TW200900418A - Isolated mtclytindecay photoprotein and use thereof - Google Patents

Abstract

The invention relates to the mtClytinDecay-141F and mtClytinDecay-182F photoproteins, to the nucleotide and amino acid sequences thereof, and to the activity and use of said mtClytinDecay-141F and mtClytinDecay-182F photoproteins.

Description

200900418 IX. INSTRUCTIONS: [Technical Field] The present invention relates to mtClytinDecay-141F and mtClytinDecay-182F photoproteins, nucleotide and amino acid sequences thereof, and photosynthetic proteins of mtClytinDecay-141F and mtClytinDecay-182F Activity and use. [Prior Art] Photoproteins 10 The phenomenon of luminescence through a living organism is related to bioluminescence. It is the result of a biochemical reaction in a cell that involves the release of chemical energy in the form of a light quanta ("cold, emission" in a chemiluminescent manner. The light produced in this way is monochromatic because it It is emitted during a discontinuous electron transition, although it can be transferred to a longer wavelength spectral region by secondary luminescers (such as the fluorescent protein of the jellyfish of the genus Jellyfish). Many and varied: in the ocean, at a depth of between 2 〇〇 and 1000 m (Mesopelagial), about 90% of all creatures emit light. The illuminating signal is used here to court, bully, and act as Inducing 20 baits. Fireflies (gl〇w-worms) and fireflies (fireflies) also use optical signals on search partners. Conversely, the meaning of bacteria, fungi, and unicellular algae is not clear. The multiplicity of a large group of individuals may be a biological clock. The multiplicity of coelenterates is bioluminescence 200900418 (bioluminescent) (Mori n et al" 1974). These creatures emit this or green light. Aequoria victoria's multi-unit aequorin (aequ〇rin) was identified as a light-producing protein in 1962 (Shimonrura et al 1 and 'as a separate protein, emitting blue light instead of The multi-tube jellyfish 5 is phenotypically observed in green light. Later, green fluorescent protein (GFP) is separated from the multi-tube jellyfish, which makes the jellyfish (medusa) multi-tube jellyfish Excited and phenotypically green (Johnson et al., 1962; Hastings et al., 1969;

Inouye et al., 1994). Other photoproteins 10 that have been identified and described are Clytin (Inouye et al., 1993), Mitrocomin (Fagan et al., 1993), and 〇belin (Illarionov et al., 1995). :SJLL Summary of some light proteins. Indicated is the name, the organism from which the protein has been separated, and the identification number of the database item (Acc. No.). Name Biometric number Obeli geniculata AAL86372 Clytin Clytia gregaria CAA49754 Multi-tube jellyfish large multi-tube jellyfish (Aequorea macrodactyla) AAK02061 Multi-tube jellyfish small multi-tube jellyfish (Aequorea parva) AAK02060 Mitrocomin Mitrocoma cellularia AAA29298 Pholasin Seaweed (Pholas dactylus) AAM18085 ?Symplectoteuthis oualaniensis AX305029 6 200900418 Summary of some photoproteins. Specified is the name of the organism from which the protein has been isolated, the name of the photoprotein, and the choice of patent and/or application. Name----- Biometric number Knee lychee Obelin W003006497 Clytia gregaria Clytin W003006497 Clytia gregaria mtClytin W02005035559 Multi-tube jellyfish multi-tube jellyfish W0200168824 US-0908909 US 6,152,358 Sea bamboo Pholasin W00028025 GB-0024357 5 Bioluminescence It is currently used technically in many ways, for example in the form of bioindicators for contamination or for the detection of proteins in biochemistry for quantification of specific compounds or in the study of cellular gene regulation. As a "reporters". Photoproteins are not only because their nucleotides differ from the amino acid sequence, but also because of their biochemical and physical properties. Physical and biochemical properties have been shown to be altered by modifying the amino acid sequence of the photoprotein. Examples of muted photoproteins have been described in the literature (US 6,495,355; US 5,541,309; US 5,093,240; Shimomura et al., 1986). 15 Light is produced by the above-mentioned photoprotein through the oxidative coelenterazine (Haddock et al., 2001; Jones et al., 1999). 7 200900418 Reporting subsystem genes (whose gene products can be easily detected with the aid of simple biochemical or histochemical methods) are often referred to as reporters (rep〇rter) or fingers (砠(11〇) The gene of at least two reporters has been distinguished. 5 丨. resistance gene (resistance senses). The resistance gene means a gene whose expression is given to cells for antibiotics or other substances (lack of resistance genes, Resistance to cell death in the growth of cardiotrophin. 2. Reporter gene. The product of the reporter gene is used as a fusion or non-fusion indicator in genetic engineering. The most commonly used reporter Genes include B. citrate-galactosidase (Alametal., 1990), alkaline phosphatase (Yang et al., 1997; Cullen et al) 1992), luciferase and other photoproteins (Shinomura, 1985; Phillips GN, 1997; Snowdowne et al., 1984). Luminescence means the emission of a photon in the visible region of the spectrum, which is emitted by an excited emitter molecule. The energy is not short-wavelength here relative to fluorescence. Radiation form The source provides a distinction between chemiluminescence and bioluminescence. Chemiluminescence refers to a chemical reaction that causes an excited molecule to emit light when the excited electron returns to an excited state. If the reaction is affected by an enzyme Catalyzed '20 This is meant to be bioluminescence. Enzymes involved in this reaction are usually referred to as luciferase. Mutant mutants are prepared by position 141 [141F] (SEQ ID NO: 1) and 8 200900418 Position 182 [182F] (Sequence Identification Number 3) was prepared by introducing mutations with the help of molecular physiology methods. For this reason, Stratagen's "quick change" method (catalog number #200521; Revision #〇 630〇lb; version 2003) is used. The primer used is sequence identification number: 9, sequence identification, numbering: 5 10. Sequence identification number: 11 and sequence identification number · · 12. The carrier is referred to as pTriplExLmtClytinDecay -UlF and pTdplEx2_ mtClytinDecay-182F. mtClytinDecay 〇 Previous literature has described photoproteins as having modified spectra and biochemical properties due to substitution of individual amino acids These proteins include Oberlin W92F (Vysotski et al., 2003) and multi-tube aequor (Shrestha et al, 2002; Ohmiya et al., 1993) ° mtClytin mutant 'mtClytinDecay, compared to mtClytin light 5 Protein or other photoproteins exhibit a modified light rdease time course. Surprisingly, mtClytinDecay photoprotein exhibits light release and kinetic deceleration kinetics (sl〇wed_d〇wn kinetics) that have not been previously described. This property allows photoproteins to be specifically used to study reactions or mechanisms with very rapid calcium release in eukaryotic cells or other systems. The light release kinetics of the previously described photoprotein mutants or wild-type photoproteins are described as "flash" kinetics because light is released within a very short time after activation (eg by Calcium) and the reaction then comes to a termination or at least becomes significantly weaker. These fast dynamic measurements require a dedicated measurement of the 200900418 instrument. The photoprotein mutant, mtClytinDecay, or its equivalent, not only enables other measuring instruments or measurement methods to be used, but also enables extremely fast kinetics to be investigated. These kinetics may occur, for example, as an ion channel comprising the P2X steroid. 5 mtClytinDecay is not only attractive because of the modified kinetics, but also surprisingly high light release. This high light release - compared to other mutant photoproteins - allows the mtClytinDecay mutant to be used as a sensitivity reporter gene. This high light release also allows the measurement instrument or method (which utilizes other photoproteins or the light release of the reporting subsystem to produce only insufficient results) to be used. The spectrum of mtClytin has been described as having a peak at 470 nm (Shimomuro et al) 1966. A summary of the spectral properties of coelenterazine has been described by Shinomuro (Shimomuro et al., 2000) 〇 mtClytin Decay photoprotein in the amine group The acid level is the most homologous to aytia 15 greSaria mtC1ytin, and has a 99% identity. [Invention] The present invention relates to mtCiytinDecay_141F having the sequence identification number: 2 and the sequence identification number: 4. And mtClytinDecay_2〇182F photoprotein. The present invention is equally related to the nucleic acid molecule described in the sequence identification number: i and the sequence identification number: 3. The present invention also relates to the functional equivalent of mtClytinDecay-141F and mtClytinDecay·182F Functional equivalents or fragments are those having comparable functional properties. 200900418 The present invention relates to mtClytin photoproteins having an amino acid mutation at position 141 which cause altered bioluminescent properties , especially bioluminescence kinetics. In this paper, mtCiytin photoproteins can also be those light White's having one or more amino acid mutations in the region of amino acid 131-151, 136-146, preferably 139-143, especially 140-142, which result in altered bioluminescent properties, in particular Bioluminescence kinetics. Furthermore, the present invention relates to mtClytin photoproteins having an amino acid mutation at position 182 which cause altered bioluminescent properties, particularly bioluminescence kinetics. In this context, mtClytin photoprotein is also It may be those photoproteins which have an amino acid mutation in the region of amino acid 172_192, Π7-187, preferably 180-184, in particular 18M83, which results in altered bioluminescent properties, in particular bioluminescence The regions having a similar motif are those having an identity of 8 〇 %, preferably 90%, especially 95% in this region. The present invention relates to the position of the amino acid at 131 -151, 136-146, preferably 139-143, especially 140-142 regions having one or more amino acid mutations that cause altered bioluminescent properties, particularly bioluminescence kinetics, of mtClytin light Protein 'and position 18 in the amino acid The region of 2 has a mutation or a combination of mutations in the region of the amino acid position 182 that result in altered bioluminescent properties. Furthermore, the invention pertains to having an amino acid mutation at position 182 (which results in a modification) The mtClytin photoprotein of bioluminescent properties, particularly modified bioluminescence kinetics, and the combination of mutations in the region of amino acid position 141. Herein, photoproteins may also be those having a similar module in the region of the present invention. The regions having a type of 200900418-like module are those in which there is a sequence of 8 〇 0 / 相同, preferably 90%, especially 95%. There are more combinations of canine mtClytin (which result in a modified bioluminescent property', especially modified bioluminescence kinetics), mutations or derivatives with mtClytin 5 or Clytin (which result in modified physicochemical properties, Is the spectral properties, calcium sensitivity or binding of the substrate). The invention also relates to a functional fragment of the mtClytin protein or a nucleic acid encoding the fragment. The invention likewise pertains to truncated functional fragments of more proteins of the invention or nucleic acids encoding such fragments. The mtClytmDecay photoprotein is useful as a reporter gene for use in cellular systems, particularly for receptors 'for ion channels, for transporters, for transcription factors or for inducible systems. mtClytinDecay photoprotein is also suitable for use as a reporter mtClytinDecay photoprotein as a reporter gene for the determination of cell organelles by using the method of describing the cell organelles (especially mitochondria) as a reporter. Inside and outside (especially granules, especially calcium,

