JP2002529085A - Forasin - Google Patents

Abstract

  (57) [Summary] The bioluminescent oxidation indicator is responsive to oxygen or one of its metabolites, producing light or radiation with altered properties when there is a change in the amount of oxygen or its metabolites present. The indicator, when modified by a covalent bond, when bound to another protein, when interacting or binding to a certain substance, or when acting as a reporter gene by binding to a specific promoter or enhancer gene, Modifications can be made to alter the light emission or emission characteristics.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to proteins capable of bioluminescence, cDNAs encoding said proteins, and their use, particularly in diagnostics and therapeutics. More specifically, a cDNA encoding forasin from the bivalve mollusc Phoras dactylus
Cloning and sequencing.

[0002] The term "bioluminescence" refers to a chemical reaction in a living organism or the organism.
Means the emission of light produced by a chemical reaction caused by this
Essential elements for chemical reactions are organic molecules, usually containing luciferin, oxygen and
Is one of its metabolites and an enzyme that catalyzes the oxidation of luciferin,
Luciferase. The chemiluminescent reactions that cause bioluminescence are listed below.
Can be Luciferin + O_TwoOr O_Two ^-Or H_TwoO_TwoOr OH or OCl^-Or OX ^- Or¹O_Two(+ Luciferase) → oxyluciferin + light

[0003] Up to three other substances may also be needed to produce light, or light of the required color and intensity. These substances are: (A) cations such as H ⁺ , Ca ²⁺ , Mg ²⁺ or transition metal cations (eg Cu ⁺ / Cu ²⁺ , Fe ²⁺ / Fe ³⁺ , La ³⁺ , and V ³⁺ ); (B) cofactors such as NAD (P) H, FMN, or ATP, and / or (c) fluorophores as energy transfer receptors.

With respect to luciferin, the following five chemical families are known. (A) aldehydes (found in freshwater mussels Latia, earthworms and in FMN in bacteria); (b) imidazolopyrazine. this is,
It is the most commonly occurring compound that causes bioluminescence in the sea (Hymenoptera, Cnidaria, Cymbidium, Annelida, Ciliate, some arthropods, In some molluscs, and some chordate phylums). (C) benzothiazole (found on beetles such as fireflies and glowworms). (D) Linear tetrapyrrole (seen in dinoflagellates, krill (euphausid shrim), and some fish)
. (E) Flavin (found in bacteria, fungi, trichomes, and some molluscs).

[0005] The chemiluminescence involved in these luciferins can produce glows or flashes with emission of violet, blue, blue-green, green, yellow, orange, or red light, and in some cases UV or IR light. This light emission can be linearly or circularly polarized. Luciferin or its products can also be detected and quantified by fluorescence or phosphorescence. There is no need to expose to UV light, visible light, or IR light because chemical reactions are directly responsible for light emission. However, some bioluminescent systems, such as the deep-sea fish Mal
The red organ of acosteus exhibits photochemiluminescence in which light can trigger or promote a chemiluminescent reaction. [Chemiluminescen
ce: Principles and Applications in Bi
ology and Medicine, AK Campbell (1988)
See Horwood / VCH Chichester, Weinheim. ]

[0006] In some bioluminescent proteins, luciferin is very strongly, ie, covalently bound, to the protein molecule and does not diffuse into the surrounding fluid as a result of the chemiluminescent reaction. In this case, the protein-luciferin complex is called a photoprotein, and the protein itself is called an apophotoprotein. Some bioluminescent proteins are proteins whose light emission or emission depends on or can vary from oxygen or one of its metabolites. These bioluminescent proteins are hereinafter referred to as “bioluminescent oxidation indicator protein” (BOIP). BOIP can be, for example, a photoprotein or a luciferin-luciferase system.

[0007] Thus, BOIPs are composed of individual cells, defined compartments of living cells such as the nucleus,
Whole organs and organisms (animals and plants, including microorganisms such as viruses and bacteria and protozoa), and substances of biological interest such as substrates, metabolites, vitamins, drugs, intracellular and extracellular signals, enzymes , Antigens, antibodies, and nucleic acids, can be used to detect and quantify oxygen or one of its metabolites. Heretofore, it was only known to use natural BOIP extracellularly.

Accordingly, the present invention is directed to a method of detecting and / or measuring oxygen or one of its metabolites in living cells (intracellularly), comprising a BOIP, such as a natural or chemical (or genetic) BOIP modified to
Or providing such a BOIP-based 'rainbow protein' into a cell, followed by light emission from the cell and / or a change in color, intensity, and / or polarization of emitted light from the cell. A method comprising detecting and / or quantifying

Further, according to the findings herein, recombinant BOIP can also be used in the method by sequencing the BOIP and identifying the cDNA encoding it, or Genetically modified recombinant B
OIP or 'rainbow protein' based on the BOIP can also be used in the above method. For example, the bivalve mollusc Phoras dactylu
s has a natural photoprotein, wherein the protein and luciferase are secreted together by the mollusc and O ₂ or one of its metabolites is present,
The photoprotein interacts with luciferase to generate light. Michel
Son's Methods in Enzymology LVII 385
-406 (1978), a description of the purification and characterization of luciferin and luciferase from Pholas Dactylus. A description of the detection of neural network activation by detection of superoxide anions can be found in Roberts Anal Biochem 160 139-148 (1987) and Mu.
J. Biol Chemilum 3 105-113 (1
989). The natural photoprotein (called forasin) consists of glycosylated apoprotein (34 kDa), to which the small organic molecule luciferin is tightly bound. This luciferin (structure unknown; Muller and Cam
pbell, J Biolum Chemilum 5 25-30 (199
0)) can be extracted from the protein component (apoforacin) or the organism by standard treatments, for example by treatment with mild acid. The forasin can be obtained from living mollusks found in anchored rock at low tide, along the southern coast of England from Plymouth to Folkstone, along the French Channel, and in the Mediterranean. Further details can be found in the Marine Animal Journal and the references cited therein.

[0010] According to a surprising finding here, forasin is an oxygen metabolite such as O ₂ ⁻ , H ₂ O ₂ , OCl ⁻ or other oxyhalide anions, or organic peroxides, even in the absence of the corresponding luciferase. Light can be generated by the addition of oxides and certain organic solvents such as dimethyl sulfide (DMSO) and dimethylformamide (DMF).

In the present invention, a cDNA encoding a (non-glycosylated) apoprotein of foracin (also referred to as 'apofolacin') has been identified. Accordingly, the present invention further provides: (a) apophotoprotein of follasin (or 'apoforacin'); (b) substantially homologous to sequence (a) or hybrid to sequence (a) under stringent conditions. (C) being substantially homologous to sequence (a) or (b), or forming a hybrid with sequence (a) or (b) under stringent conditions without degeneracy of the genetic code Array,
Or (d) an isolated, purified, or recombinant nucleic acid sequence comprising an oligonucleotide unique to any of the sequences (a), (b), or (c).

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The present invention provides a recombinant DNA encoding the apophotoprotein apophorasin and having the nucleotide sequence of the sequence shown in FIG. 4B. Three different cDNAs encoding apophorasin with different non-coding regions were isolated. These are shown in FIG. Genomic DNA without introns (gDNA) (FIG. 6) was shown to have the same basic sequence as cDNA.

