Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

• the present invention relates to a fusion protein, a nucleic acid encoding the fusion protein, and uses of the fusion protein.
• PPARs Peroxisome Proliferator-Activated Receptors
• PPARs are a group of transcription factors that were discovered to induce proliferation of intracellular peroxisomes and are considered to be closely involved in the intracellular metabolism of carbohydrates, sugars, proteins, etc., and cell differentiation.
• PPARs form the nuclear receptor superfamily together with retinoic acid receptors, vitamin D receptors, sex hormone receptors, etc.
• PPAR has three known subtypes (isoforms): ⁇ , ⁇ , and ⁇ .
• PPAR ⁇ is activated by physiological ligands such as free fatty acids and leukotriene B4, which leads to a decrease in blood triglyceride levels through peroxisome proliferation.
• PPAR ⁇ is thought to regulate many genes involved in the ⁇ -oxidation of fatty acids, and is therefore a major target for drugs to improve hyperlipidemia.
• Fibrate drugs such as fenofibrate, bezafibrate, and clofibrate are known as exogenous ligands for PPAR ⁇ .
• PPAR ⁇ is activated by physiological ligands such as prostaglandin J2, activating glucose uptake in muscles. PPAR ⁇ is thought to increase insulin sensitivity in tissues, making it one of the targets for diabetes treatment. PPAR ⁇ has also been implicated in the immune system. Thiazolidinedione drugs such as troglitazone and pioglitazone are known as exogenous ligands for PPAR ⁇ .
• PPAR ⁇ Compared to the two isoforms PPAR ⁇ and PPAR ⁇ , PPAR ⁇ has received relatively little attention. However, in recent years, it has been researched and developed as a drug discovery target for primary mitochondrial myopathy. GW501516 is known as an exogenous ligand for PPAR ⁇ .
• Non-Patent Document 1 describes a fluorescent probe for PPAR ligands.
• the document relates to a visualization technique for PPAR ligands using a probe using fluorescence resonance energy transfer (FRET) from cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP).
• FRET fluorescence resonance energy transfer
• CFP cyan fluorescent protein
• YFP yellow fluorescent protein
• the technique of Non-Patent Document 1 utilizes the relative position change between CFP and YFP due to the interaction between the agonist-dependent PPAR ligand binding region and a coactivator protein.
• Non-Patent Document 1 describes a probe for detecting agonists of the gamma subtype of PPAR.
• the probe of Non-Patent Document 1 cannot detect antagonists.
• a probe for PPAR delta ligands has not been developed.
• Non-Patent Document 2 also describes a detection system for PPAR ligands.
• the technology in this document utilizes FRET between a fluorescent PPAR ligand and a fluorescently labeled PPAR ligand binding domain.
• the above two reagents are mixed in a multi-well plate, and a candidate compound for a ligand of PPAR ⁇ (sometimes called "PPAR ⁇ ") is added. If the candidate compound is actually a ligand of PPAR ⁇ , it will compete with the binding of the fluorescent PPAR ligand to the PPAR ligand binding domain, resulting in a decrease in the FRET signal.
• the technology in Non-Patent Document 2 is intended to be used in a solution in a test tube.
• this technology cannot be used to examine the conditions for the expression of pharmacological effects caused by the living body, such as the cell membrane permeability of the candidate compound. In addition, it cannot be used to visualize the dynamics of PPAR ⁇ ligands in animals.
• the probe PPAR for retinoic acid belongs to the nuclear receptor superfamily. Nuclear receptors change their structure by binding to a ligand.
• a probe for retinoic acid (genetically encoded probes for RA: GEPRA) has been developed using the structural change of the retinoic acid receptor, one of the nuclear receptors (Non-Patent Document 3).
• GEPRA has a structure in which the ligand binding site of the retinoic acid receptor is sandwiched between CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein).
• CFP cyan fluorescent protein
• YFP yellow fluorescent protein
• the present invention relates to a fusion protein, a nucleic acid encoding the fusion protein, and uses of the fusion protein.