A bioluminescent or chemiluminescent system (especially a cell system with luciferase for use in the group with the addition of 200900418 oxygenase with lyase). mtClytinDecay photoprotein is suitable for use as a fusion protein, particularly for receptors, for ion channels, for transport proteins, for transcription factors, for proteases, for kinases, for phosphodiesterases, for hydrolases , 5 for peptidases, for transferases, for membrane proteins and for cool proteins. mtClytinDecay photoprotein is suitable for immobilization (imm〇bilizati〇n), especially through antibodies, through biotin, through magnetic or through magnetic structures. mtClytinDecay photoprotein is suitable for use as a protein in energy transfer systems, in particular FRET (fluorescence res〇nance energy i〇s ansfer's fluorescence resonance energy transfer), BRET (bioluminscence resonance energy transfer), fet (field effect transistors), Fp (flU0rescence polarization) HTRF (homogeneous time-resolved fluorescence) system. The i5 mtC1ytinDecay photoprotein is suitable for use as a marker for a receptor or a ligand', particularly for proteases, for kinases, for transferases. mtClytinDecay photoprotein is suitable for use in bacterial systems (especially for determining potency) and as a substrate for biochemical systems (especially for proteases and kinases). 2〇 mtClytinDecay photoprotein is suitable for use as a label, particularly for coupling to an antibody, coupling to an enzyme, coupling to an acceptor's coupling to an ion channel and other proteins. mtClytinDecay photoprotein is suitable for use as a reporter gene in pharmacological drug screening, particularly in HTS (high throughput screening, high 13 200900418 flux screening). mtClytinDecay photoprotein is suitable for use as a reporter gene in the description, identification and research of ion channels, especially P2X, TRP, SCN, KCN 'CNG, ACCN type. 5 mtClytinDecay photoprotein is suitable for use as a component of detection system, especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for western blotting, for confocal microscope. mtClytinDecay photoprotein is suitable for labeling for interaction analysis, in particular for protein-protein interactions, for DNA-protein interactions, for DNA-DNA interactions, for RNA-RNA interactions, for RNA-protein interaction (DNA: deoxyribonucleic acid; rna: ribonucleic acid). mtClytinDecay photoprotein is suitable for use in transgenic organisms as a 15 marker or fusion protein, particularly in mice, in the atmosphere, in hamsters and other mammals, in primates, in fish, in In insects, in plants. mtClytinDecay photoprotein is suitable for use as a marker or fusion protein in the analysis of embryo development. 2〇 mtClytinDecay photoprotein is suitable for use as a marker via a coupling regulator', particularly via biotin, via (N-hydiroxysulphosuccinimide 'N-hydroxythiosuccinimide), from CN-Br. ί mtClytinDecay photoprotein is suitable for use as a reporter for coupling to nucleic acids 14 200900418, especially to DNA, to RNA. mtClytinDecay photoprotein is suitable for use as a reporter for coupling to a protein or peptide. The mtClytinDecay photoprotein is suitable for use as a reporter for measuring intracellular or fine extracellular calcium concentration. mtClytinDecay photoproteins are useful for depicting signal cascades in cellular systems. mtClytinDecay photoprotein coupled to a nucleic acid or peptide is suitable for use as a probe, particularly for Northern blotting, for Southern blotting, for Western blotting, for ELISA, for nucleic acid sequences, For protein analysis, wafer analysis. mtClytinDecay photoprotein is suitable for use in the pharmacological formulation of markers, especially infectious, antibody, and small molecules. mtClytinDecay photoprotein is suitable for geological analysis, especially for seas, oceans and rivers. mtClytinDecay photoprotein is suitable for expression in expression systems, especially in in vitro translation systems, in bacterial systems, in yeast systems, in baculovirus systems, in viral systems, in eukaryotic systems . 2〇 mtClytinDecay photoprotein is suitable for observing tissues or cells in surgical interventions, especially invasive, non-invasive, and minimally invasive. mtClytinDecay photoprotein is suitable for use in target tumor tissues as well as other phenotypically modified tissues, particularly in histological studies, in the surgical intervention 200900418. The invention also relates to the purification of mtClytin Decay photoproteins, particularly like wild-type proteins, such as fusion proteins, such as mutated proteins. The present invention is suitable for simultaneous measurement (multiplexing) of various reported purple 5 genes in a single expression system. The invention also relates to the use of mtClytin Decay photoproteins in cosmetics, particularly bathing additives, detergents, soaps, body paints, toothpastes, and body powders. The invention also relates to the use of mtClytinDecay photoprotein for dyeing, especially food, bath additives, inks, fabrics, and plastics. The invention also relates to the use of mtClytin Decay photoprotein for paper dyeing, particularly for greeting cards, paper products, wallpapers, handicrafts. The invention also relates to the use of mtClytinDecay photoprotein for liquid dyeing, in particular water guns, sodas, beverages, ice. 15 The present invention also relates to the use of mtClytin Decay photoprotein for the manufacture of toys, particularly finger licks, cosmetics. The invention relates to nucleic acid molecules encoding a polypeptide disclosed by SEQ ID NO: 2 or a functional equivalent or functional fragment thereof. The invention relates to nucleic acid molecules encoding a polypeptide or a functional equivalent or functional fragment thereof as disclosed by SEQ ID NO: 4. The invention more particularly relates to a nucleic acid molecule or a functional equivalent or functional fragment thereof selected from the group consisting of: a) a nucleic acid molecule, encoding one comprising a sequence identification number: 2 or a sequence recognition editor. 4 polymorphic amino acid sequence revealed; 200900418 • b) nucleic acid molecule, including the right-hander