[0014] Forasin is a glycoprotein having glusamine at 11.1, fructose at 9.8, mannose at 7.1, and galactose residues at 5.2. The cDNA for apophorasin is 34, for forasin extracted from Pholas.
It has a molecular weight of 23,456 for a molecular weight of 600. This difference in molecular weight between native and recombinant apophorasin is due to glycosylation of luciferin with the native protein. The isoelectric point of the translated protein calculated by the ISOELECTRIC command of the GCG program is 3.84. The native protein has a small isoelectric point (<3.5), which is believed to be due to the presence of bound sulfate.

The three clones isolated from the library (FIG. 2) encode a unique protein (FIGS. 4 and 5), which does not have the same amino acid sequence as any known protein in the SwissProt database. Thus, the present invention not only provides cDNAs and RNAs encoding this protein, but also provides the recombinant protein itself, ie, with or without glycosylation units. Comparison of the segments of the forasin protein sequence with the proteins in the SwissProt database identified several proteins with regions of great sequence similarity to the region of the cloned protein. These include several proteins that interact with nucleotides (Table 1).

[Table 1]

[0016] Similarities are seen between Vargula luciferase and Renilla LBP, but not between other bioluminescent proteins.

[0017] The cloned protein and (a) Vargula luciferase, (b) Renil
la Sequence homology with LBP. Regions of great homology in these three proteins are shown in bold.

(Equation 1)

[0018] The three possible glycosylation sites on the protein are the common triplet sequence Asn-
Xaa-Ser / Thr (Xaa can be any residue other than proline). Thr 216 was identified as a possible site of O-linked glycosylation by a neural network trained to identify O-linked glycosylation. This amino acid sequence was also incorporated into a neural network trained to identify eukaryotic signal peptides. This confirmed that the most likely cleavage site was between positions 20 and 21 (GSG-EE).

Many families of proteins have a "characteristic" sequence of amino acids. The sequence of the clone of the present invention does not include any of these characteristic sequences present in the PROSITE database. The amino acids 170 to 185 have a calcium binding consensus sequence [DENQS
T] X [DENQST] X [DENQST] X [DENQST] X [DENQS
T] XX [DENQST]. Thirteen possible phosphorylation sites were discovered,
These sites may be a kinase phosphorylation site [RK] (2) -x- [ST], a protein kinase C phosphorylation site [ST] -x [RK], or a casein kinase II phosphorylation site [ST] -x ( 2) matches a consensus sequence for any of-[DE].

[0020] Three N-linked glycosylation sites were identified in the translated sequence of the clone. The neural network was trained to identify this type of glycosylation, which confirmed that Thr216 was a possible site for O-linked glycosylation.
To account for the presence of sugar residues, at least one of these sites must be glycosylated in the native protein. A putative signal peptide region precedes the N-terminus of the secreted protein (determined by amino acid sequence and identified as a signal peptide by the neural network). To confirm this result,
Protein sequence for PSO for motif positioning cloned protein in cellular compartment
Checked by RT. This protein sequence did not contain a transmembrane region or N-myristoylation pattern, indicating the presence of a lipid anchor. In the nucleus, mitochondria, endoplasmic reticulum, or peroxisome, target sequences or retentio
n) No sequence was found.

[0021] The fact that the clones have some sequence similarity to proteins that interact with nucleotides may suggest that forasin binds to cofactors as part of the chemiluminescent reaction. Beetle luciferase requires ATP binding for chemiluminescence. The amino acid sequence of these clones contains a P-loop binding motif ((A, G) × 4GK (S, T) or (A) × {4} GK (
T)) does not exist. However, not all ATP binding proteins contain this motif. Also, this cloned protein does not have the GXGXXG phosphate binding consensus sequence necessary for the binding of other cofactors such as nicotinamide adenine dinucleotide.

The amino acid or sugar component of forasin cannot emit light at the wavelength (490 nm) of the natural protein. This indicates that there must be a chromophore bound to this protein. However, there are proteins in which the chromophore consists of modified amino acid residues in the polypeptide. These are best characterized by the green photoprotein (GFP). This protein has a chromophore that is a ring formed by the autocatalytic cyclization of the residues Ser-dehydroTyr-Gly. Serine can be converted to threonine, which increases the intensity of the 488 nm radiation. Forasin does not have a similar amino acid sequence. Putative luciferin binding regions were identified for the two bioluminescent chemicals. Aequorin is a putative coelenterazine
terazine) binding region, which region is Vargula hiligen
Dorfii luciferase is also present in two parts. The sequence of the cloned protein has no homology to the putative luciferin binding site of aequorin, but it
The region at residues 353-411 of ula luciferase has some similarity, similar to LBP of Renilla reniformis, which also binds imidazolopyrazine. This is thought to indicate that the chemical process of forasin bioluminescence is related to imidazolopyrazine luciferin. However, the region of homology is very small. Beetle luciferase contains a small region of sequence homology that can bind to benzothiazole luciferin. This small homology results in a luciferin-like (2- (4-benzoylphenyl) thiazole-4) that photoinactivates the luciferase active site of the firefly Photinus pyralis.
It is believed that the different colors of beetle bioluminescence when using carboxylic acids can be explained. This photoinactivation is achieved by the small peptide sequence HHGF (residues 244-25).
7) directly linked to the decomposition. Therefore, this is presumed to be a luciferin binding site. The cloned protein has no sequence homology to these putative binding regions. Two strongly conserved amino acid regions are also found in the dinoflagellate Gonyaulax
It was found in both polyedra luciferase and luciferin binding protein. These regions were compared to those of the cloned protein, but showed no sequence similarity. No sequence identity could be confirmed between the bacterial luciferase and the cloned protein.

Thus, the present invention provides a cloned apophotoprotein apophorasin that has the same properties as natural (but non-glycosylated) apophorasin in terms of molecular weight, amino acid composition, potential glycosylation, large acidic pI, and intracellular arrangement. (And the cDNA encoding it) are provided. Thus, furthermore, the present invention can provide corresponding BOIPs or modified BOIPs by standard methods.

The corresponding BOIP can be produced by associating the apophotoprotein forasin with luciferin, again using standard methods. Luciferin is tightly bound in native forasin BOIP, but according to our findings, it may not be in the case of recombinant forasin BOIP. In fact, luciferin may be weakly bound to the apoprotein and may simply be present with the apoprotein. For example, 'luciferin'
Pholas dactylus (whole organism or excised light organ) containing
Methanol, water, acids or other extracts, or pure luciferin can be added to the solution, cells, or organs (FIG. 10 shows the time course of hypochlorite-induced luminescence under these circumstances). Shown). The time course of reactivation of apophorasin was determined by using partially purified recombinant forasin secreted from insect cells by Pholas d.
acid (.) acid: 0 (solid line), 1 (-)
-), 2 (---), 6 (--), or 24 hours (-), and cultured without extract (o). Protein or acid: buffer-only control sample without methanol extract () and extract-only control sample

[Outside 1] Was also processed under the same conditions. Chemiluminescence was triggered at 10 seconds (arrow) by adding 2% sodium hypochlorite. The background light is subtracted from the chemiluminescence count number. Representative curves from the hypochlorous acid trigger of native forasin are also shown (x). A representative experiment repeated twice is shown.

Luciferin binds to or remains loosely bound to apoBOIP, which forms the photoprotein, and acts like luciferase. Luciferin on the photoprotein reacts with oxygen or one of its metabolites in the presence or absence of luciferase to generate light. This light emission can be detected, quantified, or imaged by a luminometer, photographic film, or imaging camera, or the naked eye. Alternatively, light emission can be generated spontaneously by intracellular or extracellular metabolites that react with apoBOIP.

