding to form a stable catalytic center, for the proper conformation of substrate-protein interaction residues, or for both. Defects in the glycosylation modification are not related to secretion process and stability of the protein
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
N182D
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar compared to the wild-type
N182D/N404D
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
N182D/S406A
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
N404D
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar to the wild-type
S406A
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type
T184A
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type
T184A/N404D
T184A/S406A
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
additional information
T184A/N404D
site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over 2.5fold increased protein accumulation compared to the wild-type
additional information
-
biotynilation of the enzyme by attachment of an Avi-tag of a 16-residue peptide to the C-terminus of the luciferase, conjugation to the indocyanine derivative HiLyte Fluor 647 hydrazine via the glycol-chains of the enzyme
additional information
-
conjugation of prostaglandin E2 to luciferase by producing the N-hydroxysuccinimidyl ester of prostaglandin E2 by reaction of N-hydroxysuccinimde and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride and conjugation of the ester to luciferase (ratio 18/1) in 0.1 M potassium phosphate buffer, pH 7.2, with 0.15 M NaCl for 13 h on ice
additional information
-
construction of a cold-induced expression vector (pCold-ZZ-VL vector) in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of Staphylococcuss aureus protein A (ZZ-domain) and multiple cloning sites, fusing Cypridina luciferase to the ZZ-domain results in expression by Escherichia coli of a soluble cytoplasm enzyme but it is not bioluminescent (in contrast to other luciferases fused to the ZZ-domain)
additional information
-
establishment and validation of a yeast reporter assay, for detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system, using a secretory luciferase, Cypridina noctiluca luciferase CLuc, as an alternative to the conventional beta-galactosidase, the CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay, determination of the transcriptional activity of hundreds of yeast promoter fragments, overview
additional information
concept of tumor monitoring using dual luciferases, construction of the expression vector, and evaluation of tumor monitoring systems using dual luciferases from Cypridina noctiluca and firefly Photinus pyralis, overview. The enzymes are expressed in human breast cancercells MDA-MB-231, followed by inoculatin of the MDA-MB-231/FIC cell suspension subcutaneously into the back of 6-week-old male nude mice lacking T-cell function (BALB/cAJcl-nu/nu). The expressed CLuc is secreted into the blood from the cells and circulates in the living body. The blood containing CLuc can be drawn by using glass micro-hematocrit capillary tubes or a syringe
additional information
for large scale enzyme production, a simple production procedure is established using plant cell cultures. The plant cell culture tobacco BY-2 efficiently secretes luciferase, which is easily purified using a simple one-step ion-exchange chromatography method. The production yield is 20-30 mg of luciferase per liter of culture medium. The method offers a cost-effective production for Cypridina luciferase
additional information
-
for large scale enzyme production, a simple production procedure is established using plant cell cultures. The plant cell culture tobacco BY-2 efficiently secretes luciferase, which is easily purified using a simple one-step ion-exchange chromatography method. The production yield is 20-30 mg of luciferase per liter of culture medium. The method offers a cost-effective production for Cypridina luciferase
additional information
wild-type and mutant enzymes show similar secretion efficiencies, and thermostabilities at 25-37°C, overview
additional information
-
wild-type and mutant enzymes show similar secretion efficiencies, and thermostabilities at 25-37°C, overview
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is resistant to the treatment of proteases, heat, or urea
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, lyophilized Cypridina luciferin is stable for more than 1 year and the enzyme in acidic ethanol solution for at least 1 month
-
4°C, PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl, 2 months, without significant loss of activity
-
Cypridina luciferase occasionally loses almost all of its activity when chromatographed through the 90-cm Sephadex column, irrespective of whether the column is newly poured or has been used for a period. The loss also occurs with both newly dissolved and old solutions of Cypridina luciferase
-
purified native Apogon luciferase gradually loses activity on standing in the cold over a period of weeks
-
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
biotinylated enzyme is incubated with 1 microM sodium metaperiodate in 0.1 M sodium acetate buffer (pH 5.2), reaction stopped by glycerol addition, loading onto a size exclusion column, oxidized biotinylated enzyme eluted with the same buffer, pooling of bioluminescent fractions, concentration by ultrafree-0.5 centrifugal filter device, addition of 10 mM HiLyte Fluor 647 hydrazine in 0.1 M sodium acetate buffer (pH 5.2), after incubation loading onto a size-exclusion column, elution with 0.1 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl. Treatment of human Delta-like protein (Dlk-1) antibody with 2-mercaptoethylamine (cleaving hinge-region disulfide bonds between heavy chains of antibody molecules) and conjugation of half antibodies to maleimide-activated avidin and purification on a size-exclusion column results in a 200 kDa conjugate
-