The sequence described; column identification number ·" or sequence identification number: 3 C) nucleic acid molecule, Jt 25 " green B; L worker w eight-fold melon under severe conditions with a nucleic acid of a) or b) = two Its expression silk has the biological function of photoprotein; the second-severe hybridization is in an aqueous solution containing G.2xSSC-X^ saline-citric acid=15〇mM NaCl, i5mM trisodium citrate in the armpit. Executed (Sambr〇〇k et ai, Example 9). d) Nucleic acid molecules which differ from those described below because of the degeneracy of the genetic code. The nucleic acid molecule described above, wherein the sequence comprises a functional promoter 5 encoding a sequence of I(tetra), or a sequence encoding a leader or signal sequence. The invention also relates to a nucleic acid molecule, a portion of a DNA or RNA vector as described above. , 均里,, 孓 15 The present invention relates to an organism having such a carrier. The present invention relates to a photoprotein encoded by the above nucleotide sequence. / The invention relates to a method of expressing a photoprotein polypeptide according to the invention in a bacterial, eukaryotic cell or in an in vitro expression system. The invention also relates to a method of purifying/isolated a photoprotein polypeptide according to the invention. There is a use of a photoprotein according to the present invention, a nucleic acid as a marker gene or a reporter gene, particularly for pharmacological drug selection and diagnosis. The invention relates to the use of a photoprotein according to the invention and a photoprotein-encoding nucleic acid according to the invention of the invention as a marker or reporter and as a marker gene or reporter gene, respectively. The invention relates to the use of mtClytinDecay photoprotein (SEQ ID NO: 2 and sequence identification number: 4) or functional fragments or equivalents thereof 5 and encodes mtClytinDecay photoprotein or a functional fragment or equivalent thereof as Markers or reporters and their use as marker genes or reporter genes 'especially for pharmacological drug screening and diagnostics. The present invention relates to the use of nucleic acid molecules described in Sequence Identification Number: 1 and Sequence Identification Number: 3 as a marker gene or reporter gene, particularly for pharmacological drug screening and diagnostic methods. The invention also relates to the identification of a plurality of strains or monoclonal antibodies of a polypeptide according to the invention. The present invention also relates to single or multiple antibodies recognizing mtClytinDecay photoprotein (sequence identification number: 2 and sequence identification number: 4). The present invention also relates to a nucleic acid described in the preceding paragraph which contains a functional promoter 5' encoding the sequence. The invention encompasses recombinant DNA or RNA comprising the nucleic acid described above. An organism containing a carrier like the above is also in accordance with the present invention. A polypeptide encoded by a nucleic acid sequence as described above is also part of the present invention. A method of expressing the above polypeptide in a bacterial, eukaryotic cell or in vitro expression system is also in accordance with the invention. A method of purifying/isolated a polypeptide according to one of the present invention is also part of the present invention. 18 200900418 The invention relates to the use of a nucleic acid according to the invention as a marker gene or a reporter gene. The invention also relates to the use of a photoprotein according to the invention as a label or reporter. The use of a polypeptide according to the invention in combination with one or more luciferases and/or one or more photoproteins is also part of the invention. a photoprotein' or a functional fragment thereof having one or more mutations in the regions 131-151, 136-146, preferably 139143 'in particular 140-142 or having a region in positions 172_192, 177-187 Or a plurality of mutations, preferably 180-184, especially 181-183, or a combination of both, and having a modified 'especially decelerated bioluminescent signal is in accordance with the invention. A nucleic acid molecule comprising a sequence encoding a protein according to two anterior segments is also in accordance with the invention. Another part of the invention is a method of preparing a photoprotein, characterized in that one or more mutations are introduced into a photoprotein, preferably in the region defined by positions 13M51, 136-146, preferably 139-143, Is 140-142' or one or more mutations in the region of positions 172-192, 177-187 'preferably 180-184, especially 181-183, causing bioluminescence-modification', especially bioluminescence kinetics . A photoprotein prepared by the method described in the preceding paragraph is also in accordance with the present invention. The invention also relates to other photoproteins having modified light release kinetics due to one or more modifications in the amino acid sequence. 19 200900418 The invention also relates to the use of other modified photoproteins for the described use of mutants of the mtaytin Decay photoprotein. Photoproteins with modified light release kinetics (especially extended-time frames with quenching light release Ϊ = where light is released) are particularly suitable for use as a reporter gene in cell-based methods, especially in pharmacology Learn from music screening and description, especially in diagnostic methods. Photonic proteins with refractory light riding dynamics (especially with an extended time frame with (4) fast light release $ in which light is released) are particularly useful for studying ion channels. 10 15 20 The present invention also relates to codon-optimized variants of proteins according to the invention for altering biochemical or physicochemical properties, in particular for improved retrograde performance, in particular altered stability. The invention also relates to fusion of proteins according to the invention with recognition peptides for transport or localization of proteins in a cell organelle or compartment (c〇mpartment) according to the invention. The invention also relates to protein mutants according to the invention which result in changes in spectral properties, luminescence intensity, substrate specificity, use of cofactors, calcium affinity or other physicochemical or biochemical properties. The kinetics of light release can be varied as a function of experimental conditions. Figure 7 illustrates, by way of example, the light release kinetics process for mtClytinDecay. The expression of a photoprotein according to the present invention means the production of a molecule which, after the gene has been introduced into a stable 20 200900418 host cell, causes the foreign gene to be transcribed into a performance to be transcribed and translated. The expression vector contains the control signals required to express the gene in prokaryotic or eukaryotic cells. The performance carrier can in principle be constructed in two different ways. In "transcription fusi〇ns", proteins encoded by foreign genes that are transfected are synthesized like a formal, biologically active protein. To this end, the performance carrier carries all of the 5, and 3, signal signals required for performance. In "translation fusions", the protein encoded by the transgenic foreign gene is expressed as the same hybrid protein (hybrid protein heterozygous. 5, and 3 required for expression, control signal (including start codon) And a part of the sequence encoding the N-terminal region of the hybrid protein = 15 20 = born from the vector. The extra protein portion introduced is not only in many examples): it is able to stabilize the foreigner by the transplanter The protein encoded by the gene resists the decomposition of cells = enzymes, and can also be used to measure and separate the formed proteins. The performance can be temporary and stable. Bacteria, yeast, viruses, and eukaryotic cell lines. Purification of photoproteins according to the invention of white 4 white matter! Γ even after overexpression) generally means purification of protein shells. The methods and steps that have been established are for protein fg. m叮 Application Solid/liquid separation is the basic operation of _ separating eggs. This step is required when the cells are isolated from the culture 200900418 base, when the cells have been broken and the cell residue has been removed, and the crude extract is clarified, and when the sediments are separated after the sinking, etc. It occurs by centrifugation and filtration. In order to obtain intracellular protein, the cell wall must be destroyed or become permeable. Depending on the scale and the organism, a high pressure homogenizer or agitating ball mill or glass ball mill is used for this purpose. Mechanical cell pulverizers and ultrasonic processing are used, especially on a laboratory scale. 10 15 In the case of examples of extracellular proteins and examples of intracellular proteins (after cell rupture), various precipitation methods using salts (especially ammonium sulphate) or organic solvents (alcohol or acetone) represent rapid use for protein concentration. And an efficient method. When the intracellular protein is to be purified, it is necessary to remove the soluble nucleic acid (for example, streptomycin sulfate or protamine sulfate). When extracellular proteins are to be obtained, the support (e.g., starch or diatomaceous earth) is usually added prior to the addition of the precipitating agent to obtain a precipitate (which is easier to handle). The mouth ^ mm '/s · ~ team increase 沄 沄 and ion cross drag = method, gel over 遽, affinity chromatography and electrophoresis) is applied to, . The pillars of the pillars are also recorded on the scale of money. Affinity The purification factor of up to several hundred steps in the 3 step becomes feasible and is especially important. Λ 至 现 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20疋 的 的 , , , , , 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机 无机They can be removed through special, early, smear, gel filtration, dialysis and diafiltration. A large amount of protein can be used as a dry preparation. Important drying method vacuum Dry, cold east drying and spray drying. 5 Nucleotide and amino acid sequence mtClytinDecay-MlF photoprotein is encoded by the following nucleotide sequence (SEQ ID NO: 1):