Although follasin is described, the following is applicable to all BOIPs. That is, BOIP can be produced directly from natural DNA or DNA generated or amplified by the polymerase chain reaction. By dividing the DNA into two or more parts by the polymerase chain reaction, or
By adding an NA sequence to the 5 'or 3' end, the site can be inserted into the protein. For example, DNA can be expressed in bacterial, yeast, insect or human cells, or other suitable organisms that produce proteins that can be extracted and used.

In this case, the protein produced from the cloned DNA contains oxygen or a metabolite thereof, for example, a superoxide anion (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), a hydroxyl group ( OH), oxyhalide anions (OCl ⁻ , OBr ⁻ , OI ⁻ , OS)
CN ^-), nitric oxide (NO), organic peroxides, or react with the radical ROO. Changes in light emission can detect and quantify oxygen or metabolites within living cells, organelles, or on the outer or inner surface of the plasma membrane, or within organs of living organisms, At this time, there is no need to cut them out or separate the connecting part or free part. Also, enzymes that produce oxygen or its metabolites, such as chlorophyll, or oxygen or its metabolites, in living cells, organs, and whole organisms, or extracts from any of them, due to changes in light emission. Enzymes, such as oxidases and oxygenases, that react directly to add oxygen to a substrate can be detected and quantified.

BOIP may also be a cell lysate such as rabbit reticulocyte lysate or RN.
It can be made in vitro by transcription / translation in a wheat germ extract containing A polymerase. DNAs for BOIP include RNA polymerase promoters such as T7, SP6, bacterial promoters such as lac, ara, or trp, mammalian promoters such as actin, myosin, myelin protein, TK, MRT-V, SV40, CMV, RSV, metallothionein, antibodies ,
G6P dehydrogenase, which can then be amplified in vitro by the polymerase chain reaction. The poly A tail is replaced by BOIP or modified BOI
P-encoding DNA can be added to the 3 'end, and tissue-specific promoter or enhancer sequences can be added to the 5' or 3 'end, and BOIP can be added to target cells such as cardiac muscle or cancer cells. Can be specifically expressed. BOIP expression in target cells is detected and quantified by light intensity, color, or polarization, as described above.

[0029] BOIP or its DNA or RNA is delivered to living bacteria or eukaryotic cells by phage, virus, plasmid, calcium phosphate transfection, electroporation, liposome fusion, pore-forming protein, microinjection, or DNA gun. Can be captured. When cell activation or damage is initiated inside the cell or under a suitable extracellular environment, the light emission from the BOIP changes. A cell capable of producing its own light upon expression in a living organism, eg, by microinjection of a protein, RNA, or DNA, or by a transformation procedure, eg, a bacterial, microbial, animal, or plant cell (Eg, leukocytes, heart cells, or yeast, protozoa, Drosophila (Droso
phila, nematodes, polychaetes, fish, humans, mice, rats, sheep, pigs, horses, or plants) can be produced.

[0030] BOIP can be obtained by chemical means or by including a signal peptide in the BOI.
By making P genetically, it can be incorporated into a specific part of a living cell. The signal peptide can bind BOIP to the inner or outer surface of the plasma membrane, or to a specific organelle (eg, peroxisome, mitochondria, chloroplast, tonoplast, endoplasmic reticulum, Golgi apparatus, endosome, lysosome, secretory vesicle, nucleus, nucleoside). , Nuclear membrane, plasma membrane, proteosome, or gap junction, or within a tissue (eg, membrane receptor, ion channel, microtubule, cytoskeleton, nuclear skeleton, nuclear receptor, mitotic spindle, or microfibril) I do. A chemically or genetically added signal peptide usually directs normal or modified BOIP to a specific intracellular or extracellular location. For example, the N-terminal sequence MLSRLSLRLLL
Some of SRYLL or cytochrome oxidase direct BOIP to mitochondria, and N-terminal KKSALLALMYVCPGKADKE or MLLPV
PLLLLGLGLAA directs BOIP into the endoplasmic reticulum, and the C-terminal KDEL or HDEL or KEEL sequence retains BOIP there. SKL at C-terminal is B
The OIP is guided to the peroxisome and is used for PKKKRKV or this SV40 lar.
Extension of the geT antigen signal directs BOIP to the nucleus. Also, palmitoylation and / or myristoylation signals direct BOIP to the plasma membrane. B
By binding OIP to another protein that directs itself to a specific site,
IP can also be directed to the site. For example, by binding the nuclear protein nucleoplasmin or lamin B receptor to BOIP, the BOIP can be guided to the nucleus. N-terminal cytochrome oxidase directs BOIP to mitochondria. N-terminal chlorophyll directs BOIP to chloroplasts. N-terminal connexin is BOI
Leads P to the gap junction or plasma membrane. SNAP25 leads to the plasma membrane.

Other modifications of apoproteins, BOIPs, or nucleotides encoding them include, but are not limited to:

Apoproteins such as apophorasin can be glycosylated and used for detection and quantification of protein secretion and translocation through the secretory pathway.

The nucleic acid encoding BOIP when expressed inside a living cell can not only be modified, but also regulated in said cell by gene expression, for example by a promoter, enhancer or oncogene. For example, an apoprotein, such as apophorasin, can bind to a gene regulatory protein, such as a transcription factor, by genetic or chemical manipulation, and thus detect and quantify the movement of the regulatory protein or its activity in a cell. .

[0034] The BOIP or apoprotein or its DNA can bind to other proteins or DNA used in therapy, and thus
Can be detected in living cells or whole organisms, such as humans.

Apoproteins, such as apophorasin, can also be covalently modified by enzymes, for example, phosphorylation (ser / thr, his, and tyr)
Kinases and phosphatases), transglutamination (tra
to include one or more sites that can be modified by proteolysis, ADP ribosylation, glycosylation, glucosylation, halogenation, oxidation, methylation, palmitoylation, myristylation, and farnesylation. Alternatively, it can be genetically or chemically reworked.

Apoproteins such as apophorasin are antigen or intracellular signal binding sites,
For example, Ca ²⁺ , cyclic AMP, cyclic GMP, cyclic CMP, IP ₃ , IP ₄ , diacylglycerol, ATP, ADP, AMP, GTP, or any oxy or deoxyribonucleoside or nucleotide, substrate, drug, nucleus and / or gene Regulatory proteins can be genetically or chemically engineered to contain.

[0037] Also, BOIP may be located within the BOIP at the N or C terminus, or between the BOIP chimera and the energy transfer receptor, such as at a specific site such as GFP (wild-type or any mutant GFP). Can be converted into rainbow protein by building in. This is known as chemiluminescence, bioluminescence, or fluorescence resonance transition (CRET, BRET, FRET, respectively). Conversion of BOIP to a 'rainbow protein' is achieved by reaction with a cell substrate, genetic or chemical modification, or by coupling BOIP to a fluorescent element, such as the green or red fluorescent protein of the deep-sea fish Malacosteus. be able to. The result obtained is a BOIP with an altered color and / or intensity and / or polarization of the radiation. The color change can be caused by energy transfer such as resonance transfer (CRET or FRET) or electron transfer.

[0038] The initial (unmodified) BOIP is derived from the bivalve mollusc Phoras dactylus
Apophotoprotein, its DNA or RNA, or a mollusc, Rocellaria grandi, that luminesces by oxygen metabolites in a manner very similar to other BOIPs, such as Pholas dactylus
or from squid Ommastraffes, or earthworm luciferase.

