WO2008095623A2 - Luciférase sécrétée lu164m3 et son utilisation - Google Patents

Luciférase sécrétée lu164m3 et son utilisation Download PDF

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Publication number
WO2008095623A2
WO2008095623A2 PCT/EP2008/000625 EP2008000625W WO2008095623A2 WO 2008095623 A2 WO2008095623 A2 WO 2008095623A2 EP 2008000625 W EP2008000625 W EP 2008000625W WO 2008095623 A2 WO2008095623 A2 WO 2008095623A2
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WIPO (PCT)
Prior art keywords
luciferase
luciferases
secreted
lul64m3
nucleic acid
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Ceased
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PCT/EP2008/000625
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German (de)
English (en)
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WO2008095623A3 (fr
Inventor
Stefan Golz
Eugene Vysotski
Svetlana Markova
Ludmila Frank
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Bayer AG
Bayer Pharma AG
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Bayer Healthcare AG
Bayer Schering Pharma AG
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Publication of WO2008095623A2 publication Critical patent/WO2008095623A2/fr
Publication of WO2008095623A3 publication Critical patent/WO2008095623A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the invention relates to the nucleotide and amino acid sequence, as well as the activity and use of the secreted luciferase Lul64M3, as well as the use of secreted luciferases.
  • Luminescence refers to the emission of photons in the visible spectral range, this being done by excited emitter molecules. In contrast to fluorescence, the energy is not supplied from outside in the form of radiation of shorter wavelength.
  • Chemo luminescence is a chemical reaction that leads to an excited molecule that glows when the excited electrons return to their ground state. When this reaction is catalyzed by an enzyme, it is called bioluminescence.
  • the enzymes involved in the reaction are generally referred to as luciferases.
  • the luciferases are peroxidases or mono- and dioxygenases.
  • the enzyme substrates which form the starting materials for the light-emitting products are called luciferins. They vary from species to species.
  • the quantum yield of the systems is between 0.1-0.9 photons per reacted substrate molecule.
  • Luciferases can be classified according to their origin or their enzymatic properties. Luciferases can also be distinguished by their substrate specificity. The most important substrates include coelenterazines and luciferin, as well as derivatives of both substances.
  • Luciferases which are released from the cytosol into the surrounding environment by the host organism as a recombinant or wiltype protein, are sometimes called secreted luciferases.
  • Table 1 shows an overview of secretory luciferases:
  • the secreted luciferase Lu 164 is also described in Markova et al. Of 2004. reporter systems
  • Reporter or indicator genes are generally genes whose gene products can easily be detected by simple biochemical or histochemical methods. There are at least two types of reporter genes.
  • Resistance genes are genes whose expression confers on a cell resistance to antibiotics or other substances whose presence in the growth medium leads to cell death when the resistance gene is absent.
  • reporter gene The products of reporter genes are used in genetic engineering as fused or unfused indicators. The most common
  • Reporter genes include beta-galactosidase (Alam et al., 1990), alkaline
  • Luminescence refers to the emission of photons in the visible spectral range, this being done by excited emitter molecules. In contrast to fluorescence, the energy is not supplied from outside in the form of radiation of shorter wavelength.
  • Chemiluminescence is a chemical reaction that leads to an excited molecule that glows when the excited electrons return to their ground state. When this reaction is catalyzed by an enzyme, it is called bioluminescence.
  • the enzymes involved in the reaction are generally referred to as luciferases.
  • Lul64M3 is therefore kinetically different from the previously described secreted luciferases from Metridia longa. Surprisingly, Lul64M3 can be used in combination with other coelenterazine-dependent or coelenterazine-independent luciferases because of its kinetic differentiation.
  • the luciferase has an altered activity distribution of the bioluminescent reaction due to the altered kinetic properties.
  • the Lul64M3 activity to be measured per second is significantly higher than LuI 64. This higher bioluminescence allows a higher sensitivity of the measurement method used, as a lower number of cells, less activation of Lul64M3 expression, or low substrate concentration Measurement clearly above the background signal allows.
  • the invention relates to the use of Lul64M3 to improve the sensitivity, the use of low cell counts or low substrate concentrations.
