Information on EC 1.14.14.3 - alkanal monooxygenase (FMN)

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.14.14.3
-
RECOMMENDED NAME
GeneOntology No.
alkanal monooxygenase (FMN)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
an aldehyde + FMNH2 + O2 = a carboxylate + FMN + H2O + hnu
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
bacterial bioluminescence
-
-
SYSTEMATIC NAME
IUBMB Comments
alkanal,FMNH2:oxygen oxidoreductase (1-hydroxylating, luminescing)
The reaction sequence involves incorporation of a molecule of oxygen into reduced FMN, and subsequent reaction with the aldehyde to form an activated FMN.H2O complex, which breaks down with emission of light. The enzyme is highly specific for reduced FMN, and for long-chain aliphatic aldehydes with eight carbons or more.
CAS REGISTRY NUMBER
COMMENTARY hide
9014-00-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
not classified
-
-
Manually annotated by BRENDA team
firefly
-
-
Manually annotated by BRENDA team
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Vibrio harveyi ATCC BAA1116
genes lux A and luxB, of the luxCDABE operon, encoding the alpha and beta subunits of the luciferase
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
symbiotic bacterium from Kryptophanaron alfredi, flashlight fish
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
aldehyde + FMNH2 + O2
?
show the reaction diagram
-
-
-
-
?
an aldehyde + FMNH2 + O2
a carboxylate + FMN + H2O + hnu
show the reaction diagram
beetle luciferin + FMNH2 + O2
?
show the reaction diagram
-
-
-
-
ir
coelenterazine + FMNH2 + O2
CO2 + coelenteramide + FMN + light + H2O
show the reaction diagram
-
an imidazolopyrazine derivative
-
-
ir
decanal + FMNH + O2
decanoic acid + FMN + H2O + light
show the reaction diagram
decanal + FMNH- + O2
decanoic acid + FMN + H2O + hv
show the reaction diagram
decanal + FMNH2 + O2
decanoate + FMN + H2O + hn
show the reaction diagram
decanal + FMNH2 + O2
decanoate + FMN + H2O + hv
show the reaction diagram
-
-
-
-
ir
decanal + FMNH2 + O2
decanoic acid + FMN + H2O + hnu
show the reaction diagram
-
-
-
-
?
decanal + FMNH2 + O2
decanoic acid + FMN + H2O + hv
show the reaction diagram
decanal + riboflavin + O2
?
show the reaction diagram
-
riboflavin is a very poor substrate for bacterial luciferase
-
-
?
dodecanal + FMNH + O2
dodecanoic acid + FMN + H2O + light
show the reaction diagram
dodecanal + FMNH2 + O2
dodecanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
dodecanal + FMNH2 + O2
dodecanoic acid + FMN + H2O + hv
show the reaction diagram
-
-
-
-
?
dodecyl aldehyde + FMNH + O2
?
show the reaction diagram
-
-
-
-
?
fatty aldehyde + FMNH2 + O2
fatty acid + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
FMNH + O2
FMN + H2O2
show the reaction diagram
hexachlorethane + e-
tetrachlorethylene + Cl-
show the reaction diagram
-
-
-
-
?
luciferin + FMNH2 + O2
?
show the reaction diagram
-
-
-
-
ir
luciferin + O2 + ATP
oxyluciferin + AMP + CO2 + light
show the reaction diagram
myristic aldehyde + FMNH + O2
myristic acid + FMN + H2O + light
show the reaction diagram
-
-
-
-
?
n-caprinaldehyde + FMNH2 + O2
n-caprinoate + FMN + H2O + hv
show the reaction diagram
-
-
-
-
ir
n-decanal + FMNH2 + O2
n-decanoate + FMN + H2O + hn
show the reaction diagram
nonanal + FMNH2 + O2
nonanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
octanal + FMNH + O2
octanoic acid + FMN + H2O + light
show the reaction diagram
octanal + FMNH2 + O2
octanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
pentachlorethane + e-
trichlorethylene + Cl-
show the reaction diagram
-
-
-
-
?