Thus, BOIP, apoBOIP, or the nucleic acids encoding it, whether modified or not, can be used in a range of biology and studies as described below. (A) Detection, localization, and measurement of signals in a substrate, such as a living cell, organ or organism, or extracellular fluid. (B) Detection, localization, and measurement of oxygen and its metabolites in substrates, such as living cells, organs or organisms, or extracellular fluids, water (seawater and freshwater), soil, or the atmosphere. (C) normal cells, such as microorganisms (protozoa, yeast, fungi, mold, bacteria,
Virus) detection and localization. (D) abnormal cells, such as cancer cells, hyperactive cells of rheumatoid arthritis and other inflammatory diseases, cells infected with pathogens such as viruses and other infectious agents,
Detection of cells damaged by physical, chemical, or biological attack, cells damaged by perfusion or reperfusion injury, or cells damaged by oxygen or one of its metabolites And orientation. (E) Measurement and localization of enzymes, especially those that produce oxygen or its metabolites, and other tumor responses in cells or biological fluids. (F) DNA and RNA binding assays. (G) Immunoassays and other protein binding assays. (H) in genetic engineering, development of transformed animals and plants and microorganisms, and in horticulture, agriculture, medicine, and veterinary medicine. And / or (i) in genetically engineered entertainment, by incorporating into light sticks, greeting cards, or toys that generate various colors, intensities, fluctuations, flashes, and glows of light; In beverages such as beer, wine, spirits, cola and other soft drinks.

Thus, the present invention further provides apoproteins, such as forasin apoproteins (or apophorasins) in unglycosylated and glycosylated form, and their BOIPs, such as forasin, alone (ie, the isolated natural protein itself). (E.g., excluding native folacin itself), or in combination with one or more of the following: This binds to a target or signal peptide, glycosylate, a site that can be modified by an enzyme, an antigen or intracellular signal binding site, a promoter, an enhancer, or an oncogene, or a pharmaceutically active substance, and the like. The present invention further provides nucleic acid sequences encoding any of these proteins, vectors containing nucleic acid sequences encoding any of these proteins, hosts transformed with such vectors, any of these proteins. A recombinant cassette comprising living cells comprising or expressing the same, such as bacterial, insect, eukaryotic, prokaryotic, primitive or plant cells, and the rainbow proteins described herein and the nucleic acid sequences encoding them. .

Hereinafter, the present invention will be described with reference to the following non-limiting examples. In these examples, the methods cited are known to those skilled in the art and / or could be followed by reference to the protocols set forth in the references cited below. See these references. Books 1. Campbell, AK. (1988). Chemiluminesce
nnce: principles and applications in b
iology and medicine, pp608. Horwood / VC
H. Chichester and Weinheim. 2. Campbell, AK (1994). Rubycon: the fif
th dimension of biology. pp304. Duckwo
rth, London. Paper 1. See Campbell, AK Patel, A .; (1983) Biochem
J. 216: 185-194. A homogeneous immuno
assay for cyclic nucleides based o
n chemiluminescence energy transfer. 2. Roberts, PA. Knight, J Campbell, AK. (1
987) Anal. Biochem. 160: 139-148. Pholasi
na-biluminescent indicator for det
ecting activation of single neurotroph
ils. 3. Mueller, T. Davies, EV Campbell, AK. (1
989). Biolum. Chemilum. 3: 105-113. Pho
lasin: Chemiluminescence Detections Most
ly superoxide Anion Released from Ac
activated Human Neutrophils. 4. Mueller, T Campbell, AK. (1989) J. Am. Biol
um. Chemilum. 5: 25-30. The Chromophore
of Pholasin: A Highly Luinescent Prot
ein. 5. Sala-Newby, G and Campbell, AK (1991)
Biochem. J. 279: 727-732. Engineering a
bioluminescent indicator for cyclic
AMP dependent protein kinase. 6. Campbell, AK, Trewavas, AJ and Knight
, MR (1996). Calcium imaging shows diff
erential sensitivity to cooling and
communication in luminous transgenic
plants. Cell Calcium 19: 211-218. 7. Kendall, JM, Sala-Newby, G Badminton,
M. Campbell AK and Remold, CR (1996). F
ree Ca ²⁺ in the endoplasmic reticulum
of living cells measured using aequ
orin as a pseudo luciferase. Biochem.
J 318: 383-387 8. Badminton, MN, Campbell, AK and Rembo
ld, CR (1996). J. Biol. Chem. 271: 31210312
14． Differential regulation of nuclea
r and cytosolic Ca ²⁺ in HeLa cells. 9. Sala-Newby, GB, Taylor, KT, Badminton,
MN, Remold, CR and Campbell, AK (1998). Imaging bioluminescent indicators s
hows Ca ²⁺ and ATP permeability thresh
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element. Immunology. 93: 4: 601-609 10. Sala-Newby, GB, Kendall, JM, Jones, H,
Taylor, KM, Badminton, MN, Llewelllyn, DH
and Campbell, AK (1999). Targeting biol
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Example 1 Production of BOIP in Bacteria Apophorasin-encoding c or genomic DNA, with or without cDNA encoding a signal peptide, was ligated to restriction sites such as BamHl at each end.
Amplified by PCR. The cDNA was run on an agarose gel and the full-length DNA was eluted and purified. The DNA was then cut with BamH1 to generate sticky ends and ligated to an expression plasmid also cut with BamH1 such as pET3a. After the ligation reaction, the sealed plasmid was replaced with standard E
. E. coli K12 strain such as JM109 was transformed and colonies were picked for large plasmid preparation selection. After confirming that the plasmid has the correct sequence for apophorasin and is in the correct orientation, the plasmid is transformed into E. coli. E. coli, for example, used to transform standard or other expression strains of BL21 (DE3). One colony was picked from the agar plate and grown in standard LB broth for 2 hours. IPTG was added as inducer and allowed to grow for a further 2 hours. Apophorasin could then be extracted by disrupting the bacteria by lysozyme digestion or sonication in a standard salt culture, for example 50 mM HEPES pH7 +/- 1 mM ascorbate. Because apophorasin is not glycosylated, it tends to aggregate and form inclusion bodies. These inclusions can be disrupted by 8M urea or guanidium chloride and subsequently dialyzed. When the pH of the PAGE gel is alkaline, aggregation of unglycosylated and glycosylated apophorasin and intact foralasin also tends to occur. Signal peptide eg β
-The lactamase signal directs the BOIP into the periplasm, resulting in the ability to secrete the expressed protein from the external fluid of the cell.

Example 2 Production of BOIP in Insect Cells c or genomic DNA encoding apophorasin was inserted into a plasmid suitable for conversion to baculovirus when infected to insect cells. Since forasin is secreted by Pholas itself, there is a signal peptide at the N-terminus. Removal of this by PCR allows for cytosolic expression in insect cells. If the signal peptide is left alone or changed to the honeybee melittin signal peptide, apophorasin is secreted into the external culture. Next, the virus containing DNA for apophorasin was purified and stored until needed. Next, aliquots were added to the fresh insect cells and cultured for 3-7 days. Apophorasin was then separated from the supernatant if a signal peptide was used, and from the cells if not. Apophorasin can then be purified by ammonium sulfate precipitation, gel filtration, and DEAE chromatography. Glycosylation states that when the molecular weight is 34 Kda,
It can be evaluated by performing AGE. Removal of glycosylation by the enzyme returned the protein to apophorasin size of 23.5 Kda. This apophoracin can be stored frozen or lyophilized and activated by the addition of luciferin as described in Example 3 to produce foracin.

