EP2126057A2 - Luciférase secrétée mluc7 et son utilisation - Google Patents

Luciférase secrétée mluc7 et son utilisation

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Publication number
EP2126057A2
EP2126057A2 EP08707330A EP08707330A EP2126057A2 EP 2126057 A2 EP2126057 A2 EP 2126057A2 EP 08707330 A EP08707330 A EP 08707330A EP 08707330 A EP08707330 A EP 08707330A EP 2126057 A2 EP2126057 A2 EP 2126057A2
Authority
EP
European Patent Office
Prior art keywords
luciferase
luciferases
lui
mluc7
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08707330A
Other languages
German (de)
English (en)
Inventor
Stefan Golz
Eugene Vysotski
Svetlana Markova
Anna Tumenceva
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AXXAM SpA
Original Assignee
AXXAM SpA
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Filing date
Publication date
Application filed by AXXAM SpA filed Critical AXXAM SpA
Publication of EP2126057A2 publication Critical patent/EP2126057A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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 MLuc7, 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.
  • 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.
  • 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 efficiency of the systems is between 0.1-0.9 photons per converted
  • 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 LuI 64 is also described in Markova et al. Of 2004.
  • 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. Among the most common reporter genes is beta-galactosidase (Alam et al., 1990), alkaline phosphatase (Yang et al., 1997, Cullen et al., 1992), luciferases, and other photoproteins (Shinomura, 1985, Phillips GN, 1997; Snowdowne et al., 1984).
  • 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.
  • MLuc7 a new luciferase was surprisingly identified (hereinafter referred to as MLuc7) and cloned, whose biochemical and physicochemical properties differed significantly from the previously identified luciferases.
  • the expression of the secreted luciferase MLuc7 turned out surprisingly, that the luciferase aus utilizat an altered temporal resolution of the bioluminescent reaction (kinetics).
  • the kinetics differences are substrate independent for the substrates examined and shown in FIGS. 8 and 9.
  • the course of the bioluminescent reaction for MLuc7 and Lu 164 is shown in FIG. The significantly faster kinetics of MLuc7 can be clearly seen.
  • MLuc7 shows after only a few seconds a drop in the measured luminescence per second as LuI 64. After 60 seconds 70-80% of the integral signal of 300 seconds are already detected. Lu 164 shows a much slower drop of the bioluminescence signal per second, so that even after 300 seconds, a clear signal on background is measurable. MLuc7 is therefore kinetically different from the previously described secreted luciferases from Metridia longa. Due to this property, MLuc7 can surprisingly be used in combination with other coelenterazine-dependent or coelenterazine-independent luciferases, since a kinetic differentiation is possible. activity
  • MLuc7 per second significantly higher than LuI 64. This higher bioluminescence allows a higher sensitivity of the measuring method used, as a lower
  • the invention relates to the use of MLuc7 to improve the sensitivity, the use of low cell counts or low S stratkonzentrationen.
  • the altered kinetic properties of MLuc7 allow a differentiated kinetic evaluation of bioluminescence.
  • different intervals can be used for the evaluation.
  • FIG. 13 shows the summed bioluminescence signals for intervals of 10 seconds each. Within the first 60 seconds (the exact time depends on the amount of used
  • Luciferase and substrate ab shows MLuc7 a significantly higher bioluminescence than LuI 64. After this period, the bioluminescence of MLuc7 decreases faster than LuI 64, so that LuI 64 has a higher bioluminescence signal.
  • FIG. 14 shows the summed bioluminescence of the luciferases MLuc7 and Lu 164 for a period of 300 seconds under the selected experimental conditions.
  • FIG. 15 shows the summed bioluminescence signals for intervals of 60 seconds each.
  • the length and selection of the measurement intervals can therefore 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 bioluminescent activity of MLuc7.
  • the invention relates to the kinetic evaluation of measurements of the bioluminescence activity of Lul64, Lu22, LuAL, Lu39, Lu45, Lul ⁇ 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 is particularly suitable for multiplex reactions due to its altered properties.
  • the luciferase MLuc7 shows a significantly faster kinetics compared to other luciferases, which allows a combination with other luminescent or non-luminescent measuring methods (readouts).
  • the luminescent systems do not mutually inhibit each other or outshine the respective signals.
  • 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 when both systems use independent substrates.
  • MLuc7 significantly shortens the time between measurements due to its fast kinetics. Inactivation of the reaction is not necessary. Since the luciferase MLuc7 is a secreted luciferase, a combination with intracellular systems (such as Firefly luciferase) is possible.