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hn
show the reaction diagram
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hnu
show the reaction diagram
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hv
show the reaction diagram
tetradecanal + FMNH2 + O2
tetradecanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
undecanal + FMNH2 + O2
undecanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
an aldehyde + FMNH2 + O2
a carboxylate + FMN + H2O + hnu
show the reaction diagram
beetle luciferin + FMNH2 + O2
?
show the reaction diagram
-
-
-
-
ir
coelenterazine + FMNH2 + O2
CO2 + coelenteramide + FMN + light + H2O
show the reaction diagram
-
an imidazolopyrazine derivative
-
-
ir
decanal + FMNH + O2
decanoic acid + FMN + H2O + light
show the reaction diagram
fatty aldehyde + FMNH2 + O2
fatty acid + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
luciferin + O2 + ATP
oxyluciferin + AMP + CO2 + light
show the reaction diagram
n-caprinaldehyde + FMNH2 + O2
n-caprinoate + FMN + H2O + hv
show the reaction diagram
-
-
-
-
ir
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hn
show the reaction diagram
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hnu
show the reaction diagram
RCHO + FMNH2 + O2
RCOOH + FMN + H2O + hv
show the reaction diagram
-
-
-
-
?
tetradecanal + FMNH2 + O2
tetradecanoate + FMN + H2O + hn
show the reaction diagram
-
-
-
-
ir
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-deaza-FMNH2
-
can replace FMNH2
2',3'-Diacetyl-FMNH2
-
as substitute for FMNH2
2-Thio-FMNH2
-
as substitute for FMNH2
3'-Carboxymethyl-FMNH2
-
as substitute for FMNH2
-
4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate
-
model bioluminescence emitter molecule, binding and fluorescence quantum yield studies, complexed with the enzyme in a 1:1 molcular ratio
Iso-FMNH2
-
-
additional information
-
the 4a-hydroperoxy-4a,5-dihydroFMN intermediate luciferase transforms from a low quantum yield IIx to a high quantum yield IIy fluorescent species on exposure to excitation light
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,4-benzoquinone
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-
1-aminodecanal
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-
1-aminododecanal
-
-
1-Decanol
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-
1-Dodecanol
1-Heptanol
-
-
1-nonanol
-
-
1-Octanol
-
-
1-Tetradecanol
-
-
1-undecanol
-
-
2,2-diphenylpropylamine
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-
2,3-Dichloro-(6-phenylphenoxy)ethylamine
2,4-Dinitrofluorobenzene
-
i.e. Sanger's reagent
2-Bromodecanal
-
protection by dithiothreitol or mercaptoethanol
2-diethylaminoethyl-2,2-diphenylvalerate
2-methyl-1,4-benzoquinone
-
-
2-methyl-5-isopropyl-1,4-benzoquinone
-
-
5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate
-
binding and fluorescence quantum yield studies of the substance as a model, complexed with the enzyme in a 1:1 molcular ratio leading to 80% and 90% inhibition of wild-type and mutant C106A at 0.01 mM, respectively, binds to the active site
8-Anilino-1-naphthalenesulfonate
acetone
-
-
aliphatic alcohols
-
-
Aliphatic alkanes
-
-
-
Amino group reagents
benzylalcohol
-
-
butanoic acid
-
IC50: 13.6 mM
Butanone
-
-
Chloroform
-
-
Decanoic acid
diethylether
dodecanamide
-
inhibits bacterial luciferase luminescence reaction. By injecting the dodecaneamide into the bacterial luciferase system, the luminescence intensity decreases to about half of the initial intensity