GA.CAGATAAAAAATTCACTCCTTAGATTATTTAGTQAATAAGAGAAAAAAA.GGATAAGAAA

TCAAGATGCAAAGGTTTACAAATCGTCTTCTTTCCATGTCGGCTTTACGTGCAAGATCAAG

ATTGGAACGCACGGCAAATTTTCACACCAGCATACTCTTGGCTACAGATTCAAAATACXSCG

GTCAAA.CTCGATCCTGATT1TGCAAATCCAAAATGGATCAACAGACACAAATTTATGTTCA

ACTTTTTGGACATAAACGGTAAGGGGAAAATCACATTAGATGAAATCGTCTCCAAAGCTTC

AGACGACATTTGTGCTAAACTGGATGCAACACCAGAACAGACCAAACGTCACCAGGATGCT

GTTGAAGCCTTTTTCAAGAAAATGGGCATGGATTATGGTAAAGAAGTTGCATTCCCAGAAT

TTATTAAGGGATGGGAAGAGTTGGCCX3AACACGACTTGGAACTCTGGTCTCAAAAC3AAAG TACATTGATCCGTGAATGGGGAGATGCTGTTTTCGACATTTTCGACAAAGACGCAAGTGGC TCAATCAGTTTAGACGAATGGAAGGCTTACGGACGAATCTCTGGAATCTGTCCATCAGACG AAGACGCTGAGAAGACGTTCZ ^ ACATTGTGATTTGGACAACAGTGGCAAACTTGATGTTGA · TGAGATGACCAGGCAACATTTAGGCTTCTTCTACACATTAGATCCAACTTCTGATGGTCTT TATGGCAATTTTGTTCCCTAAGAAGCGTTCAGTTAAAAACGCTAAACATTGTTCAGTTGTA AAATTATATTCATTTTCATTTCGTAAAATTAGTATTTATAAATTTGTATCATAAATTGTAT CC ^ TGTTGTAGACTAAATAAGACTCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA - 10 3, · which generates a (SEQ ID. No: 2) The amino acid sequence of: 23200900418

MQRFTNRLLSMSALRARSRLQRTANFHTSILLATDSKYAVKLDPDFANPKWINRH

KFMFNFLDINGKGKIILDEIVSKASDDICAKLDATPEQTKRHQDAVEAFFKKMGM

DYGKEVAFPEFIKGWEELAEHDLELWSQNKSTLIREWGDAVFDIFDKDASGSISLD

EWBCAYGRISGICPSDEDAEKTFKHCDLDNSGKLDVDEMTRQHLGFFYTLDPTSDG

LYGNFVP mtClytinDecay-182F photoprotein is encoded by the following nucleotide sequence (SEQ ID NO: 3):

GACAGATAAAAAATTCACTCCTTAGATTATTTAGTGAATAAGAGAAAAAAAGGATAAGAAA

TCAAGATGCAAA.GGTTTACAAATCGTCTTCTTTCCATGTCGGCTTTACGTGCAAGATCAAG

ATTGCAACGCACGGCAAATTTTCACACCAGCATACTCTTGGCTACAGATTCAAAATACGCG

GTCAAACTCGATCCTGATTTTGCAAATCCAAAATGGATCAACAGACACAAATTTATGTTCA

ACTTTTTGGACATAAACGGTAAGGGGAAAATCACATTAGATGAAATCGTCTCCAAAGCTTC

AGACGACATTTGTGCTAAACTGGATGCAACACCAGAACAGACCAAACGTCACCAGGATGCT

GTTGAAGCCTTTTTCAAGAAAATGGGCATGGATTATGGTAAAGAAGTTGCATTCCCAGAAT

TTATTAAGGGATGGGAAGAGTTGGCCGAACACGACTTGGAACTCTGGTCTCAAAACAAAAG

TACATTGATCCGTGAATGGGGAGATGCTGTTTTCGACATTTTCGACAAAGACXiCAAGTGGC

TCAATCAGTTTAGACGAATGGAAGGCGTTTGGACGAATCTCTGGAATCTGTCCATCAGACG

AAGACGCTGAGAAGACGTTCAAACATTGTGATTTGGACAACAGTGGCAAACTTGATGTTGA

TGAGATGACCAGGCAACATTTAGGCTTCTGGTACACATTGGATCCAACTTCTGATGGTCTT

TATGGCAATTTTGTTCCCTAAGAAGCGTTCAGTTAAAAACGCTAAACATTGTTCAGTTGTA

AAATTATATTCATTTTCATTTCGTAAAATTAGTATTTATAAATTTGTATCATAAATTGTAT

CCATGTTGTAGACTAAATAAGACTCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA -3' . This generates an amino acid sequence (SEQ ID NO: 4):