Escherichia coli cell culture with expression vector is cooled on ice-water bath, expression induced, incubation for 18 h at 15°C, cell concentration by centrifugation, solution in 50 mM Tris-HCl (pH 7.6) and 10 mM EDTA, disruption by sonication, centrifugation, SDS-PAGE
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loading of recombinant prostaglandin E2 luciferase onto size-exclusion column, elution with 0.1 M potassium phosphate buffer, pH 7.2, and 0.15 M NaCl, fractions with bioluminescent activities (Cypridina luciferin solution, 0.1 M Tris-HCl, pH 7.4, 0.3 M sodium ascorbate, 0.2 M Na2SO3) are combined and stored at -30°C
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native enzyme by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
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native enzyme from photogenic organs by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
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Ni affinity column chromatography, SA beads column chromatography, monomeric avidin column chromatography, and TSK G300SW gel filtration
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recombinant secreted enzyme from Nicotiana benthamiana BY-2 cell medium by ultrafiltration, buffer exchange gel filtration, anion exchange chromatography, and dialysis
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of a chimeric protein G-luciferase enzyme in COS-1 and CHO cells
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expression of human Delta-like protein (Dlk-1) antigen in HEK-293 cells, immunization of mice (BALB/c) with HEK293-Dlk-1 cells or with the expression vector that encodes full-length Dlk-1 cDNA
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recombinant expression from vector p35SHSPG in Nicotiana benthamiana BY-2 cells, luciferase is efficiently secreted from cells with its native signal peptide, two potential N-glycosylation sites are modified with plant-type oligosaccharide chains. Recombinant expression of the enzyme in Arabidopsis thaliana roots in the cell wall of intact cells and the apoplast of plasmolyzed cells but not in the protoplasm. Cluc contains its own secretion signal peptide, which results in efficient secretion and folding of Cluc
recombinant expression of wild-type and mutant enzymes in COS-1 cells and in Pichia pastoris strain GS115
recombinant mutant enzyme lacking N-glycosylation motifs is produced using Expi293F cells, silkworms, and tobacco BY-2 cells. The relative activity of the produced Dmt CLucEX is almost the same as that of the wild-type CLucEX, whereas recombinant CLuc deficient in N-glycosylation binding sites (Dmt CLucCO) is approximately 20% of that of wild-type CLucCO. In the case of the silkworm system, the relative activity of mutant CLuc purified using only anti-FLAG affinity gel is 25% of that of wild-type CLucSW. However, further purification reveals that approximately 75% of the FLAG purified recombinant protein is aggregated and its enzyme activity is lost. In the BY-2 cells, attempt to express Dmt CLucBY result in no recombinant protein production despite the use of 4 independent transgenic callus clones. The productivity of CLuc mutated in the N-glycosylation sites differs depending on the expression host
Staphylococcus aureus ZZ-domain gene of protein A is amplified by PCR using pEZZ18 plasmid, the gene for Cypridina luciferase (obtained by PCR) is fused to the C-terminus of the ZZ-domain in the vector to give the expression vector pCold-ZZ-VL, the vector is then expressed in Escherichia coli BL21 grown in Luria-Bertani broth at 37°C
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
diagnostics
molecular biology
analysis
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intramolecular bioluminescence resonance energy transfer system consisting of a fusion protein of the enzyme and enhanced yellow fluorescent protein for investigating peptide processing in living cells
analysis
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dual reporter assay using one luciferase for monitoring gene expression and a second as an internal control, based on secreted luciferases from Cypridina noctiluca and Gaussia princeps that do not require lysis or special equipment. The assay can be carried out sequentially, the activities are high and can be measured in a small volume and a simple procedure. Development of a one-tube reporter assay for the two enzymes
analysis
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use of enzyme as a reporter in a screening system of Saccharomyces cerevisiae to improve secretion efficiency in yeast
analysis
Cypridina noctiluca luciferase is utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays
analysis
the secreted Cypridina luciferase (CLuc) is used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase is used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Tumor monitoring systems using dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals, e.g. BALB/cAJcl-nu/nu mice. Continuous non-invasive measurement of CLuc activity for tumor growth allows us to determine a suitable time to assess tumor spatial distribution and growth, thereby, reducing the animal stress and the experimental cost
analysis
the thermostable enzyme can be used for various research applications, including in vivo imaging and high throughput reporter assays
diagnostics
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Cypridina luciferase is used as bioluminescence reporter enzyme in yeast, bacterial, and mammalian cell-based assays, enzyme activity is measured with a luminometer by addition of native or synthetic luciferin, also known as Vargulin, 0.5 microM in 10 mM Tris-HCl, pH 7.4
diagnostics
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far-red luminescence imaging technology to visualize tumor specific antigens on cell surfaces in vitro (human hepato-cellular carcinoma cell line Huh-7) and in living bodies (mice xenografted with human Delta-like protein-positive Huh-7 cells), based on a far-red fluorescent indocyanine derivative conjugated to biotinylated Cypridina lucife