  • luciferases from Metridia longa are also suitable for combination with intracellular systems such as the firefly luciferase. However, an inactivation step is necessary to lower the residual bioluminescence to a low level.
  • the invention relates to the use of MLuc7 in multiplex approaches in which MLuc7 is used in combination with one or more reporter genes or readouts. According to the invention, the use of Mluc7 in approaches for the measurement of multiple target genes.
  • the invention relates to the use of LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, and Lu52 in multipex approaches in which LuI 64, Lu22, LuAL, Lu39, Lu45, LuI6 and Lu52 in combination with one or more reporter genes or measurement techniques (readouts) is used. Also useful in the present invention is the use of LuI 64, Lu22, LuAL, Lu39, Lu45, LuI6 and Lu52 in approaches to measuring multiple target genes.
  • the invention relates to the use of secreted luciferases in multipexen
  • 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.
  • luciferases Use of the luciferases on the basis of substrates and reaction conditions is possible.
  • the substrates Firefly and Cypridina luciferin are not used under the chosen reaction conditions of all three luciferases or only to a small extent as substrates.
  • the invention relates to the use and combination of different
  • the invention relates to the use and combination of different substrates for producing bioluminescence by LuI 64, Lu22, LuAL, Lu39, Lu45, Lul6 and Lu52.
  • the invention relates to the use and combination of different
  • the invention relates to the use and combination of different substrates for producing bioluminescence by proteins according to the invention.
  • Bioluminescence reaction at temperatures between 10 and 50 0 C measured.
  • For this supernatants from a transient transfection of CHO cells with MLuc7 were used.
  • the result shows a dependence of the bioluminescent reaction of MLuc7 on the reaction temperature. This dependence can be used both to optimize and tailor the reaction in reporter gene applications, as well as to distinguish and combine different bioluminescent systems.
  • the invention relates to the use and combination of temperature dependence for the development and optimization of measurement methods for MLuc7.
  • the invention relates to the use and combination of
  • the invention relates to the use and combination of temperature dependence for the development and optimization of measurement methods for secreted luciferases.
  • the invention relates to the use and combination of temperature dependence for the development and optimization of measurement methods for proteins according to the invention.
  • the bioluminescent reaction was measured at KCl concentrations between 1 and 400 mM.
  • the supernatant from a transient transfection of CHO cells with MLuc7 were used.
  • the result shows a dependence of the bioluminescent reaction of MLuc7 on the ion concentration in the reaction medium. This dependence can be both for
  • the invention relates to the use and combination of the ionic dependence of the bioluminescent reaction for the development, optimization and use of measurement methods for MLuc7.
  • the invention relates to the use and combination of the ion-dependence of the bioluminescent reaction for the development, optimization and use of measuring methods for Lul64, Lu22, LuAL, Lu39, Lu45, Lul6 and Lu52.
  • the invention relates to the use and combination of the ion dependence of the bioluminescent reaction for the development, optimization and use of
  • the invention relates to the use and combination of the ion dependence of the bioluminescent reaction for the development, optimization and use of measuring methods for proteins according to the invention.
  • bioluminescent proteins affected by these factors are currently unknown. It is also possible to speculate about a development-stage-specific expression of bioluminescent proteins, but such is to be assumed.
  • a targeted examination of individuals of selected stages of development can therefore lead to the identification of bioluminescent proteins that are not expressed or significantly less expressed in other development stages and are therefore only partially accessible to expression cloning.
  • the invention relates to the study of bioluminescent organisms of specific developmental stages for the identification of novel bioluminescent proteins.
  • RNA from Metridia longa was isolated using the Straight A's mRNA isolation kit (Novagen) according to the manufacturer.
  • the isolated poly A mRNA was detected using the PowerScript Reverse Transcriptase (Clontech) in cDNA using the SMART cDNA Library Construction Kit (Clontech)
  • the vector pTriplEx2 (Clontech) was used, the cDNA fragments were integrated into the Sfil A-B interfaces.
  • the resulting expression vectors were transformed by means of electroporation into E. coli XLI-Blue.
  • the E. coli transformants were added
  • coelenterazine (native) was added to a final concentration of 10 ⁇ M and the bioluminescence determined in a luminometer.
  • the cDNA of the bioluminescence-positive clones was sequenced using the ALFexpress II system according to the manufacturer's instructions (TermoSequenase Cy5 Dye Terminator Kit (GE Healthcare)).
  • the invention relates to the secreted luciferase MLuc7 having the amino acid sequence represented by SEQ ID NO: 2.
  • the invention also relates to the nucleic acid molecule represented in SEQ ID NO: 1.