MQRFmRLLSMSALRARSRLQRTANFHTSILLATDSKYAVKLDPDFANPKWINRH

KFMFNFLDINGKGKITLDEIVSKASDDICAKLDATPEQTKRHQDAVEAFFKKMGM

DYGKEVAFPEFIKGWEELAEHDLELWSQNKSTLIREWGDAVFDIFDKDASGSISLD

EWKAFGRISGICPSDEDAEKTFKHCDLDNSGKLDVDEMTRQHLGFWYTLDPTSDG

LYGNFVP mtClytin photoprotein is described in patent application 24 200900418 W02005035559 and is encoded by the following nucleotide sequence (SEQ ID NO: 5): 5'-

GACAGATAAAAAATTCACTCCTTAGATTATTTAGTGAATAAGAGAAAAAAAGG

ATAAGAAATCAAGATGCAAAGGTTTACAAATCGTCTTCTTTCCATGTCGGCTTT

ACGTGCAAGATCAAGATTGCAACGCACGGCAAATTTTCACACCAGCATACTCT

TGGCTACAGATTCAAAATACGCGGTCAAACTCGATCCTGATTTTGCAAATCCA

AAATGGATCAACAGACACAAATTTATGTTCAACTTTTTGGACATAAACC3GTAA

GGGGAAAATCACATTAGATGAAATCGTCTCCAAAGCTTCAGACGACATTTGTG

CTAAACTGGATGCAACACCAGAACAGACCAAACGTCACCAGGATGCTGTTGAA

GCCTTTTTCAAGAAAATGGGCATGGATTATGGTAAAGAAGTTGCATTCCCAGA

ATTTATTAAGGGATGGGAAGAGTTGGCCGAACACGACTTGGAACTCTGGTCTC

AAAACAAAAGTACATTGATCCGTGAATGGGGAGATGCTGTTTTCGACATTTTC

GACAAAGACGCAAGTGGCTCAATCAGTTTAGACGAATGGAAGGCTTACGGAC

GAATCTCTGGAATCTGTCCATCAGACGAAGACGCTGAGAAGACGTTCAAACAT TGTGATTTGGACAACAGTGGCAAACTTGATGTTGATGAGATGACCAGGCAACA,

TTTAGGCTTCTGGTACACATTGGATCCAACTTCTGATGGTCTTTATGGCAATTTT

GTTCCCTAAGAAGCGTTCAGTTAAAAACGCTAAACATTGTTCAGTTGTAAAATT

ATAITCATTTTCATTTCGTAAAATTAGTATTTATAAATTTGTATCATAAATTGTA

TCCATGTTGTAGACTAAATAAGACTCGGCAAAAAAAAAAAAAAAAAAAAAAA ^ AAAAAAA-3'.

This generated (SEQ ID. No: 6) in the amino acid sequence: MQRFIT ^ LSMSALRARSRLQRTANFHTSILLATDSKYAVKLDPDFANPKWINRH KF3VIFNFLDINGKGKITLDEIVSKASDDICAKLDATTEQTKRHQDAVEAFFKKMGM DYGKEVAFPEFIKGWEELAEHDLELWSQNKSTLIREWGDAVFDIFDKDASGSISLD EWKAYGRISGICPSDEDAEKTFKHCDLDNSGKLDVDEMTRQHLGFWYTLDPTSDG LYGNFVP mtClytin light putative signal peptide protein having the following sequence (SEQ ID. No: 7):

MQRFTNRLLSMSALRA 25 10 200900418 and has the following nucleic acid sequences: 5, - ATGCAAAGGTTTACAAATCGTCTTCTTTCCATGTCGGCTTTACGTGCA - 3' (SEQ ID NO: 8) The following primers were used to prepare the mtClytinDecay-141F mutant: 5 Sequence ID: 9, 5, -GACGAATGGAAGGCGTTTG<?ACGAATCTCTG- 3, Sequence ID: 10, -CAGAGATTCG, CCAAACGCCTTCCATTCGTC-3, The following primers were used to prepare the mtClytinDecay-182F mutant: Sequence number 1J Identification number 1 1 : 5, -CAACATTTAGGCTTCTTCTACACATTAGATCCAAC-3, Sequence identification number 1 2 : 5 , -GTTGGATCTAATGTGTAGAAGAAGCCTAAATGTTG-3, 10 These sequences can be found in the sequence listing. [Examples] Examples Example 1 15 Mutants were prepared by inserting mutations at positions 141 or 182 with the aid of molecular-biological methods. Stratagen (USA)'s "quick change" method is used for this purpose. The primers used are (sequence identification number: 9 + 10) and (sequence identification number: 11 + 12). The cDNA was inserted into the SfilA-SfilB cleavage site of the pTriplEx2 vector. These vectors are described as 20 pTriplEx2-mtClytinDecay-141F and pTriplEx2-mtClytinDecay-182F 〇 Figure 1 depicts the plastid map of the pTriplEx2-mtClytin Decay-141F vector. Figure 2 depicts the plastid map of the pTripLEx2-mtClytinDecay_182F vector. 26 200900418 Example 2 The vector used to prepare the constructs described below was the pcDNA3.1(+) plastid of Clontech. The derivative of this vector is referred to as pcDNA3-mtClytinDecay-141F and pcDNA3-mtClytinDecay, 5 182F°pcDNA3-mtClytinDecay-141F and pcDNA3, and the mtClytinDecay-182F vector is used to express mtClytinDecay-141F in eukaryotic cell systems. And mtClytinDecay-182F. Figure 3 depicts the plastid map of the pcDNA3-mtClytin Decay-141F vector. 1 〇 Figure 4 depicts the plastid map of the pcDNA3-mtClytin Decay-182F vector. Example 3 Bacterial Expression 15 Bacterial expression was performed in E. coli by transforming bacteria by expressing plastids with pTriplEx2-mtClytinDecay-141F or pTriplEx2-mtClytinDecay-182F. The transformed bacteria were cultured at 37 ° C for 3 hours in LB medium and the performance was induced according to the manufacturer's information. The induced bacteria were collected by centrifugation, resuspended in 5 mM Tris/HCl 2 (pH 9.0) + 5 mM EDTA and recovered by ultrasonic destruction. The lysate was then centrifuged at 13 000 rpm (16 000 rcf) for 15 minutes and the supernatant was removed. The supernatant was cultured in the dark for 3 hours with coelenterazine murine with Tris/HCl pH 9.0). Bioluminescence was immediately measured in a illuminometer after the addition of 5 mM calcium chloride. The measurement integration time is 60 seconds. Figure 6 depicts the kinetics of mtClytinDecay bioluminescence measurements in bacteria. 5 Example 4 Eukaryotes are shown in transient or stability experiments. Constitutive eukaryotic expression is expressed in CHO cells by pcDNA3-mtClytinDecay-141F, pcDNA3-mtClytinDecay-182F and pcDNA3 · 1 (+) The body is transferred to ίο and the cells are administered. For this purpose, 10,000 cells per well are divided into flats.