Since apophorasin tends to aggregate in insect supernatants, this protein can be added as quickly as possible to a non-aggregation buffer such as 50 mM HEPES pH 6, 1-1.
It is important to be within 0 mM ascorbate.

Next, generation of forasin can be realized as described in the third embodiment.

Example 3 In order to produce foracin and generate light emission , apophoracin must first be converted to foracin by luciferin. Luciferin can be extracted from native forasin by mild acid treatment, or can be extracted from light organs or whole organisms isolated from Phoras dactylus by methanol, mild acid, or alkali treatment. After homogenization, the extract is centrifuged or filtered to remove particulate matter. Further purification can be performed by TLC or HPLC. Luciferin is best stored dry, but can also be stored at -70 ° C. Integrity and concentration can be assessed by measuring fluorescence or absorbance. Details are as follows. (A) Separation of luciferin Four protocols (1 to 4) were defined to extract and separate luciferin, which causes light emission of forasin. Luciferin is available from
small organic moieties that are tightly bound to apophoracin when separated from L. rus, but some are not bound to apophoracin. Thus, the extraction procedure separates either form of luciferin. 1. The organism Phoras dactylus or its light organs is brought to pH on ice.
6. Homogenize in 50 mM sodium phosphate. Foracin is precipitated with saturated sodium sulfate (stirred at 4 ° C.) and centrifuged at about 15,000 g for 30 minutes at low temperature. Next, the supernatant is allowed to fall through a SEP-PAK silica column that binds to luciferin. The column is washed with 5 ml of ethyl acetate and a further 5 ml
Wash with methanol. Active fraction containing luciferin (active fra
ctions) are assayed by reactivation of apophorasin or chemiluminescence in DMSO, DMF, or NaOCl. Luciferin can be concentrated and further purified by TLC or HPLC with standard solvents. The obtained is dried and stored at -70 ° C. 2. The organism Phoras dactylus or its light organs is homogenized in cold acetone on ice, filtered through a Buchner funnel, further extracted with methanol: acetone (1: 1), the residual powder is extracted three times with methanol, Combine the extracts. These are then concentrated on a Rotavaporator and left on ice for 1 hour to allow further precipitation to occur. Next, the suspension is re-filtered and concentrated. Next, the resulting solution containing luciferin is lowered in a SEP-PAK silica column that binds to luciferin. The column is washed with 5 ml of ethyl acetate and then with another 5 ml of methanol. The active fraction containing luciferin was purified by reactivation of apophorasin or DMSO, DMF or NaO
Assay by chemiluminescence in Cl. Luciferin can be concentrated and further purified by TLC or HPLC with standard solvents. The resulting can be dried and stored at -70 ° C. 3. The organism Phoras dactylus or its light organ is homogenized in cold acetone on ice and filtered through a Buchner funnel to obtain acetone powder. Next, extraction is performed twice with methanol: acetone (1: 1) for 10 minutes, and further three times with methanol. The combined extracts are concentrated on a rotary evaporator. Leave on ice for 1 hour to allow further precipitation to occur, refilter and concentrate. Residual powder 5
Dissolve in 0 mM sodium phosphate pH 6.0, 10 mM ascorbate and add
Ultrafiltration at 4 ° C. through a 0 kD Amicon membrane to remove forasin. Next, the solution containing luciferin is lowered in the SEP-PAK silica column that binds to luciferin. The column is washed with 5 ml of ethyl acetate and then with another 5 ml of methanol. Active fractions containing luciferin are assayed by reactivation of apophorasin or chemiluminescence in DMSO, DMF, or NaOCl. Luciferin can be concentrated and further purified by TLC or HPLC with standard solvents. The result is dried and stored at -70 ° C. 4. The organism Phoras dactylus or its light organs is placed on ice for 5 hours.
Homogenize with methanol and 100 mM HCl in 0 mM HEPES buffer and incubate on ice for 2 hours. After centrifugation at about 15,000 g for 30 minutes at low temperature, the supernatant is passed down a SEP-PAK silica column that binds to luciferin. The column is washed with 5 ml of ethyl acetate and then with another 5 ml of methanol. The active fraction containing luciferin is reactivated with apophorasin,
Alternatively, assay by chemiluminescence in DMSO, DMF, or NaOCl. Luciferin can be concentrated and further purified by TLC with standard solvents. The result is dried and stored at -70 ° C. Usually, method 4 gives the most luciferin. Luciferin is characterized by its absorption and fluorescence spectra and chemiluminescence by DMSO, NaOCl, and apophorasin. (B) Formation of forasin from apophorasin and luciferin A small amount of luciferin sample (1 to 10 μl) was added to an appropriate buffer
HEPES pH 6-7.5, +/- 0.1% gelatin, +/- 1-10 mM ascorbate, or 500 mM NaCl, 10 mM TES, 1 mM ED.
TA, 1 mM mercaptoethanol pH 6-7.5) to apophorasin. The resulting mixture was kept at room temperature for no more than 24 hours and forasin was assayed by adding oxygen metabolites such as NaOCl, or luciferase to the sample. When apophorasin is expressed intracellularly, luciferin is added externally, microinjected into individual cells, or added by liposomes to allow luciferin to reach the cells. Light was detected and quantified by a standard luminometer, imaging camera (sensitized or CCD), or silicon chip.

Example 4 Generation of BOIP In Vitro c or genomic DNA encoding apophorasin, with or without a signal peptide, is amplified by PCR with 5 'primers containing DNA encoding T7 RNA polymerase. did. The DNA product was purified and precipitated. After dissolving the DNA in 10 mM Tris / 1 mM EDTA pH 7, it was added to a standard in vitro transcription / translation system such as rabbit reticulocyte lysate or wheat germ agglutinin and kept at 30 ° C. for 30-60 minutes. The apophorasin can then be purified and activated to produce forasin as described in Example 3.

Example 5: Targeting BOIP Ex Vivo BOIP can also be used to include a signal peptide that places the BOIP on the inner or outer surface of the plasma membrane or within a particular organelle. Manipulation by chemical means or genetic engineering can be incorporated into specific parts of living cells. These organelles include, for example, peroxisomes, mitochondria, chloroplasts, tonoplasts, endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, secretory vesicles, nuclei, kernels, proteosomes, or gap junctions, or microtubules, cytoskeletons, nuclei There are tissues such as the skeleton, nuclear receptors, or the mitotic spindle.
A chemically or genetically added signal peptide usually directs normal or modified BOIP to a specific intracellular or extracellular location. For example, the array MLSRL
SLRLLSRYLL or a portion of the cytochrome oxidase on the N-terminus is
The IP leads to mitochondria, the KKSALLALMYVCPGKADAKE or MLLPVPLLLLGLLGLAA or the N-terminal ER protein calreticulin leads the BOIP to the endoplasmic reticulum, and the C-terminal KDEL or HDEL sequence
Is held in place. CKL at the C-terminus leads BOIP to peroxisomes,
Extension of PKKKRKV or this SV40 large T antigen signal directs BOIP to the nucleus and palmitoylation and / or myristoylation signal (MGCVCSSNPD = LCK N-terminal acylation motif from tyrosine kinase) directs BOIP to the plasma membrane. By binding BOIP to another protein that directs itself to a particular site, BOIP is also directed to that site. For example, by binding the nuclear protein nucleoplasmin or lamin B receptor to BOIP, the BOIP is directed to the nucleus. N-terminal cytochrome oxidase is B
Guide OIP to mitochondria. N-terminal chlorophyll directs BOIP to chloroplasts. Connexin at the N-terminus also leads BOIP to gap junctions or the plasma membrane,
AP25 leads to the plasma membrane.