dodecanenitrile
-
-
dodecanoic acid
Dodecanol
-
-
dodecylamine
-
-
Enflurane
-
-
Ethoxyformic anhydride
Fluroxene
-
-
Halothane
hexadecanoic acid
-
IC50: 0.00067 mM
hexanoic acid
-
IC50: 3.4 mM
Imidazole reagents
iodoacetamide
-
-
Isoflurane
-
-
methanol
-
bacterial luciferase luminescence intensity decreases to the steady state depending on the methanol concentration
Methoxyflurane
N,N-Diethyl-2,4-dichloro-(6-phenylphenoxy)ethylamine
-
-
N,N-Dimethylaniline
-
-
n-butanol
-
-
n-decanal
N-ethylmaleimide
-
protection by substrates
n-Heptanol
-
-
n-Hexanol
-
-
N-phenacylthiazolium bromide
-
; highly selective inhibition
octadecanoic
-
IC50: 0.00063 mM
-
Octanoic acid
-
IC50: 2.9 mM
Paraldehyde
-
-
Pargyline
-
-
pifithrin-alpha
-
highly selective inhibitor in vivo and in vitro
potassium iodide
-
quenches the fluorescence of FMN effectively at 0.2 M, and enhances the decay of wild-type and HFOOH enzymes, the wild-type enzyme forms an inactive complex with KI
Proteases
-
trypsin, chymotrypsin
-
reduced riboflavin
-
-
SKF-525A
-
-
sulfhydryl reagents
tetradecanoic acid
-
IC50: 0.00068 mM
trans-2-decenal
-
-
Tridecanoic acid
-
-
Undecanal
-
-
undecane
-
-
Urea
-
denaturation curve, thermodynamics, wild-type and mutants, overview
urethane
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-fluoroethylamine
-
alphaH44A mutant, works as catalytic base
ammonia
-
alphaH44A mutant, works as catalytic base
cyanomethylamine
-
alphaH44A mutant, works as catalytic base
ethanolamine
-
alphaH44A mutant, works as catalytic base
ethylamine
-
alphaH44A mutant, works as catalytic base
halogenated aromatic hydrocarbons
-
stable aryl hydrocarbon receptor ligands from crude extracts of environmental samples, that activate the enzyme, detailed overview
-
imidazole
-
alphaH44A mutant, works as catalytic base
methanol
-
bacterial luciferase luminescence intensity slightly increases during the initial stage of the methanol injection
methylamine
-
alphaH44A mutant, works as catalytic base
omega-carboxypentylflavin
-
as substitute for FMNH2
polyaromatic hydrocarbons
-
labile aryl hydrocarbon receptor ligands from crude extracts of environmental samples, that activate the enzyme, detailed overview
-
Propylamine
-
alphaH44A mutant, works as catalytic base
Sodium acetate
-
activates mutant E328A
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.001 - 0.01
aldehydes
-
-
0.0003 - 0.0173
decanal
0.0009
FMN
-
in the presence of different flavin concentrations, 0.001 mM Fre oxidoreductase, 10 mM decanal, and 0.01 mM NADPH and 0.005 mM luciferase
0.00015 - 0.0234
FMNH
0.00018 - 0.0584
FMNH2
0.0012 - 0.009
n-decanal
-
depending on buffer system
0.0001
O2
-
-
0.0013
riboflavin
-
in the presence of different flavin concentrations, 0.001 mM Fre oxidoreductase, 10 mM decanal, and 0.01 mM NADPH and 0.005 mM luciferase
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000015 - 0.0000317
FMNH
0.1
FMNH2
Vibrio harveyi
-
purified enzyme
additional information
additional information
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000482
1-Dodecanol
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.000356
1-Tetradecanol
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.115
2,2-diphenylpropylamine
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.03
8-Anilino-1-naphthalenesulfonate
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
13.6
butanoic acid
Photinus pyralis
-