It was placed in DMEM-F12 medium in a 96-well microtiter plate and cultured overnight at 37 °C. Transfection was performed using the Fugene 6 kit (Roche) and based on the manufacturer's information. The transfected cells were cultured in DMEM-F12 medium at 37 ° C overnight. The medium was then removed and replaced with 15 50 μL of coelenterazine (10E-07 Μ 肠 配 配 in PBS). The cells were cultured at 28 ° C for 24 hours' and then ATP (adenosine triphosphate) was added until a final concentration of 5 μΜ. After the addition, the measurement is started directly in a illuminometer. The integration time is 1 second, with a total measurement time of 90 seconds. 20 Figure 7 depicts the results of measuring mtClytinDecay bioluminescence in CHO cells. 28 200900418 Example 5 Figure 5 depicts the arrangement of mtClytinDecay-141F, mtClytinDecay-182F and mtClytin (wild type; wt) at the amino acid level. 5 Example 6 Kinetic analysis of mtClytinDecay expressed in CHO cells To perform kinetic analysis of mtClytinDecay-141F bioluminescence, CHO (Chinese hamster ovary) cells were transfected with pCDNA3-mtClytinDecay-141F or pcDNA3 (without Incorporated CDNA). The transfection and measurement were carried out as described in Example 4. The measurement data is recorded for a period of one second for a period of 60 seconds. Figure 7 depicts the results of a kinetic analysis measuring mtClytinDecay in CHO cells. 15 Example 7 Use of mtClytinDecay in multiplex experiments mtClytinDecay-141F and mtClytin-182F photoprotein are suitable for use as a component of multiplexing readout processes, many of which report gene (eg luciferase or light) Protein) was used in a 20 experimental reaction mixture. To this end, CH0 cells expressing mtClytinDecay-141F or mtClytinDecay-182F were mixed with CH0 cells expressing wild-type mtClytin photoprotein in a 1:1 ratio (or 1:2, 1:3, ...). Cells expressing wild-type mtClytin additionally exhibit a G-protein-coupled receptor [e.g., neuromedin U receptor 2]. Fine 29 200900418 The cell mixture was applied to a 96-, 384- or 1536-well microtiter plate and cultured at 37 ° C for 24 hours. The cells were then administered coelenterazine (as described in Example 4). The addition of a 〇_protein receptor agonist results in intracellular calcium release, which can be read by wild-type mtClytin (light release through wild-type multi-tube aequogen). Subsequent addition of an agonist [the activation of a CHO-endogenous receptor (e.g., Ατρ)] activates a cell type of mtClytinDecay-141F or mtClytin-182F. 1〇 Literature/Patent Case US 6,495,355 US 5,541,309 US 5,093,240 US-0908909 15 US 6,152,358 GB-0024357 W003006497 W0200168824 W02005035559

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Fogarty KE, Rizzuto R. Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling. Biochem J. 2001 Apr l; 355 (Pt 1): 1-12 Claros, MG, Vincens, P. (1996); Computational method to ίο predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem 241, 779-786 Cullen Bryan R., Malim Michael H., Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods in Enzymology. 216:362ff 15 Fagan TF, Ohmiya Y, Blinks JR, Inouye S, Tsuji FI.

Cloning, expression and sequence analysis of cDNA for the Ca(2+)-binding photoprotein, mitrocomin, FEBS Lett. 1993 Nov l;333(3):301-5

Hastings, J.W. and Morin, J.G. (1969) Comparative 2〇 biochemistry of calcium-activated photoproteins from the ctenophore, Mnemiopsis and the coelenterates Aequorea, Obelia, and Pelagia. Biol. Bull. 137, 402.

Haddock SH, Rivers TJ, Robison BH. Can coelenterates make coelenterazine? Dietary requirement for luciferin in 31 200900418 cnidarian bioluminescence. Proc Natl Acad Sci USA 2001 Sep 25;98(20):11148-51

Inouye S, Tsuji FI. (1994) Aequorea green fluorescent protein. Expression of the gene and fluorescence 5 characteristics of the recombinant protein. FEBS Lett 1994

Mar 21;341(2-3):277-80

Inouye S., Tsuji FI. Cloning and sequence analysis of cDNA for the Ca(2+)-activated photoprotein, clytin. FEBS Lett. 1993 Jan ll;315(3);343-6. io Illarionov BA, Bondar VS, Illarionova VA, Vysotski ES.

Sequence of the cDNA encoding the Ca(2+)-activated photoprotein obelin from the hydroid polyp Obelia longissima. Gene. 1995 Feb 14;153(2):273-4.

Jones K, Hibbert F, Keenan M. Glowing jellyfish, 15 luminescence and a molecule called coelenterazine. Trends

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Johnson, FH, Shimomura, O., Saiga, Y., Gershman, LC, Reynolds, GT, and Waters, JR (1962) Quantum efficiency of Cypridina luminescence, with a note on that of Aequorea. J. 20 Cell. Comp. Physiol. 60, 85-103.

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Shimomura O., Bioluminescence in the sea: photoprotein systems. Symp Soc Exp Biol. 1985; 39:351-72 Shimomura O. and Teranishi K. (2000) Luminescence 15, 51-58. 15 Shimomura O. Isolation and properties of various Molecular forms of aequorin. Biochem J. 1986 Mar l;234(2):271-7. Shimomura, O. and Johnson, FH (1966) in: Bioluminescence in Progress (Johnson, FH and Haneda, Y., Eds.) Pp.496-521, Princeton University Press, Princeton, NJ. 2〇Shrestha S, Paeng IR, Deo SK, Daunert S. Cysteine-free mutant of aequorin as a photolabel in immunoassay development. Bioconjug Chem. 2002 Mar-Apr; 13 (2): 269-75 Snowdowne KW, Borle AB. Measurement of cytosolic free calcium in mammalian cells with aequorin. Am J Physiol. 33 200900418 1984 Nov;247(5 Pt l): C396-408.

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Yang Te-Tuan, Sinai Parisa, Kitts Paul A. Kain Seven R.,

Quantification of gene expression with a secreted alkaline phosphatase reporter system. Biotechnique. 1997 23(6)1 llOff 15 [Simple description of the diagram]. Figure 1: Figure 1 depicts the plastid map of the pTriplEx2-mtClytinDecay-141F vector. Figure 2: Figure 2 depicts the plastid map of the 20-body of pTripffix2-mtClytinDecay-182F. Figure 3: Figure 3 depicts the plastid map of the pcDNA3-mtClytinDecay-141F vector. Figure 4: Figure 4 depicts the plastid map of the pcDNA3-mtClytinDecay-182F vector. 34 200900418 Figure 5: Figure 5 depicts the arrangement of photoprotein, mtClytin'mtClytinDecay-MIF and mtaytinDecay_182F at the amino acid level. Figure 6: Figure 6 depicts the bioluminescent reaction of mtClytinDecay after bacterial performance. Χ_axis: in seconds; γ axis: relative light units. 5 Figure 7: Figure 7 depicts the bioluminescent response of mtClytinDecay after eukaryotic cell expression. X-axis: time in seconds; gamma axis: relative light unit; information in the box corresponds to the final concentration of ATP (adenosine triphosphate) in the experimental reaction mixture. 10 [Key component symbol description] None 35 200900418 Sequence table

<X10> Bayer Healthcare AG <120> Isolated mtClytinDecay photoprotein and use thereof, <130> BHC 07 1 034 5 <160> 12 <170> Patent In version 3-3 <210> 1 <;211> 912