In order to direct folacin to specific sites in living cells, DNAs encoding these target sequences were added by PCR. In the case of cytosolic apophorasin, the natural signal peptide can be removed and BOIP can be conjugated to large N- or C-terminal proteins, such as firefly luciferase or aequorin, to keep BOIP out of the nucleus. This also allows ATP and oxygen metabolites, or Ca2 + and oxygen metabolites, to be measured in the same cell at the same time by intensity, color or polarization of different bioluminescence indicators. Multiple bioluminescence index is also 3
It can be created from DNA encoding one or more bioluminescent proteins by PCR, or by using restriction enzyme sites. By simple sieving of the transformed bacteria, multiple rainbow proteins of two or more colors can be separated.

The DNA is then added to the transcription / translation system in vitro, as described in Example 4, in the presence of the target organelle (eg, microsomes against the endoplasmic reticulum, which glycosylate apophorasin). .

The new DNA can be inserted into a plasmid by standard methods, transformed into Bacteria, or used in eukaryotic cells such as H
It can also be infected or injected with eLa or COS.

The addition of luciferase as described in Example 3 forms forasin, which can be detected by light emission. Thus, cells may be exposed to external oxygen metabolites, changes in oxygen concentration, the addition of stimulants such as TNF, EGF, hormones or drugs, or pathogens such as bacteria, viruses, complement,
Changes in oxygen metabolite production upon exposure to antibodies, toxins, and attack by cells of the immune system can be detected by a luminometer or imaging camera.

Example 6: Creating a covalent modification site in BOIP (a) Protein kinase A (RRAS or Kemptide), protein kinase C (
MARCKS), MAP kinase, ERK, ER-nuclear signal kinase IREI
P or phosphatase encoding sites were added to the N or C terminus or inserted by PCR at various sites within apophorasin,
And expressed as described in Next, forasin was generated by the addition of luciferin as described in Example 3. Addition of a catalytic subunit to protein kinase A, or cyclic A
Activation by MP resulted in phosphorylation or dephosphorylation of the modified forasin and a change in light emission (intensity, color, or polarization). Prescreening is required to select the appropriate protein and discard those that have lost all activity. (B) Proteases (thrombin, enterokinase, HIV protease,
A site encoding caspase) was added to the N- or C-terminus of apophorasin by PCR.
Or inserted at various sites within the protein and expressed as described in Examples 1-5. Next, forasin was produced by the addition of luciferin as described in Example 3.

Example 7: Incorporation of BOIP into "rainbow protein" A cDNA encoding apophorasin was ligated to another protein using the cDNA encoding the protein. For example, wild-type GFP, the S65T mutant of green photoprotein, YGFP, or EGFP is linked to the N- or C-terminus of apophorasin by PCR, or by cleaving one or both proteins by multi-step PCR. I let it. Middle, has a protease site (alpha-thrombin or enterokinase) and IP binding site for ₃ or 15 amino acid sequence type IP ₃ kinase, (IP ₄ binding site) 'active' peptide appears. A peptide containing 6 lysine residues can also be added to the C-terminal of GFP by PCR. The protein is expressed and fluorescein is covalently linked to these lysines by the addition of fluorescein isothiocyanate. Addition of luciferin produces forasin as described in Example 3. The color change is caused by chemiluminescent resonance energy transfer. In the absence of fluorescein, the rainbow protein emits blue-green light (508 nm) but changes to blue (490 nm) when the reactive substance binds to the reactive peptide or when thrombin or enterokinase is added. I do. Using a 6 amino acid linker, the color is green (5
30 nm), changing from green to bluish green and then blue as the specific reactive sequence binds to each analyte. Using rhodamine instead of fluorescein produces a rainbow protein that varies from red to green to blue.

Preliminary sieving is required to select the appropriate rainbow protein and discard those that have lost all activity.

Other proteins that bind to apophorasin can be, for example, any of the following, chemically or genetically linked: 1. Firefly or any benzothiazole luciferase at the N or C terminus gives two colors for ATP and oxygen metabolites. 2. Any imidazole pyrazine luciferase, for example, coelenterazine systems (Decapoda shrimp, fish, squid, Renilla, flower worm, Trichinella, Radiolaria, or subradiator), and Vargula classification (ostracod, Porichthys and similar fish, Cyprinidae, and Vargula). 3. Any tetrapyrrole luciferase, such as dinoflagellate, krill, or stomatoid fish. 4. Bacterial luciferase or other aldehydes or flavin luciferases, such as polycapillaris. 5. Any GFP, such as wild type, S65T, enhanced GFP, blue GFP,
Yellow GFP, Renilla GFP, Ptilocarpus GFP, and Pennatura GFP, all worm GFPs, or all luminal GFPs. 6. Crocodile fish (Malactosteus, Aristostos)
mias, Photostomas). 7. Phycobiliproteins (phycoerythrin and phycocyanobilin). 8. Blue fluorescent lumazine protein of the bacterium Photobacterium. 9. Y Vibrio yellow flavin fluorescent protein. 10. Any lysine or arginine or other amino acid side chain to which a fluorophore can be covalently attached. In this case, the rainbow protein can emit more than two colors. For example, rhodamine on the forasin-linker-GFP chimera varies from red to green to blue.

Preliminary sieving may be required to select chimeras that have not lost all bioluminescent activity.

A “reactive” peptide is a binding site for any analyte, protein or D
NA, metabolite, substrate vitamin, enzyme such as protein kinase C or phosphatase, ion channel, ion pump, antigen, antibody, nucleotide or nucleoside such as ATP, GTP, ADP, AMP, adenosine, cAM
P, cCMP, cCCP, or their deoxy equivalents, and inositol phosphates such as IP ₃ or IP ₄ , lipids such as diacylglycerol, phosphatidylinositol biphosphate, phosphates, cations such as Ca ²⁺ , K ⁺ or Na ⁺ or Cu ^{2 +} , Or Zn ²⁺ , or an anion such as Cl ⁻ , sulfate, or a gas such as NO, O ₂ or H ₂ , or a protein binding site such as calmodulin, kinesin, dynein, tubulin, or myosin. .

When foracin is triggered by oxygen metabolites, Pholas luciferase, or peroxidase, foracin oxyluciferin converts GFP to
Energy transfer to fluorescein through the reaction, producing yellow radiation. Upon addition of thrombin for 3 hours, GFP fluorescein is cleaved from foracin and light emission returns to the native foracin blue. Complete the chimera addition of IP _3, the energy transfer efficiency is changed. As a result, the proportion of emitted light from yellow to blue changes. This ratio is directly related to the analyte concentration or amount and can be plotted against it. This light is provided with a two-wavelength luminometer or a ratio meter (rat
iometric) can be detected by an imaging camera and the ratio of blue light to green light can be measured.

Alternatively, any fluorophore can be used, and all binding sites with the correct properties as shown in the examples above will work properly if simple sieving is used to select the correct chimera. I do.