  • Lul64M3 The altered kinetic properties of Lul64M3 allow a differentiated kinetic evaluation of bioluminescence. In a continuous measurement over (for example) 90 seconds, different intervals can be used for the evaluation. By choosing the measuring window, a distinction can therefore be made between the luciferases.
  • the shape of the kinetic curve may vary depending on the experimental conditions chosen.
  • the length and selection of the measurement intervals can be based on the respective experimental conditions and can be used flexibly.
  • the total measuring time can also be flexibly selected on the basis of the data shown.
  • the invention relates to the kinetic evaluation of measurements of the bioluminescence activity of Lul64M3.
  • the invention relates to the kinetic evaluation of measurements of the bioluminescence activity of LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6 and Lu52.
  • the invention relates to the kinetic evaluation of measurements of the bioluminescence activity of secreted luciferases.
  • the invention relates to the kinetic evaluation of measurements of the bioluminescence activity of proteins according to the invention.
  • the luciferase due to its altered properties, is particularly suitable for multiplex reactions.
  • the luciferase Lul64M3 shows a significantly faster kinetics compared to other luciferases, which enables a combination with other luminescent or non-luminescent measuring methods (readouts).
  • the luminescent systems do not mutually inhibit each other or the respective signals outshine.
  • the luminescence After activation of the first system (for example, by substrate addition), the luminescence must be returned to the initial level before the second reaction can be started. This is also necessary if both systems use independent substrates. Due to its fast kinetics, Lul64M3 significantly shortens the time between measurements. Inactivation of the reaction is not necessary. Since the luciferase Lul64M3 is a secreted luciferase, a combination with intracellular systems (such as Firefly luciferase) is possible.
  • FIG. 6 shows by way of example the combination of secreted luciferases (on the example of the luciferase LuI 64) from Metridia longa with the intracellular luciferase Firefly.
  • the luciferases can be expressed either in one or different cellular systems.
  • the luciferases Firefly and Lu 164 were each in vectors
  • CRE promoters stably expressed in CHO cells.
  • the cells were co-cultivated for measurement and the cAMP-dependent promoters by the addition of forskolin for 4
  • the invention relates to the use of Lul64M3 in multiplexed approaches using Lul64M3 in combination with one or more reporter genes or readouts.
  • the use of Lul64M3 in approaches for measuring several target genes is also according to the invention.
  • the invention relates to the use of Lul64, Lu22, LuAL, Lu39, Lu45, Lul6 and Lu52 in multipex approaches in which Lul64, Lu22, LuAL, Lu39, Lu45, Lul6 and Lu52 in combination with one or more reporter genes or readouts is used. Also useful in the invention is the use of LuI 64, Lu22, LuAL, Lu39, Lu45, Lul6, and Lu52 in approaches to measuring multiple target genes.
  • the invention relates to the use of secreted luciferases in multipex approaches in which secreted luciferases are used in combination with one or more reporter genes or readouts.
  • the invention also relates to the use of secreted luciferases in batches for measuring a plurality of target genes.
  • the invention relates to the use of proteins according to the invention in multipex approaches in which proteins according to the invention are used in combination with one or more reporter genes or measurement techniques (readouts).
  • the invention also relates to the use of proteins according to the invention in batches for measuring a plurality of target genes.
  • the mutations and deletions were inserted using molecular biological methods.
  • the invention relates to the secreted luciferase Lul64M3 with the amino acid sequence represented by SEQ ID NO: 2.
  • the invention also relates to the nucleic acid molecule shown in SEQ ID NO: 1.
  • the invention also relates to functional equivalents of the secreted luciferase Lul64M3.
  • Functional equivalents are those proteins that have comparable physicochemical or biochemical properties.
  • mutants of the Lul64M3 protein or for such encoding nucleic acids are according to the invention.
  • the secreted luciferase Lul64M3 is suitable as a reporter gene for the technique of "high content screening” (HCS) .
  • HCS is an encroachment for modern microscopy technologies for cell analysis.
  • the characteristic feature of HCS methods is the quantitative detection of several parameters at the cellular or subcellular level
  • the secreted luciferase Lul64M3 is suitable as reporter gene for cellular systems especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.