  • the invention also relates to functional equivalents of the secreted luciferase
  • MLuc7 Functional equivalents are those proteins that have comparable physicochemical or biochemical properties. Also functional fragments of the MLuc7 protein or for such encoding nucleic acids are according to the invention.
  • mutants of the MLuc7 protein or nucleic acids coding for such are erf ⁇ ndungshiel.
  • the secreted luciferase MLuc7 is suitable as reporter gene for the technique of
  • HCS High content screening
  • the secreted luciferase MLuc7 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 MLuc7 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 MLuc7 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 MLuc7 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,
  • the secreted luciferase MLuc7 turns out to be a marker protein, especially in the FACS (fluorescence activated cell sorter) sorting.
  • the secreted luciferase MLuc7 is suitable as a fusion protein especially for receptors, for ion channels, for transporters, for transcription factors, for Proteinases, kinases, phosphodiesterases, hydrolases, peptidases, transferases, membrane proteins, glycoproteins.
  • the secreted luciferase MLuc7 is suitable for immobilization, in particular by antibodies, by biotin, by magnetic or magnetizable carriers.
  • the secreted luciferase MLuc7 is suitable for systems of energy transfer especially the fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), field effect transistor (FET), fluorescence polarization (FPRF), homogenous time-resolved fluorescence (HTRF) ) Systems.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • FET field effect transistor
  • FPRF fluorescence polarization
  • HTRF homogenous time-resolved fluorescence
  • the secreted luciferase MLuc7 is suitable for labeling substrates or
  • the secreted luciferase MLuc7 is suitable for expression in bacterial systems especially for titer determination, as substrates for biochemical systems especially for proteinases and kinases.
  • the secreted luciferase MLuc7 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 MLuc7 is suitable as a reporter gene in the pharmacological search for active substances, especially in HTS (High Throughput Screening).
  • the secreted luciferase MLuc7 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 MLuc7 is suitable as a marker for the analysis of interactions specifically for protein-protein interactions, for DNA
  • RNA-RNA Interactions for RNA-protein interactions (DNA: deoxyribonucleic acid; RNA: ribonucleic acid).
  • the secreted luciferase MLuc7 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 MLuc7 is suitable as a marker or fusion protein for the analysis of embryonic development.
  • the secreted luciferase MLuc7 is useful as a marker via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
  • the secreted luciferase MLuc7 is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.
  • the secreted luciferase MLuc7 is suitable as a reporter coupled to proteins or peptides.
  • nucleic acid or the peptide of the coupled protein MLuc7 is suitable as
  • the protein MLuc7 is suitable as a marker of pharmacological formulations especially of infectious agents, of antibodies, of "small molecules”.
  • the protein MLuc7 is suitable for geological investigations especially for ocean, groundwater and river currents.
  • the protein MLuc7 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 MLuc7 specifically as a wild-type protein, as a fusion protein, as a mutagenized protein.
  • the invention also relates to the use of MLuc7 in the field of cosmetics, especially bath preparations, lotions, soaps, body colors, toothpaste, body powders.
  • the invention also relates to the use of Mluc7 for coloring foodstuffs, bath additives, ink, textiles, plastics.
  • the invention also relates to the use of Mluc7 for coloring paper, especially greetings cards, paper products, wallpaper, craft articles.
  • the invention also relates to the use of Mluc7 for staining
  • Liquids especially for water pistols, for fountains, for drinks, for ice cream.
  • the invention also relates to the use of Mluc7 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 MLuc7.
  • the invention relates to methods for expression of the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in vitro
  • 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
  • the invention relates to the secreted luciferase MLuc7 having the amino acid sequence represented by SEQ ID NO: 2 and the nucleotide sequence represented by SEQ ID NO: 1.
  • a protein MLuc7 characterized in that its
  • Sequence comprising 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. Mutants and derivatives of secretory luciferases
  • FIG. 3 shows the alignment of the luciferases MLu7, the metridia luciferases (LuI 64, Lu22, LuAL, Lu39, Lu45, LuI6 and Lu52) and of the gaussia luciferase.
  • the luciferase MLuc7 represents a significantly shorter polypeptide than the other analyzed luciferases.
  • the luciferases Lu22 and Gaussia luciferase also comprise significantly shorter polypeptides.
  • 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 which are changes or deletions in the range of amino acids 23 to 78 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives which are changes or deletions in the range of amino acids 23 to 78 of the luciferases Lul64, Lu22, LuAL, Lu39,
  • Lu45, LuI6, Lu52 and Gaussia luciferase with altered biochemical or physicochemical properties of the luminescent reaction.