[0061] Example 8: Two to put make in "Rainbow protein" Rukoto Apoforashin for subject the BOIP, by using the cDNA and PCR, bound to firefly luciferase, after them Example 2 It was expressed in insect cells as described. Addition of luciferin as described in Example 3 produces folacin. In the presence of firefly luciferin (1 mM), ATP, and oxygen metabolites,
This chimera emits blue and yellow simultaneously and these lights can be measured separately by using a two-wavelength luminometer or imaging camera.

Example 9 Expression of BOIP in Mammalian Cells HeLa cells were infected with c or genomic apophorasin in an expression plasmid having a CMV promoter. After culturing for 3 days to express apophorasin, luciferin was added to generate forasin. Expression of foracin increased in rabbits was confirmed using a polyclonal antibody. By adding oxygen metabolites to the outside of the cells, the permeability of the plasma membrane to oxygen metabolites could be evaluated. As oxygen metabolites penetrate into the cytosol, light emission increases.

Example 10: Expression of BOIP in plants c or genomic DNA encoding apophorasin was inserted into a plasmid having a cauliflower mosaic virus promoter and Agrobacteri
um tumicificans. Next, these were added to tobacco leaves to generate seedlings, and seedlings expressing apophorasin were selected. The plants were grown and fruited, and seedlings were grown from the seeds. Addition of luciferin produced folacin as described in Example 3. During the growth and development of this plant, for example, when exposed to wind, contact, cold, or peroxide, or hormones, it produces light, indicating the production of oxygen metabolites in living plants. Was. The cell-specific promoter, built on the apophorasin cDNA prior to the production of the transformed plant, allows the detection of oxygen metabolites in specific cells throughout living plants.

Example 11: Detection of oxidative damage in vitro Detection of oxygen metabolites when adding the drug or other substance of interest by adding folacin to rat, mouse or human serum or plasma. Measurement becomes possible.

Example 12: Detection of ROM in cardiac cells Reperfusion to cause oxygen metabolite damage in cardiac myocytes was presented.
Foracin has made it possible to test it for the first time. Plasmids containing apophorasin cDNA and the CMV promoter were used to infect isolated cardiac myocytes in culture. It was expressed within 1 to 3 days, and forasin was produced by adding luciferin as described in Example 3. Reintroducing normal oxygen after placing these cells in a hypoxic condition resulted in light emission, indicating that oxygen metabolites were formed inside the cells. The use of an imaging camera makes it possible to visualize and count the cells emitting light, so that the digital or analog properties of this phenomenon can be evaluated.

Example 13: Detection of ROM in the nuclear and endoplasmic reticulum (ER) Nucleoplasmin DNA or calreticulin DNA (C) linked to forasin DNA to direct apophorasin to the nuclear or ER or CMV promoter for expression, respectively. HeLa cells in culture were infected with plasmid-containing apophorasin cDNA with or without KDEL at the end). 1
Expression occurred within ~ 3 days, and forasin was produced by the addition of luciferin as described in Example 3. Extracellular addition of oxygen metabolites or hypoxia /
Oxygen shock produced light, measured by a luminometer, indicating that oxygen metabolites rapidly penetrated these organelles. The number of cells permeable to oxygen metabolites could be counted by imaging with a photon counting imaging camera. The location of forasin can be determined by imaging living cells, or by immunofluorescence with a forasin antibody on partially fixed cells or GFP forasin in living cells. The use of rainbow proteins allows two or more analytes to be detected together.

Example 14: Use of forasin as a protein label Forasin can be used as a label in allogeneic or heterogeneous immunoassays. First, apophorasin was covalently bound to an antibody against HIV, and forasin was generated by the addition of luciferin as described in Example 3. This antibody was then used in a standard chemiluminescent immunoassay procedure. The addition of the HIV antigen increased the binding to the antibody and increased the amount of added HIV-dependent light emission. The amount of HIV in a blood sample can be assessed by relating the forasin light emission in the sample to a standard curve.

Example 15: Apophorasin was covalently linked to an oligonucleotide probe to detect the presence of the follasin cystic fibrosis gene as a DNA label . Addition of the probe to DNA by standard Southern blot allows the probe to be bound if the gene is present. Forasin can be produced by the addition of luciferin as described in Example 3. Hypochlorite in barbital buffer pH 9 (
With the addition of 10 mM), forasin flashes and the gene can be visualized with a photon counting imaging camera.

Example 16: Forasin protein-protein interaction in a protein complex detection system can be detected by making apophorasin in one half of the protein complex detection system and making GFP in the other. The binding allows the yeast to grow.

[0070] Example 17: folacin folacin in genetic engineering entertainment can chemiluminescence wide pH range including acidic pH e.g. 3-4 (3-10). Thus, folacin can make them luminous in addition to drinks such as beer, cola, soft drinks, and distilled spirits. The food can also be made luminescent by adding folacin to the ingredients of the cake, sugar coating, popcorn, applying folacin or apophoracin to the food, or genetically introducing it into the food source. Forasin can be used in a wide range of toys and other recreational equipment such as water guns, greeting cards, and pens.

[0071] Rainbow proteins can also be used as an alternative to forasin alone, and can provide a variety of colors, such as rainbows, and color changes.

Example 18 Forasin in Transgenic Animals Transgenic animals such as nematodes, mice, or plants can be produced from apophorasin cDNA by standard methods. Active folacin is produced by injecting luciferin into the plant or by culturing the whole plant in luciferin. Thus, oxygen or its metabolites can be detected, measured, and imaged in healthy organs or from whole organisms. Folacin can also be used in DNA therapy and diagnosis in humans.

Example 19: Apoprotein from the luminous squid Ommastrophes Apoprotein from the luminous squid Ommastrophes was used in place of apophorasin, and the methods of Examples 1 to 18 were performed.

Example 20: Apoprotein from the mollusc Rocellaria Apoprotein from the mollusc Rocellaria was used in place of apophorasin, and the methods of Examples 1-18 above were performed.

Example 21: The methods of Examples 1 to 18 were carried out using earthworm luciferase instead of apophorasin as earthworm luciferase BOIP.

Example 22 Genomic DNA from Pholas, Rocellaria, Ommastrophes, or earthworms was used in place of the recombinant proteins of Examples 1-18,
The methods of these examples were performed in a similar manner.

[Brief description of the drawings]

FIG. 1. Three different cDNA encoding apophorasins are shown as clones 40, 3, and 5. Bold nucleotides indicate codons used to start and stop translation.

FIG. 2 shows the three sequences of FIG. 1 arranged to show sequence similarity. This figure is Crus
tal. The positions indicated by asterisks are the same for all three clones. Codons for translation initiation and termination are highlighted in bold.

FIG. 3 shows oligonucleotides used for complete sequencing of positive clones. These were obtained from a cDNA library. The position of the oligonucleotide in clone 40 is shown. Oligonucleotides are shown in bold. The Bluescript plasmid is shown in italics.

FIG. 4 shows the protein sequence described by DNA sequence coding for apophorasin. FIG. 4A shows the complete sequence of the positive clone 40 obtained from the Pholas dactylus light organ library. The first 20 amino acids at the N-terminus are signal peptides, which are BI
When generating an OP, it can be saved or eliminated. FIG. 4B shows the cDNA encoding apophorasin with untranslated 5 ′ and 3 ′ ends. Untranslated regions are also shown.

FIG. 5. Foracin with signal peptide (FIG. 5B) and without (FIG. 5B)
5A shows the protein sequence for FIG. 5A).