  • the secreted luciferase Lul64M3 is suitable as reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeasts, in bakulo, in plants
  • the secreted luciferase Lul64M3 is suitable as a reporter gene for cellular systems in combination with bioluminescent or chemiluminescent systems especially systems with luciferases, with oxygenases, with phosphatases.
  • the secreted luciferase Lul64M3 is suitable as a reporter gene for cellular systems in combination with bioluminescent or chemiluminescent systems especially systems with photoproteins and ionic indicators, especially aequorin, clytin, obelin, berovine and bolinopsin.
  • the secreted luciferase Lul64M3 turns out to be a marker protein, especially in the FACS (fluorescence activated cell sorter) sorting.
  • the secreted luciferase Lul64M3 is suitable as a fusion protein especially for receptors, ion channels, transporters, transcription factors, proteinases, kinases, phosphodiesterases, hydrolases, peptidases, transferases, membrane proteins, glycoproteins.
  • the secreted luciferase Lul64M3 is suitable for immobilization, in particular by antibodies, by biotin, by magnetic or magnetizable carriers.
  • the secreted luciferase Lul64M3 is suitable for energy transfer systems such as FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), FET (field effect transistor), FP (fluorescence polarization), HTRF (Homogeneous time-resolved fluorescence ) Systems.
  • the secreted luciferase Lul64M3 is suitable for labeling substrates or ligands specifically for proteases, for kinases, for transferases, for transporters, for ion channels and receptors.
  • the secreted luciferase Lul64M3 is suitable for expression in bacterial systems especially for titer determination, as substrates for biochemical systems especially for proteinases and kinases.
  • the secreted luciferase Lul64M3 is useful as a marker specifically coupled to antibodies coupled to enzymes coupled to receptors coupled to ion channels and other proteins.
  • the secreted luciferase Lul64M3 is suitable as a reporter gene in the pharmacological drug discovery, especially in HTS (High Throughput Screening).
  • the secreted luciferase Lul64M3 is suitable as components of detection systems especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for Western blot, for confocal microscopy.
  • ELISA enzyme-linked immunosorbent assay
  • the secreted luciferase Lul64M3 is useful as a marker for the analysis of interactions specifically for protein-protein interactions, for DNA-protein interactions, for DNA-RNA interactions, for RNA-RNA interactions, for RNA-protein interactions (DNA
  • the secreted luciferase Lul64M3 is useful as a marker or fusion protein for expression in transgenic organisms, especially in mice, in rats, in hamsters and other mammals, in primates, in fish, in worms, in plants.
  • the secreted luciferase Lul64M3 is suitable as a marker or fusion protein for the analysis of embryonic development.
  • the secreted luciferase Lul64M3 is useful as a marker via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
  • the secreted luciferase Lul64M3 is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.
  • the secreted luciferase Lul64M3 is suitable as a reporter coupled to proteins or peptides.
  • the nucleic acid or the peptide of the coupled protein Lul64M3 is suitable as probe especially for Northern blots, for Southern blots, for Western blots, for ELISA, for nucleic acid sequencing, for protein analyzes, chip analyzes.
  • the protein Lul64M3 is suitable as a marker of pharmacological formulations especially of infectious agents, of antibodies, of "small molecules".
  • the protein Lul64M3 is suitable for geological investigations specifically for marine, groundwater and river currents.
  • the protein Lul64M3 is suitable for expression in expression systems especially in in vitro translation systems, in bacterial systems, in yeast systems, in Bakulo systems, in viral systems, in eukaryotic systems.
  • the invention also relates to the purification of the protein Lul64M3 specifically as a wild-type protein, as a fusion protein, as a mutagenized protein.
  • the invention also relates to the use of Lul64M3 in the field of cosmetics especially bath preparations, lotions, soaps, body colors, toothpaste, body powders.
  • the invention also relates to the use of Lul64M3 for coloring especially food, bath additives, ink, textiles, plastics.
  • the invention also relates to the use of Lul64M3 for coloring paper, especially greetings cards, paper products, wallpaper, craft items.
  • the invention also relates to the use of Lul64M3 for coloring liquids especially for water pistols, for fountains, for drinks, for ice cream.
  • the invention also relates to the use of Lul64M3 for the manufacture of toys, especially finger paint, make-up, water pistols.