IC50: 13.6 mM
0.0132
Decanoic acid
Photinus pyralis
-
IC50: 0.0132 mM
0.00008
dodecanenitrile
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.0012
dodecanoic acid
Photinus pyralis
-
IC50: 0.0012 mM
0.00003
dodecylamine
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.00067
hexadecanoic acid
Photinus pyralis
-
IC50: 0.00067 mM
3.4
hexanoic acid
Photinus pyralis
-
IC50: 3.4 mM
0.1
N,N-Dimethylaniline
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.095
N-phenacylthiazolium bromide
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.00063
octadecanoic
Photinus pyralis
-
IC50: 0.00063 mM
-
2.9
Octanoic acid
Photinus pyralis
-
IC50: 2.9 mM
1
Pargyline
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.017
pifithrin-alpha
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.09
SKF-525A
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.00068
tetradecanoic acid
Photinus pyralis
-
IC50: 0.00068 mM
0.005
trans-2-decenal
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
0.000122
undecane
Vibrio harveyi
-
in 50 mM phosphate buffer, pH 7.0 at 23C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.105
-
substrate tetradecanal, uncoupled enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
-
optimum for reaction velocity
6.5
-
optimum for binding of FMNH2
8.1
-
optimum for quantum yield
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
temperature dependence of thermodynamic parameters
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
gene expression kit of dioxin-responsive chemical-activated luciferase
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75430
-
wild-type enzyme, analytical ultracentrifugation
76360
-
mutant beta-H82A, analytical ultracentrifugation
76740
-
mutant beta-H81A, analytical ultracentrifugation
76750
-
mutant beta-H81A/E89D, analytical ultracentrifugation
76860
-
mutant alpha-A81H, analytical ultracentrifugation
77000 - 78000
84000
-
renatured enzyme, osmometry
158000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
homodimer
-
2 * 77000, SDS-PAGE; 2 * 77800, calculated from amino acid sequence
monomer
-
1 * 78000, produced by gene fusion of luxA and luxB genes
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bacterial luciferase/FMN complex, by the hanging drop method, at 2.3 A resolution. Crystals of recombinant luciferase are grown at room temperature prior to soaking with millimolar concentrations of FMN. Belongs to space group P212121. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. Mobile loop that is crystallographically disordered, appears to be a boundary between solvent and the active center. Within this portion of the protein, there is a single contact between Phe272 of the R subunit and Tyr151 of the beta subunit
structure is determined in absence of substrate at low-salt concentrations
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
-
-
348582
6 - 9.5
-
-
348582
additional information
-
the enzyme exhibits enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature
671414
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
stable below
30 - 37
-
half-life of about 1020 min at 37C. At 37C, the native enzyme retains around 60% of the bioluminescence of the enzyme expressed at 30C
40
-
wild-type enzyme and mutanbt A75G: loss of 60% activity within 50 min, mutants C106V and C106V/A75G show increased thermolability loosing 99% and 90% activity, respectively
47
-
the enzyme is inactivated in 15 min at 47?