  • mutants or derivatives are the changes or deletions in the range of amino acids 13 to 88 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the range of the amino acids 13 to 88 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered biochemical or physicochemical properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the range of amino acids 33 to 68 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered kinetic properties of the luminescence reaction.
  • mutants or derivatives are the changes or deletions in the range of amino acids 33 to 68 of the luciferases LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6, Lu52 and Gaussia luciferase with altered biochemical or physicochemical properties of the luminescence reaction.
  • the invention particularly relates to:
  • a nucleic acid molecule selected from the group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence disclosed by SEQ ID NO: 2;
  • nucleic acid molecules comprising the sequence shown in SEQ ID NO: 1;
  • nucleic acid molecules whose complementary strand hybridizes with a nucleic acid molecule from a) or b) under stringent conditions and which encode luciferases;
  • nucleic acid molecules which differ from those mentioned under c) due to the degeneration of the genetic code
  • nucleic acid molecules which have a sequence identity of at least 70, 75,
  • SEQ ID NO: 1 80, 85, 95%, 98%, 99% to SEQ ID NO: 1 and their protein products are luciferases;
  • nucleic acid molecules which have a sequence identity of at least 65%
  • SEQ ID NO: 1 show and which luciferases encode; g) fragments of the nucleic acid molecules according to a) - f), wherein the fragments encode functional luciferases.
  • a nucleic acid according to item 1 which contains a functional promoter 5 'to the photoprotein coding sequence.
  • oligonucleotides having more than 10 consecutive nucleotides which are identical or complementary to a partial sequence of a nucleic acid molecule according to item 1.
  • reporter gene (s) is the firefly luciferase, or luciferases from the organism Metridia longa.
  • luciferases selected from the group consisting of LuI 64, Lu22, LuAL, Lu39, Lu45, LuI 6 and Lu52.
  • SEQ ID NO: 2 (MLuc7 amino acid sequence)
  • MDIKFIF ALVCIALVQ ANPTVNND VNRGKMPGKKLPLEVLIEMEANAFKAGC TRGCLICLSKIKCTAKMKQYLPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFK EMEPMEQFIAQVDLCADCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQ KEAHNIKGLAGDR
  • SEQ ID NO: 3 (Lul64 - nucleotide sequence - coding)
  • SEQ ID NO: 4 (Lul64 amino acid sequence)
  • FIG. 1 A first figure.
  • FIG. 1 shows the vector map of the construct pcDNA3-MLuc7.
  • FIG. 2 shows the vector map of the construct pASM-MLuc7.
  • FIG. 3 shows an alignment of different secreted luciferases at the amino acid level.
  • FIG. 4 shows the substrate specificity of the luciferases Lu 164, Lu22 and MLuc7.
  • Axis Coelenterazine, Y-axis: relative light units (RLU).
  • RLU relative light units
  • FIG. 5 shows the temperature dependence of the luciferase MLuc7.
  • X-axis temperature in 0 C
  • Y-axis relative light units (RLU).
  • RLU relative light units
  • FIG. 6 shows the dependence of the luciferase MLuc7 on the calcium chloride concentration in the reaction buffer.
  • X-axis KCl concentration in mM
  • Y-axis relative light units (RLU).
  • RLU relative light units
  • FIG. 7 shows the bioluminescence measurement of MLuc7 at a constant MLuc7 amount and different concentrations of coelenterazine.
  • X-axis Coelenterazine concentration in ⁇ M.
  • Y axis : relative light units (RLU).
  • the figure shows the kinetics of the bioluminescent reaction of MLuc7 with three different coelenterazines.
  • X-axis time in seconds.
  • Y axis :: relative light units (RLU).
  • the figure shows the kinetics of the bioluminescent reaction of Lu 164 with three different coelenterazines.
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • FIG. 10 shows the result of the bioluminescence measurement of MLuc7 (black) and LuI 64 (gray) for a measurement of 300 seconds with an integration time of 1.5 seconds.
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • FIG. 11 shows the result of the bioluminescence measurement of MLuc7 at a constant substrate concentration and decreasing concentration of Mluc7 by dilution of the cell supernatant.
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • Figure 12 shows the result of bioluminescence measurement of LuI 64 at a constant substrate concentration and decreasing concentration of Lu 164 by dilution of the cell supernatant.
  • X-axis time in seconds.
  • Y axis relative
  • FIG. 13 shows the result of the kinetic evaluation of the bioluminescence measurement of Mluc7 and LuI 64 in sections of 10 in each case
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • FIG. 14 shows the result of the kinetic evaluation of the bioluminescence measurement of Mluc7 and LuI 64 with an integration time of 300
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • FIG. 15 shows the result of the kinetic evaluation of the bioluminescence measurement of Mluc7 and LuI 64 in sections of 60 in each case Seconds of integration time.