<212> DNA 10 <213> Artificial sequence <220><223> mtClytin mutant <400> 1 gacagataaa aaattcactc cttagattat ttagtgaata agagaaaaaa aggataagaa 60 15 atcaagatgc aaag^tttac aaatcgtctt ctttccatgt cggctttacg tgcaagatca 120 agattgcaac gcacggcaaa ttttcacacc agcatactct tggctacaga ttcaaaatac 180 gcggtcaaac tcgatcctga ttttgcaaat ccaaaatgga tcaacagaca caaatttatg 240 ttcaactttt tggacataaa cggtaagggg aaaatcacat tagatgaaat cgtctccaaa 300 gcttcagacg acatttgtgc taaactggat gcaacaccag aacagaccaa acgtcaccag 360 20 gatgctgttg aagccttttt caagaaaatg ggcatggatt atggtaaaga agttgcattc 420 ccagaattta ttaagggatg ggaagagttg gccgaacacg acttggaact ctggtctcaa 480 aacaaaagta cattgatccg tgaatgggga gatgctgttt tcgacatttt cgacaaagac 540 gcaagtggct caatcagttt agacgaatgg aaggcttacg Gacgaatctc tggaatctgt 600 ccatcagacg aagacgctga gaagacgttc aaacattgtg atttggacaa cagtggcaaa 660 720 25 cttgatgttg atgagatgac caggcaacat ttaggcttct tctacacatt agatccaact 1 200900418 5 10 tctgatggtc tttatggcaa ttttgttcc c taagaagcgt tcagttaaaa acgctaaaca ttgttcagtt gtaaaattat attcattttc atttcgtaaa attagtattt ataaatttgt atcataaatt gtatccatgt tgtagactaa ataagactcg gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa < 210 > 2 < 211 > 228 < 212 > PRT < 213 > artificial sequence < 220 > < 223 > mtClytin mutation Body <400> 2 Met Gin Arg Phe Thr Asn Arg Leu Leu Ser Met Ser Ala Leu Arg Ala 7$0 840 900 912 10 15

Arg Ser Arg Leu Gin Arg Thr Ala Asn Phe His Thr Ser lie Leu Leu 15 20 25 30

Ala Thr Asp Ser Lys Tyr Ala Val Lys Leu Asp Pro Asp Phe Ala Asn 35 40 45

Pro Lys Trp He Asn Arg His Lys Phe Met Phe Asn Phe Leu Asp lie 50 55 60 20 Asn Gly Lys Gly Lys Xle Thr Leu Asp Glu lie Val Ser Lys Ala Ser 65 70 75 80

Asp Asp lie Cys Ala Lys Leu Asp Ala Thr Pro Glu Gin Thr Lys Arg 85 90 95

His Gin Asp kla. Val Glu Ala Phe Phe Lys Lys Met Gly Met Asp Tyr 25 100 105 110 2 200900418

Gly Lys Glu Val Ala Phe Pro Glu Phe lie Lys Gly Trp Glu Glu Leu 115 120 125

Ala Glu His Asp Leu Glu Leu Trp Ser Gin Asn Lys Ser Thr Leu lie 130 135 140

Arg Glu Trp Gly Asp Ala Val Phe Asp lie Phe Asp Lys Asp Ala Ser X45 150 155 160

Gly Ser lie Ser Leu Asp Glu Trp Lys Ala Tyr Gly Arg l Ser Gly 165 170 175

He Cys Pro Ser Asp Glu Asp Ala Glu Lys Thr Phe Lys His Cys Asp 10 180 185 190

Leu Asp Asn Ser Gly Lys Leu Asp Val Asp Glu Met Thr Arg 6Xn His 195 200 205

Leu Gly Phe Phe Tyr Thr Leu Asp Pro Thr Ser Asp Gly Leu Tyr Gly 210 215 220 15 Asn Phe Val Pro 225 <210> 3 <211> 914 20 25 <212> ϋΝΛ <213> Artificial sequence <; 22Q > < 223 > mtClytin mutation Zhao < 400 > 3 60 120 gacagataaa aaattcactc cttagattat ttagtgaata agagaaaaaa aggataagaa atcaagatgc aaaggtttac aaatcgtctt ctttccatgt cggctttacg tgcaagatca 3 200900418 agattgcaac gcacggcaaa ttttcacacc agcatactct tggctacaga ttcaaaatac 180 gcggtcaaac tcgatcctga ttttgcaaat ccaaaatgga tcaacagaca caaatttatg 240 ttcaactttt tggacataaa cggtaagggg aaaatcacat tagatgaaat cgtctccaaa 300 gcttcagacg acatttgtgc taaactggat gcaacaccag aacagaccaa acgtcaccag 5 gatgctgttg aagccttttt caagaaaatg ggcatggatt atggtaaaga agttgcattc 360 420 ccagaattta ttaagggatg ggaagagttg gccgaacacg acttggaact ctggtctcaa 480 aacaaaagta cattgatccg tgaatgggga gatgctgttt tcgacatttt cgacaaagac 540 gcaagtggct caatcagttt agacgaatgg aaggcgtttg gacgaatctc tggaatctgt 600 ccatcagacg aagacgctga gaagacgttc aaacattgtg attt ggacaa cagtggcaaa 10 cttgatgttg atgagatgac caggcaacat ttaggcttct ggtacacatt ggatccaact 660 720 tctgatggtc tttatggcaa ttttgttccc taagaagcgt tcagttaaaa acgctaaaca 780 ttgttcagtt gtaaaattat attcattttc atttcgtaaa attagtattt ataaatttgt 840 atcataaatt gtatccatgt tgtagactaa ataagactcg gcaaaaaaaa aaaaaaaaaa 900 9X4 aaaaaaaaaa aaaa 15 < 210 > 4 < 211 > 228 < 212 > PRT <213> Artificial sequence <220> 20 <223> mtClytin mutant <400> 4

Met Gin Arg Phe Thr Asix Arg Leu Leu Ser Met Ser Ala Leu Arg Ala X5 10

Arg Ser Arg Leu Gin Arg Thr Ala Asn Phe His Thr Ser lie Leu Leu 25 20 25 30 4 200900418

Ala Thr Asp Ser Lys Tyr Ala Val Lys Leu Asp Pro Asp Phe Ala Asn. 35 40 45

Pro Lys Trp He Asn Arg His Lys Phe Met Phe Asn Phe Leu Asp He 50 55 60 5 Asn Gly Lys Gly Lys lie Thr Leu Asp Glu lie Val Ser Lys Ala Ser 65 70 75 80

Asp Asp lie Cys Ala Lys Leu Asp Ala Thr Pro Glu Gin Thr Lys Arg 85 90 95

His Gin Asp Ala Val Glu Ala Phe Phe Lys Lys Met Gly Met: Asp Tyr 10 100 105 110

Gly Lys Glu Val Ala Pile Pro Glu Phe lie Lys Gly Trp Glu GXu Leu 115 120 125

Ala Glu His Asp Leu Glu Leu Trp Ser Gin Asn Lys Ser Thr Leu He 130 135 140 15 Arg Glu Trp Gly Asp Ala Val Phe Asp lie Phe Asp Lys Asp Ala Ser 145 150 155 160

Gly Ser lie Ser Leu Asp Glu Trp Lys Ala Phe Gly Arg lie Ser Gly 165 170 175 lie Cys Pro Ser Asp Glu Asp Ala Glu Lys Thr Phe Lys His Cys Asp 20 180 185 190