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
citrate stabilizes against inactivation by proteases, heat, urea
diphosphate stabilizes against inactivation by proteases, heat, urea
inactivation by lyophilization
labile to proteases
no inactivation by repeated freezing/thawing
phosphate stabilizes against inactivation by proteases, heat, urea
repeated freezing/thawing causes inactivation of immobilized enzyme
-
sulfate stabilizes against inactivation by proteases, heat, urea
the wild-type enzyme belongs to the group of luciferases with slow decay, mutant E175G is turned into a luciferase with fast decay, the decay rate of the enzyme is determined by residue Glu175
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme exhibits enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature
-
671414
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50 mM potassium phosphate buffer, pH 7.0, protein concentration 1 mg/ml , -20C, 0.1 M phosphate buffer, pH 7, 0.1 mM dithiothreitol, 1 mM EDTA
-20C, phosphate buffer
-
-80C, 0.5 mM dithiothreitol
-
0-4C, immobilized enzyme, 0.1 mM dithiothreitol, 20% loss of activity in 3 days
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity methods
DEAE-Sepharose column chromatography, Superdex 200 gel filtration, and Mono Q column chromatography
-
nickel affinity column
-
on a nickel affinity column, to more than 90% purity
on nickel affinity column, to more than 90% purity
-
preparation of enzyme with modified subunits
-
preparation of subunits
-
recombinant enzyme from Escherichia coli
recombinant enzyme from Escherichia coli strain JM101 to over 95% purity
-
recombinant wild-type and mutant C106A enzymes from Escherichia coli strain JM101 to over 95% purity
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21
-
recombinant wild-type and mutant enzymes from Escherichia coli strain JM101 to homogeneity
-
recombinant wild-type and mutant enzymes from Escherichia coli strain JM109 to over 85% homogeneity
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified from the pJHD500 plasmid and ligated into a pET21b vector, expressed from pZCH2 in an Escherichia coli BL21 (lambdaDE3) cell line after growth to an OD600 of 0.5
cloning of the cDNA encoding the destabilized enzyme into an adenoviral expression plasmid and transfection of Vero, Hep-2, Chang, A-549, COS-1, and HeLa cells, luciferase expression is linear with respect to viral multiplicity of infection, protein synthesis inhibiting drugs, e.g. shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin A, and cycloheximide, reduce bioluminescence respresenting the antiviral activity
-
coexpression of luciferase and cytochrome P-450; expression in Pseudomonas putida
-
expressed in Escherichia coli BL21(DE3) and HEK-293T cells
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli from pJHD500, ligated into a pET21b vector. Luciferase subcloned from pZCH2 into a pASKIBA-3c vector with the restriction sites XbaI and XhoI. The resulting luciferase containing a strep-II tag on the C terminus of the beta-subunit (pZCB4) expressed
-
expressed in Escherichia coli JM109 (native enzyme) and BL21(DE3) cells (luciferase-mOrange fusion enzyme)
-
expressed in NIH-3T3 cells
-
expressed in Pseudomonas putida mt-2
-
expression analysis, cloning into expression vector pPL2lux useable as a reporter system based on the luciferase activity of the enzyme, overview
-
expression in different Escherichia coli strains, which are wild-type, or deficient in gene clpA, clpB, and clpX encoding Hsp chaperones, respectively
expression in Escherichia coli
-
expression in Escherichia coli strain JM101
-
expression in transfected Leishmania amazonensis strain LV79 amastigotes, transfection by electroporation
-
expression of fused luxA and luxB genes and also luxF gene in Escherichia coli and Nicotiana plumbaginifolia
-
expression of fused luxA and luxB genes in Escherichia coli
expression of fused luxA and luxB genes in Saccharomyces cerevisiae, Bacillus subtilis, plant cells, plasmid expression vector and in Escherichia coli
-
expression of luxA gene in Escherichia coli
-
expression of seperated luxA and luxB gene in Escherichia coli JM109
-
expression of the enzyme in Mycobacterium tuberculosis under control of the inducible/repressible promotor of the alanine dehydrogenase from Mycobacterium tuberculosis strain H37Rv, usage of a mycobacterial-Escherichia coli shuttle vector
-
expression of wild-type and mutant C106A enzymes in Escherichia coli strain JM101
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expression of wild-type and mutant enzymes in Escherichia coli
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expression of wild-type and randomly generated mutants in Escherichia coli strain BL21
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gene Gluc, expression in Mycobacterium smegmatis using an hsp60 promoter, subcloning in Escherichia coli strain DH5alpha
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gene lux, improvement of a tagging system for luciferase by construction of a highly active, constitutive promoter resulting in a 100fold higher recombinant activity compared to native activity
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gene luxA, expression of wild-type and mutant enzymes in Escherichia coli strain JM109
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gene luxAB, expression of wild-type and mutant enzymes in Escherichia coli strain BL21
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gene luxAB, functional coexpression in Saccharomyces cerevisiae with NADPH-specific FMN reductase FRP from Vibrio harveyi, subcloning in Escherichia coli
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genes luxA and luxB, expression under the control of a consensus-type promoter, lacUV5, in Escherichia coli, activity declines abruptly upon entry into the stationary growth phase, while the levels of luciferase proteins remian constant, the phenomenon, termed ADLA, i.e. abrupt decline of luciferase activity, is caused by a decrease in the availability of flavin mononucleotide
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ligated into pET21-b vector, expressed from pZCH2 in Escherichia coli BL21 (lambdaDE3) cell line