  • the invention relates to organisms containing a vector according to the invention.
  • the invention relates to organisms expressing a polypeptide of the invention.
  • the invention relates to organisms expressing a functional equivalent of Lul64M3.
  • the invention relates to methods for expression of the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems.
  • the invention also relates to methods for purifying / isolating a polypeptide of the invention.
  • the invention relates to peptides having more than 5 consecutive amino acids, which are recognized immunologically by antibodies against the fluorescent proteins according to the invention.
  • the invention relates to the use of the fluorescent proteins according to the invention as marker and reporter gene, in particular for the pharmacological drug discovery and diagnostics.
  • the invention relates to the secreted luciferase Lul64M3 with the amino acid sequence represented by SEQ ID NO: 2 and the nucleotide sequence represented by SEQ ID NO: 1.
  • a protein Lul64M3 characterized in that its sequence comprises the sequence shown in SEQ ID NO: 2, as well as functional fragments thereof.
  • the invention further provides a nucleic acid molecule which encodes a protein comprising the sequence shown in SEQ ID NO: 1, as well as functional fragments thereof.
  • a component of the invention is a recombinant RNA or DNA vector which comprises a nucleic acid as described in the preceding section.
  • a component of the invention is a method for expressing a polypeptide according to the invention in bacteria, eukaryotic cells, or in in vitro translation systems.
  • a component of the invention is the use of a nucleic acid according to the invention as marker or reporter gene also in combination with one or more other markers or reporter genes.
  • Also part of the invention is the use of a protein according to the invention as marker or reporter gene also in combination with one or more other markers or reporter gene proteins.
  • FIG. 2 shows the alignment of the luciferases Lul64M3 and Lul64.
  • the luciferase Lul64M3 represents a significantly shorter polypeptide than the wild-type enzyme Lul64.
  • mutants or derivatives of the luciferases Lu 164, Lu22, LuAL, Lu39, Lu45, LuI6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the region of the amino acids 18 and 58 of the luciferases Lu 164, Lu22, LuAL, Lu39, Lu45, Lu16, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives which are changes or deletions in the region of the amino acids 23 and 48 of the luciferases Lul64, Lu22, LuAL, Lu39, Lu45, Lu 16, Lu52 and Gaussia luciferase with altered biochemical or physicochemical properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the region of amino acids 13 and 88 of the lucerases Lul64, Lu22, LuAL, Lu39, Lu45, LuI6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the region of the amino acids 33 and 68 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • SPCS Single Plate Clone Selection
  • Secretory luciferases such as the luciferases of the invention, are useful as reporter genes for the identification of cell lines with recombinant genes.
  • stable reporter gene lines with a secretory luciferase e.g. Lul64M3 or Lu 164 made.
  • the luciferase expression can be regulated via promoters with responsive elements.
  • CRE elements cAMP responsive element
  • the change in cAMP concentration can be determined by the modulation of a recombinant or endogenous target gene (target).
  • target a recombinant or endogenous target gene
  • GPCR Gs-coupled G protein-coupled receptor
  • the cell line described above is transfected with the cDNA of the GPCR to be expressed.
  • the transfected cells are then seeded on microtiter plates (eg 384 or 1536 well plates) and the transfected cells are selected.
  • microtiter plates eg 384 or 1536 well plates
  • the GPCR is activated by the addition of the agonist, resulting in an increase in intracellular cAMP concentration in a Gs-coupled receptor. This cAMP increase in concentration leads to increased reporter gene expression.
  • the substrate in Coulenterazine Lul64M3 and Lul64
  • bioluminescence measurement without lysing the cells.
  • the medium-substrate mixture can be removed and the underlying cells are removed.
  • the removed cells are then available for further cultivation, cultivation and measurement.
  • conventional systems using intracellular reporter gene such as the Firefly luciferse
  • duplication or multiplication of the seed plate prior to measurement is necessary because the measurement destroys the cells and access to a copy plate becomes necessary. This can lead to duplication errors that can be avoided with the SPCS method.
  • the SPCS method allows 1.
  • FIG. 5 shows the result of a clone identification by means of the SPCS method.
  • LuI 64 As a secreted luciferase LuI 64 was used in the vector pASM.