Leu Asp Asn Ser Gly Lys Leu Asp Val Asp Glu Met Thr Arg Gin His 195 200 205

Leu Gly Phe Trp Tyr Thr Leu Asp Pro Thr Ser Asp Gly Leu Tyr Gly 210 215 220 25

Asn Phe Val Pro 5 200900418 225 <210> 5 <211> 912

≪ 212 > DNA 5 < 213 > artificial sequence < 400 > 5 gacagataaa aaattcactc cttagattat ttagtgaata agagaaaaaa aggataagaa 60 atcaagatgc aaaggtttac aaatcgtctt ctttccatgt cggctttacg tgcaagatca 120 agattgcaac gcacggcaaa ttttcacacc agcatactct tggctacaga ttcaaaatac 180 10 gcggtcaaac tcgatcctga ttttgcaaat ccaaaatgga tcaacagaca caaatttatg 240 ttcaactttt tggacataaa cggtaagggg aaaatcacat tagatgaaat cgtctccaaa 300 gcttcagacg acatttgtgc taaactggat gcaacaccag aacagaccaa acgtcaccag 360 gatgctgttg aagccttttt caagaaaatg ggcatggatt atggtaaaga agttgcattc 420 ccagaattta ttaagggatg ggaagagttg gccgaacacg acttggaact ctggtctcaa 480 15 aacaaaagta cattgatccg tgaatgggga gatgctgttt tcgacatttt cgacaaagac 540 gcaagtggct caatcagttt agacgaatgg aaggcttacg gacgaatctc tggaatctgt 600 ccatcagacg aagacgctga gaagacgttc aaacattgtg atttggacaa cagtggcaaa 660 cttgatgttg atgagatgac caggcaacat Ttaggcttct ggtacacatt ggatccaact 720 tctgatggtc tttatggcaa ttttgttccc taagaagcgt tcagttaaaa acgctaaaca 780 20 ttgttcagtt gtaaaattat Attcattttc atttcgtaaa attagtattt ataaatttgt 840 atcataaatt gtatccatgt tgtagactaa ataagactcg gcaaaaaaaa aaaaaaaaaa 900 aaaaaaaaaa aa 912 <210> €

<211> 228 25 <212> PRT 6 200900418 <213> Artificial sequence <400> 6

Met Gin Arg Phe Thr Asn Arg Leu Leu Ser Met Ser Ala Leu Arg Ala 1 5 10 15 5 Arg Ser Arg Leu Gin Arg Thr Ala Asn Phe His Thr Ser He Leu Leu 20 25 30

Ala Thr Asp Ser Lys Tyr Ala Val Lys Leu Asp Pro Asp Phe Ala Asn 35 40 45

Pro Lys Trp lie Asn Arg His Lys Phe Met Phe Asn Phe Leu Asp He 10 50 55 60

Asn Gly Lys Gly Lys lie Thr Leu Asp Glu He Val Ser Lys Ala Ser 65 70 75 80

Asp Asp lie Cys Ala Lys Leu Asp Ala Thx Pro Glu Gin Thr Lys Arg 85 90 95 15 His Gin Asp Ala Val Glu Ala Phe Phe Lys Lys Met Gly Met Asp Tyr 100 105 110

Gly Lys Glu Val Ala Phe Pro Glu Phe He Lys Gly Trp Glu Glu Leu 115 120 125

Ala Glu His Asp Leu Glu Leu Trp Ser Gin Asn Iiys Ser Thr Leu lie 20 130 135 140

Arg Glu Trp Gly Asp Ala Val Phe Asp lie Phe Asp Lys Asp Ala Ser 145 150 155 X60

Gly Ser lie Ser Leu Asp Glu Trp Lys Ala Tyr Gly Arg lie Ser Gly 165 170 175 25

He Cys Pro Ser Asp Glu Asp Ala Glu Lys Thr Phe Lys His Cys Asp 200900418 180 185 190

Leu Asp Asn Ser Gly Lys Leu Asp Val Asp Glu Met Thr Arg Gin His 195 200 205

Leu Gly Phe Trp Tyr Thr Leu Asp Pro Thr Ser Asp Gly Leu Tyr Gly 5 210 215 220

Asn Phe Val Pro 225 <210> 7 <211> 16 10 <212> PRT <213> Artificial sequence <400>

Met Gin Arg Phe Thr Asn Arg Leu Leu Ser Met Ser Ala Leu Arg Ala 1 5 10 15 15 <2io> 8 <211> 48 <212> DNA <213> Artificial sequence <400> 8 20 atgcaaaggt Ttacaaatcg tcttctttcc atgtcggctt tacgtgca <210> 9 <211> 31 <212> DNA <213> Artificial sequence 25 <220> 200900418 <223> Introduction <400> 9 gacgaatgga aggcgtttgg acgaatctct g <210> 10 5 <211> 31 <212> DNA <213><220> Artificial sequence <223> Introduction 10 <400> 10 cagagattcg tocaaacgcc ttccattcgt c <210> 11 <21X> 35 <212> DNA 15 <213><220> Artificial sequence <223> primer <400> 11 caacatttag gcttcttcta cacattagat ccaac 20 <2io> 12 <211> 35 <212> DNA <213><;220> Artificial sequence 25 <223> Intro 200900418 <400> 12 35 gttggatcta atgtgtagaa gaagcctaaa tgttg 10

Claims (1)

2009〇〇4lg 10 15 20, the scope of patent application: a nucleic acid molecule or a functional fragment thereof, selected from the group consisting of: a nucleic acid molecule encoding a polypeptide comprising a sequence identification number: 2 or Sequence identification number: amino acid column disclosed in 4; nuclear I molecule, including the sequence described in sequence identification number: 丨 or SEQ ID NO: 3; $ fragment containing a nucleic acid molecule according to a) or b) The nucleic acid is divided into a functional photoprotein of the nucleic acid molecule and the polypeptide (4) or a polypeptide of the amino acid mainly composed by sequence recognition. ~·4 is the main amino acid 182-polypeptide or a functional fragment thereof, and the patent is included in the patent. The species contains the sequence identification number: 2, the characteristic of photoprotein. The polypeptide of the sequence described. Or a sequence photo number: 4 in a photoprotein or a functional fragment thereof: 2 is the predominant position 131 to 152 which in turn encodes a modified bioluminescence time course. ^ There is a dog change, and it has a photoprotein or a functional number: 4 is the main position! 72 to ^%, in the sequence identification, a modified bioluminescence time course. a mutated, and having a nucleic acid molecule, including according to the scope of the patent application 4 b) c) 2. 3. 4. 5. 36 6. 15 200900418 7. 8. 9. 10. 11. 12. 13. 14. 15. The sequence of the 16. or 5 protein. Such as the application of a patent range of a functional promoter 5 /. , a nucleic acid that contains a numerator sequence, a double = DNA or RNA vector, comprising a nucleic acid according to item 7 of the & patent patent. Another method is to purify and isolate the photoprotein polypeptide. 10 items were expressed: species, according to the scope of the patent application, or the use of the reporter gene. As 禚δ-hexyl: species according to the scope of the patent application, or 6 items - the use of its reporter gene (the method of protein 'is characterized by - or multiple mutations in the sequence identification number: 2 In the area defined by =52, the bioluminescence time history is determined according to the method of claim 14 of the patent application scope = 艮 = the photoprotein of the second, third, fourth, fifth or fifteenth patent range is private or Use of the report. - Use of the light egg according to the scope of patent application No. 2, 3, 4, 5 or 15. 17. 2009 200900418 White as a marker or reporter for the use of other reporter genes. 18. mtClytin photoprotein A variant having a modified bioluminescence time course.