FIG. 6 shows the sequence of apophorasin relative to genomic DNA. Two gDNA clones are identified, but no introns. This figure shows the sequence of cDNA from cDNA clone 40 and gDNA amplified by both rTth DNA polymerase XL and BioXAct polymerase. PCR products and pGE
The sequence of the MT insert was aligned with the sequence of the cDNA of clone 40, indicating that the former sequence was the same as the latter.

FIG. 7 shows oligonucleotides used for screening and expression. Shown in FIG. 7A are degenerate oligonucleotides for library screening.
The one shown in B is a non-degenerate oligonucleotide. FIG. 7C shows the oligonucleotides used for protein expression.

FIG. 8 shows major restriction sites in DNA for the production of forasin.

FIG. 9 is a schematic representation of FIG. 8, positioned in the sequence (translation region) of FIG. 4A.

FIG. 10 shows the time course of chemiluminescence induced by hypochlorite.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4B065 1/21 C12P 21/02 C 4C085 5/10 C12Q 1/02 4H045 C12P 21/02 1/66 C12Q 1/02 G01N 21/78 C 1/66 33/483 C G01N 21/78 33/68 33/483 C12N 15/00 ZNAA 33/68 5/00 A F-term (reference) 2G045 AA35 CB01 CB17 DA20 DA36 FA19 FB12 GC10 GC15 2G054 AA02 AA06 CA21 CE02 EA03 GA04 4B024 AA11 BA80 CA03 CA04 CA07 CA09 DA01 DA02 DA06 EA02 EA04 FA02 FA18 GA11 GA25 HA14 4B063 QA01 QA05 QAQQAQQAQQQ1QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ directly at the Atlas at Attachment AA35 CB01 CB17 DA20 DA36 FA19 FB12 GC10 GC15 2G054 AA02 AA06 CA21 CE02 EA03 GA04 4B024 AA11 BA80 CA03 CA04 CA07 CA09 DA01 DA02 DA06 EA02 EA04 FA02 FA18 GA11 GA25 HA14 4B063 QA01 QA05 QA13 QA12 QR48 QR56 QR74 QR77 QR80 QS34 QX02 4B064 AG01 CA02 CA10 CA11 CA19 CC01 CC24 DA13 4B065 AA26X AA58X AA72X AA88X AA90X AA90Y AB01 AC14 BA02 BD50 CA24 CA46 4C085 HH11 HH1 3 JJ01 KA27 KB82 KB92 LL17 LL18 4H045 AA10 AA20 AA30 BA10 BA41 CA50 EA50 FA74 GA01

Claims (30)

[Claims] 1. A sequence encoding apophotoprotein of foracin (or 'apofolacin'); (b) a sequence substantially homologous to sequence (a) or under stringent conditions (a) And (c) substantially homologous to sequence (a) or (b), or hybridizing under stringent conditions to sequence (a) or (b). An array to form without
Or (d) an oligonucleotide unique to any of the sequences (a), (b) or (c), wherein the isolated, purified or recombinant nucleic acid sequence is characterized by: 2. The sequence according to claim 1, wherein the sequence encoding apophorasin is as shown in FIG. 4B. 3. The sequence encoding apophorasin is shown in FIGS. 1, 2, 3, 4A,
10. The method as claimed in claim 1, wherein the signal is one of:
The sequence described in 1. 4. The sequence according to claim 1, wherein the apophorasin is not glycosylated. 5. The sequence according to claim 1, wherein the apophorasin is glycosylated. 6. An isolated, purified or recombinant cassette comprising a sequence encoding the apophorasin protein according to any one of claims 1 to 5. 7. A sequence comprising an apophotoprotein-encoding sequence, wherein expression of the apophotoprotein in a substrate cooperating with luciferin therefor signals the presence of oxygen or oxygen metabolites in said substrate. A separation, purification, or recombinant cassette, characterized in that: 8. An apophotoprotein having a sequence encoding the apophotoprotein, wherein the expression of the apophotoprotein in a substrate cooperating with luciferin thereto is achieved by the absence of the corresponding luciferase in the substrate. Or a separation, purification, or recombinant cassette characterized in that it indicates the presence of oxygen metabolites. 9. The method according to claim 1, wherein the nucleic acid sequence is operatively linked to a nucleotide that enables expression and secretion of apophorasin in a cell host. Recombined cassette. 10. DNA or RNA according to any one of claims 1 to 9. 11. An isolated, purified, or recombinant polypeptide comprising an apophotoprotein of foracin (apophoracin), or a mutant or a variant of apophotoprotein having substantially the same activity as apophoracin. 12. The isolated, purified or recombinant polypeptide according to claim 11, which has the amino acid sequence of FIG. 4 or FIG. 13. The apophorasin according to claim 11 or 12, when expressed by the recombinant DNA or RNA according to claim 10. 14. The apophorasin according to claim 13, which is not glycosylated. 15. A cell, a plasmid, characterized in that it is genetically produced to produce an apoprotein and comprises a sequence according to any one of claims 1 to 10 so that it can be expressed. A virus, or living organism. 16. A vector comprising the sequence according to any one of claims 1 to 10. 17. A host cell transformed by, or infected with, the vector of claim 16. 18. B as defined herein, characterized in that it comprises the apophotoprotein according to any one of claims 11 to 14 together with luciferin.
OIP. 19. The BOIP according to claim 18, wherein the luciferin is obtained from Pholas dactylus. 20. A method for producing a BOIP as defined herein, comprising binding an apophotoprotein, such as a recombinant apophorasin, to a luciferin against the protein, such as luciferin obtained from Pholas dactylus. Method. 21. A BOIP, an apophotoprotein thereof, or a BOIP, characterized in that it has a chemically or genetically modified sequence according to any one of FIGS. 2 to 6 or the sequence according to FIG. A nucleic acid sequence encoding any of these. 22. Extracellular detection and / or detection of oxygen or one of its metabolites
Or a method of measuring a BOIP, such as a natural or chemically or genetically modified BOIP
Alternatively, the 'rainbow protein' based on the BOIP is supplied extracellularly, and then light emission from the BOIP and / or changes in the color, intensity and / or polarization of the emitted light are detected and detected. And / or quantifying, wherein the apophotoprotein comprises recombinant apophorasin. 23. A method for detecting and / or measuring (intracellularly) oxygen or one of its metabolites in living cells, comprising a BOIP, eg, naturally or chemically or genetically modified BOIP
Alternatively, a “rainbow protein” based on the BOIP is supplied into a cell, and then the color, intensity, and / or polarization of light emitted from the BOIP and / or emitted light from the BOIP is changed. Detecting and / or quantifying the following. 24. The method according to claim 22 or 23, wherein the BOIP has a signal peptide that directs it to a predetermined site outside or inside a cell. 25. The method according to claim 22 or 23, wherein the test sample is cultured with the cells according to claim 15 or with a membrane product obtained from said cells. 26. The method according to any one of claims 22 to 24, wherein the light emission occurs in the absence of luciferase. 27. Use of a sequence or protein according to any one of claims 1 to 21 in the detection, diagnosis or measurement of oxygen or its metabolites. 28. A diagnostic kit comprising the sequence or protein according to any one of claims 1 to 21. 29. A method for obtaining a substantially homologous source of apophorasin, wherein the cell has been introduced to express a polynucleotide encoding apophorasin as defined in any one of claims 1 to 10. And then harvesting the cultured cells. 30. A method, use, or kit according to any one of claims 20 to 27, each substantially as described herein with reference to the Examples.