  • the target gene of the clone to be identified was a Gs-coupled G-protein coupled receptor in the vector pcDNA3.
  • the chosen representation allows the induction factor for each clone to be determined directly at the chosen time and the corresponding clone to be isolated from the same hole.
  • the agonist, the culture conditions or the measurement systems parameters of later measurement methods may be included in the identification of the recombinant clones.
  • the invention relates to the use of Lul64M3 in the identification of cell clones by the SPCS method.
  • the invention relates to the use of the secreted luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6 and Lu52 in the identification of cell clones according to the SPCS method.
  • the invention relates to the use of secreted luciferases in the identification of cell clones according to the SPCS method.
  • the invention relates to the use of proteins according to the invention in the identification of cell clones by the SPCS method.
  • the invention relates to the use of bioluminescent or non-bioluminescent reporter genes in the identification of clones according to the SPCS method.
  • the use of several measuring methods in an experimental approach allows the coupled measurement of different parameters or pharmacological target genes.
  • different reporter systems can be combined for this purpose.
  • the secreted luciferase Lul64M3, as well as the secreted luciferases from Metridia longa, can be combined with various intracellular and secreted reporters.
  • the combination of secreted and intracellular reporter genes is exemplified below.
  • the secreted luciferase LuI 64 was combined with the intracellular luciferase Firefly.
  • the luciferases were cloned into vectors which regulate cAMP-dependent expression of the luciferases (pASM vectors).
  • Both vectors were transfected into independent CHO cell lines and the cell lines stably cultured.
  • both cell lines were co-cultivated on microtiter plates and subsequently stimulated with forskolin.
  • forskolin leads to an increase in the intracellular cAMP concentration, which leads to the expression of the respective luciferase in both cell lines.
  • the microtiter plate was placed in a bioluminescence meter, which allows the addition of liquids within the meter (schematic representation in Figure 6 and 7). After the addition of the substrate of the secreted luciferase (here coelenterazine), the measurement was started.
  • the bioluminescence was measured for a time interval of 60 seconds. Subsequently, the second luciferase substrate (here firefly luciferin) was added in combination with a detergent (here Triton) to disrupt the cells. The addition of Triton stops the bioluminescence reaction of the secreted luciferase and lowers luminescence to the background level. The bioluminescence reaction of the firefly luciferase, which is uninfluenced by this, can subsequently be measured. The total measurement time was 150 seconds under the present conditions. This method can be carried out with Lul64M3, the secretory luciferases from Metridia longa, or other secretory luciferases or reporter genes.
  • Lul64M3 the secretory luciferases from Metridia longa, or other secretory luciferases or reporter genes.
  • the invention relates to the use of Lul64M3 in multiplex approaches.
  • the invention relates to the use of Lul64, Lu22, LuAL, Lu39, Lu45, Lul6 and Lu52 in multiplex approaches.
  • the invention relates to the use of secreted luciferases in multiplex approaches.
  • the invention relates to the use of proteins according to the invention in multiplex mixtures.
  • SEQ ID NO: 1 (Lul64M3 nucleotide sequence)
  • SEQ ID NO: 3 (Lul64 - nucleotide sequence - coding)
  • FIG. 1 A first figure.
  • FIG. 1 shows the vector map of the construct pcDNA3-Lul64M3.
  • FIG. 2 shows an alignment Lul64M3 with LuI 64 at the amino acid level.
  • the figure shows the result of the bioluminescence measurement of Lul64M3 at a constant substrate concentration and decreasing concentration of Lul64M3 by dilution of the cell supernatant.
  • the figure shows the result of the bioluminescence measurement of LuI 64 at a constant substrate concentration and decreasing concentration of LuI 64 by dilution of the cell supernatant.
  • X-axis time in seconds.
  • Y axis relative light units (relative light units
  • the figure shows the use of secretory luciferases to identify clones according to the SPCS system.
  • the reporter gene used was the secreted luciferase LuI 64
  • the figure shows the schematic sequence of a cell-based multiplex approach using a secretory luciferase in combination with a non-secretory luciferase.