  • X-axis time in seconds.
  • Y axis relative light units (RLU).
  • Figure 19 shows the representation of the preferred water depth of individuals of the species Metridia longa, depending on the status of development.
  • X axis :
  • the plasmid pcDNA3.1 (+) from Clontech was used for constitutive 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-MLuc7.
  • the derivative of the vector pcDNA3 was called pcDNA3-MLuc7.
  • the cloning was carried out using standard molecular biological methods.
  • the vectors pcDNA3-Mluc7 and pASM-MLuc7 were used to express MLuc7 in eukaryotic systems.
  • Fig. 1 shows the plasmid map of the vector pcDNA3-Mluc7.
  • Fig. 2 shows the plasmid map of the vector pASM-MLuc7.
  • the constitutive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-MLuc7, pcDNA3-Lu 164 and pcDNA3 (without cDNA insertion) in transient experiments.
  • 10,000 cells per well in DMEM-Fl 2 medium were plated on 96-well microtiter plates and incubated overnight at 37 ° 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 takes place after substrate addition with an imaging
  • buffer A pH 7.4
  • Composition 130mM NaCl, 5mM KCl, 20mM Hepes, 1mM MgC12 x 6H2O and 5mM NaHCC ⁇
  • the transfected cells were selected with 2 mg / ml geneticin and the bioluminescence activity of the clones or supernatants was determined.
  • FIG. 3 shows the alignment of the secreted luciferases at the amino acid level.
  • Cypridina luciferase was not included in the alignment because its sequence identity to the remaining luciferases is too low.
  • 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 luciférase sécrétée MLuc7.
EP08707330A 2007-02-06 2008-01-26 Luciférase secrétée mluc7 et son utilisation Withdrawn EP2126057A2 (fr)

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DE102007005803A DE102007005803A1 (de) 2007-02-06 2007-02-06 Sekretierte Luziferase MLuc7 und deren Verwendung
PCT/EP2008/000624 WO2008095622A2 (fr) 2007-02-06 2008-01-26 Luciférase secrétée mluc7 et son utilisation

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WO2012071631A1 (fr) 2010-12-03 2012-06-07 Gene Stream Pty Ltd Molécules électroluminescentes améliorées
US8435777B1 (en) 2012-01-20 2013-05-07 Cayla CPG-free gene for a new secreted reporter protein
RU2495929C1 (ru) * 2012-05-30 2013-10-20 ФЕДЕРАЛЬНОЕ ГОСУДАРСТВЕННОЕ БЮДЖЕТНОЕ УЧРЕЖДЕНИЕ НАУКИ Институт биофизики Сибирского отделения Российской академии наук УКОРОЧЕННАЯ МУТАНТНАЯ ЛЮЦИФЕРАЗА ИЗ Metridia longa ДЛЯ ПРИМЕНЕНИЯ В КАЧЕСТВЕ БИОЛЮМИНЕСЦЕНТНОГО РЕПОРТЕРА В ЖИВЫХ КЛЕТКАХ
CN103160528B (zh) * 2013-03-07 2014-06-11 西北农林科技大学 增强型单体细菌荧光素酶基因luxAB及其应用
CN104178463B (zh) * 2013-04-27 2017-06-20 西北农林科技大学 一种制备增强型单体细菌荧光素酶luxAB的方法
US10737111B2 (en) * 2014-12-16 2020-08-11 Rensselaer Polytechnic Institute X-optogenetics / U-optogenetics
CN111534529A (zh) * 2020-05-09 2020-08-14 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一种报告基因细胞株及其构建方法和应用
RU2757736C1 (ru) * 2020-08-17 2021-10-21 Федеральное государственное бюджетное научное учреждение "Федеральный исследовательский центр "Красноярский научный центр Сибирского отделения Российской академии наук" (ФИЦ КНЦ СО РАН, КНЦ СО РАН) Мутантная копеподная люцифераза для применения в качестве биолюминесцентного репортера in vitro и in vivo

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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

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RU2009130108A (ru) 2011-02-10
WO2008095622A2 (fr) 2008-08-14
JP2010517543A (ja) 2010-05-27
WO2008095622A3 (fr) 2008-10-02
US20100105090A1 (en) 2010-04-29
TW200846469A (en) 2008-12-01
DE102007005803A1 (de) 2008-08-07
CA2677424A1 (fr) 2008-08-14
CN101821384A (zh) 2010-09-01
KR20090116733A (ko) 2009-11-11

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