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luxCDABE operon, genetic organization, overview
overexpression in Escherichia coli
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overexpression of mutant in XL1 blue MRF' cell line
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overexpression of wild-type and mutant enzymes in Escherichia coli strain JM101
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stable expression, using a bicistronic expression vector, of wild type luxA and luxB, WTA/WTB, codon-optimized luxA and wild type luxB, COA/WTB, and codon-optimized versions of both luxA and luxB genes, COA/COB, in HEK-293 cells, expression analysis, method evaluation and optimization, highest bioluminescence by expression of both codon-optimized genes, overview; stable expression, using a bicistronic expression vector, of wild type luxA and luxB, WTA/WTB, codon-optimized luxA and wild type luxB, COA/WTB, and codon-optimized versions of both luxA and luxB genes, COA/COB, in HEK-293 cells, expression analysis, method evaluation and optimization, highest bioluminescence by expression of both codon-optimized genes, overview
the bacterial luciferase lux gene cassette consists of five genes, luxCDABE. The lux operon is re-synthesized through a process of multibicistronic, codon-optimization to demonstrate self-directed bioluminescence emission in a mammalian HEK-293 cell line in vitro and in vivo, overview. To overcome the limitations by FMNH2 supply, co-expression of a constitutively expressed flavin reductase gene frp from Vibrio harveyi is performed leading to a 151fold increased increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D232G
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random mutagenesis, 63% of wild-type luminescence activity
E175G
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random mutagenesis, the single point mutation leads to increased decay rate of the enzyme, 0.9% of wild-type luminescence activity
E175G/N199D
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random mutagenesis, 0.1% of wild-type luminescence activity
K202R
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random mutagenesis, 95% of wild-type luminescence activity
M190T
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random mutagenesis, 29% of wild-type luminescence activity
T198S
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random mutagenesis, 84% of wild-type luminescence activity
A74F
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site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme
A74G
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
A75G
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site-directed mutagenesis, activity similar to the wild-type enzyme
A75G/C106V/V173A
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site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173C
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site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173S
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site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173T
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site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A81H
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site-directed mutagenesis, residue of the alpha-subunit, mutant shows 13% of wild-type activity
alphaDELTA262-290beta
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four times higher affinity for FMN than wild type
alphaF114A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114D
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114Y
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF117A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117D
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117Y
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF327A
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site-directed mutagenesis, mutant activity is similar to the wild-type enzyme
alphaF46A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46D
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46Y
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF49A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49D
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49Y
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF6A
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site-directed mutagenesis, mutant activity is similar to the wild-type enzyme
alphaH44A
alphaR107A
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lower affinity for FMNH
alphaR107E
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lower affinity for FMNH
alphaR107S
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lower affinity for FMNH
C106A
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site-directed mutagenesis, catalytic properties are similar to the wild-type enzyme, mutant shows 60% of wild-type quantum yield
C106V
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site-directed mutagenesis, highly reduced ability to stabilize the reaction intermediate due to interaction between Val106 and Ala75 side chains, and therefore highly reduced activity and increased thermal lability compared to the wild-type enzyme
C106V/A75G
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site-directed mutagenesis, mutation of Ala75 restores about 90% of the activity abolished by mutation of Cys106, shift in the light emission spectrum to that of Photobacterium phosphoreum possessing Val and Gly at positions 106 and 75, respectively
D262A
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90% reduced activity with octanal, 36% reduced activity with decanal, activity with dodecanal as the wild-type
D265A
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activity with octanal as the wild-type, 81% reduced activity with decanal, complete loss of dodecanal activity
D271A
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complete loss of octanal and decanal activity, 18% reduced activity with dodecanal
E328A