  • the figure shows the result of a cell-based multiplexing approach using a secretory luciferase in combination with a non-secretory luciferase using the kinetics of the various measurement sections. Indicated are the relative light units (relative light units
  • the plasmid pcDNA3.1 (+) from Clontech was used for consumptive expression.
  • the vector pASM contains cAMP responsive elements (CRE), which regulate the promoter activity as a function of the cAMP concentration.
  • CRE cAMP responsive elements
  • the derivative of the vector was named pASM-Lul64.
  • the derivative of the vector pcDNA3 was designated as pcDNA3-Lul64M3 or pcDNA3-Lul64M3.
  • the cloning was carried out using standard molecular biological methods.
  • the vectors pcDNA3-Lul64, pcDNA3-Lul64M3 and pASM-Lul64 were used to express Lul64M3 and Lul64 in eukaryotic systems.
  • FIG. 1 shows the plasmid map of the vector pcDNA3-Lul64M3.
  • Consumptive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-Lul64M3, pcDNA3-Lul64 and pcDNA3 (without cDNA insertion) in transient experiments.
  • 10 000 cells per hole in DMEM-F 12 medium were plated on 96-well microtiter plates and incubated overnight at 37 0 C.
  • the transfection was carried out using the Fugene 6 kit (Roche) according to the manufacturer's instructions.
  • the transfected cells were incubated overnight at 37 ° C in DMEM-F12 medium.
  • the measurement of the bioluminescence results after substrate addition with an imaging system.
  • buffer A pH 7.4
  • the measurement of diluted supernatants was carried out in buffer A (pH 7.4) with the composition 130mM NaCl, 5mM KCl, 20mM Hepes, 1mM MgC12 x 6H2O and 5mM NaHCO3
  • the transfected cells were selected with 2 mg / ml geneticin and the bioluminescence activity of the clones or supernatants was determined.
  • FIG. 2 shows the alignment of the secreted luciferases at the amino acid level.
  • Christopoulos TK Verhaegent M, Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem. 2002, Sep; 74 (17): 4378-85.

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Abstract

La présente invention concerne la séquence de nucléotides et d'acides aminés, ainsi que l'activité et l'utilisation de la Lu164M3, ainsi que l'utilisation de luciférases sécrétées.
PCT/EP2008/000625 2007-02-06 2008-01-26 Luciférase sécrétée lu164m3 et son utilisation Ceased WO2008095623A2 (fr)

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DE102007005804.9 2007-02-06
DE200710005804 DE102007005804A1 (de) 2007-02-06 2007-02-06 Sekretierte Luziferase Lu164M3 und deren Verwendung

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013544100A (ja) * 2010-12-03 2013-12-12 ジーン・ストリーム・ピーティワイ・リミテッド 改良光放射分子
CN116536354A (zh) * 2022-09-27 2023-08-04 中国水产科学研究院黄海水产研究所 一种欧洲海鲈npffr2重组质粒、细胞系及其应用

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Publication number Priority date Publication date Assignee Title
AT508478B1 (de) 2009-06-26 2012-01-15 Tdw Delaware Inc Sensorarray für die inspektion der innenwand eines rohres

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10058091A1 (de) 2000-11-23 2002-06-06 Bayer Ag Isolierte Luziferasen Lu164, LuAL und Lu22, sowie deren Verwendung
WO2006061906A1 (fr) * 2004-12-09 2006-06-15 Nec Soft, Ltd. Gene codant pour une nouvelle luciferase

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013544100A (ja) * 2010-12-03 2013-12-12 ジーン・ストリーム・ピーティワイ・リミテッド 改良光放射分子
EP2646548A4 (fr) * 2010-12-03 2014-05-14 Gene Stream Pty Ltd Molécules électroluminescentes améliorées
AU2011335901B2 (en) * 2010-12-03 2016-12-15 Gene Stream Pty Ltd Improved light-emitting molecules
US10428317B2 (en) 2010-12-03 2019-10-01 Gene Stream Pty Ltd Light-emitting molecules
US11661587B2 (en) 2010-12-03 2023-05-30 Promega Corporation Light-emitting molecules
CN116536354A (zh) * 2022-09-27 2023-08-04 中国水产科学研究院黄海水产研究所 一种欧洲海鲈npffr2重组质粒、细胞系及其应用

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