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme, the activity is rescued by addition of sodium acetate, but not by phosphate, at pH 6.0-8.0 with increasing activity at lower pH
E328D
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328F
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site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme
E328H
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328L
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328Q
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site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
F261A
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site-directed mutagenesis, residue of the alpha-subunit, 0.19% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261D
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site-directed mutagenesis, residue of the alpha-subunit, 0.004% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261S
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site-directed mutagenesis, residue of the alpha-subunit, 0.13% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261Y
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site-directed mutagenesis, residue of the alpha-subunit, 2-3% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
G275A
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site-directed mutagenesis, residue of the alpha-subunit, 27% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275F
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site-directed mutagenesis, residue of the alpha-subunit, 6-7% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275I
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site-directed mutagenesis, residue of the alpha-subunit, 15% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275P
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site-directed mutagenesis, residue of the alpha-subunit, 0.04% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G284P
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site-directed mutagenesis, residue of the alpha-subunit, 1-2% of the wild-type activity
H285A
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26% reduced activity with octanal, 74% reduced activity with decanal, complete loss of dodecanal activity
H81A
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site-directed mutagenesis, residue of the beta-subunit, mutant shows 59% of wild-type activity
H81A/E89D
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site-directed mutagenesis, residues of the beta-subunit, mutant shows 13% of wild-type activity
H82A
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site-directed mutagenesis, residue of the beta-subunit, mutant shows 22% of wild-type activity
K274A
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89% reduced activity with octanal, 21% reduced activity with decanal, 81% reduced activity with dodecanal
K283A
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complete loss of octanal and decanal activity, 96% reduced activity with dodecanal, does not significantly impede binding of decanal, results in destabilization of intermediate II, results in a loss in quantum yield comparable with that of the loop deletion mutant, binds reduced flavin more weakly
K286A
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92% reduced activity with octanal, complete loss of decanal activity, 87% reduced activity with dodecanal, does not significantly impede binding of decanal, increase in exposure of reaction intermediates to a dynamic quencher, results in a loss in quantum yield comparable with that of the loop deletion mutant, binds reduced flavin more weakly
R291A
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77% reduced activity with octanal, 58% reduced activity with decanal, 71% reduced activity with dodecanal
V173A
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173C
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173F
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173H
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173I
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173L
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173N
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173S
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173T
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site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
W277A
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11% reduced activity with octanal, 50% reduced activity with decanal and dodecanal
Y151A
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151D
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151K
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151R
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151T
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151W
least active mutant, binds reduced flavin with wild-type affinity, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
quick refolding within several min at room temperature or 25C of thermoinactivated enzyme requires ATP and the DnaK-DnaJ-GrpE-system encoding the Hsp70 chaperone but is independent of chaperone ClpA, 80% activity after reactivation, refolding depends on the Escherichia coli strain used for recombinant expression of the luciferase, overview
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slow refolding at room temperature or 35C of thermoinactivated recominant enzyme requires the DnaK-DnaJ-GrpE-system encoding the Hsp70 chaperone, 7-8% activity after refolding over 10 min, refolding depends on the Escherichia coli strain used for recombinant expression of the luciferase, overview
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
diagnostics
expression of the bacterial luciferase system in mammalian cells for generation of bioreporters for in vivo monitoring and diagnostics technology, method evaluation and optimization, overview; expression of the bacterial luciferase system in mammalian cells for generation of bioreporters for in vivo monitoring and diagnostics technology, method evaluation and optimization, overview
molecular biology
Show AA Sequence (525 entries)
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