• a fusion protein comprising a cyan fluorescent protein, a ligand binding domain of a peroxisome proliferator-activated receptor, and a yellow fluorescent protein, a ligand-binding domain of a peroxisome proliferator-activated receptor between a cyan fluorescent protein and a yellow fluorescent protein, and no coactivator peptide of the peroxisome proliferator-activated receptor;
• the fusion protein is not limited to, the following aspects.
• a method for detecting a ligand for a peroxisome proliferator-activated receptor comprising: Contacting the fusion protein according to any one of [1] to [5] with a biological sample; If the fluorescent signal from the cyan fluorescent protein changes, it is determined that a ligand for the peroxisome proliferator-activated receptor is present in the biological sample.
• the method comprising: [12] The method according to [11], wherein the ligand is an agonist or antagonist of a peroxisome proliferator-activated receptor.
• the fusion protein of the present invention enables more accurate detection of PPAR ligands. Furthermore, the fusion protein of the present invention can detect not only agonists but also antagonists as ligands.
• FIG. 1 is a schematic diagram of the present invention.
• FIG. 2 shows the response of PPAR ligand probes for each subtype ( ⁇ , ⁇ , ⁇ ) to agonists.
• the graph shows the time course of the signal change rate (vertical axis) in response to agonists for each subtype for a probe (d0) using the full-length fluorescent protein YPet as a FRET acceptor and a probe in which the N-terminus of YPet is truncated by 1 to 5 amino acids (d1 to d5).
• the average of the responses from three cells was plotted.
• the thick line indicates the signal at d0.
• Figure 3 shows the response of PPAR probes to antagonists.
• ⁇ indicates the response of mPPARP-A d0
• ⁇ indicates the response of mPPARP-D d3
• ⁇ indicates the response of mPPARP-G d3.
• ⁇ was examined for the response to one type of antagonist, while ⁇ and ⁇ were examined for the response to two types of antagonist. The average response of three cells was plotted for each. 4 shows the response of the probe without sFATT to an agonist, where " ⁇ " indicates the response of PPARP-A d0, “ ⁇ ” indicates the response of PPARP-D d3, and " ⁇ ” indicates the response of PPARP-G d3.
• the present invention includes, but is not limited to, the following aspects. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one aspect" in this specification, it means that the aspect is not limited, i.e., is non-limiting.
• the present invention relates to a fusion protein.
• the fusion protein of the present invention includes, but is not limited to, a cyan fluorescent protein, a ligand-binding domain of a peroxisome proliferator-activated receptor, and a yellow fluorescent protein, and includes the ligand-binding domain of a peroxisome proliferator-activated receptor between the cyan fluorescent protein and the yellow fluorescent protein, and does not include a coactivator peptide of the peroxisome proliferator-activated receptor.
• Peroxisome proliferator-activated receptors are a group of transcription factors that were discovered to induce proliferation of peroxisomes within cells and are thought to be closely involved in the intracellular metabolism of carbohydrates, sugars, proteins, etc. and cell differentiation.
• PPARs belong to the nuclear receptor superfamily, whose members include retinoic acid receptors, vitamin D receptors, and sex hormone receptors.
• the PPAR ⁇ protein has, for example, the amino acid sequence set forth in SEQ ID NO:2.
• the PPAR ⁇ protein has, for example, amino acids encoded by a nucleic acid having a base sequence set forth in SEQ ID NO:1.
• the PPAR ⁇ protein may be a mutant of a protein having the amino acid sequence set forth in SEQ ID NO:2 or the amino acid encoded by a nucleic acid having a base sequence set forth in SEQ ID NO:1.
• the definition of "mutant" is described later in this specification.
• the "mutant” may have an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from the amino acid sequence set forth in SEQ ID NO:2.
• the “mutant” may have an amino acid sequence encoded by a nucleic acid having at least 70% sequence identity to the base sequence set forth in SEQ ID NO:1, or an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from the base sequence set forth in SEQ ID NO:1.
• the "mutant” may have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO:1 or its complementary sequence.
• the “mutant” maintains the biological activity of the PPAR ⁇ protein having the amino acid sequence set forth in SEQ ID NO: 2.
• the biological activity of the PPAR ⁇ protein means, for example, the activity of binding to a ligand.
• the ligand may include both agonists and antagonists. For example, it may include those used in the examples of the present specification.
• the PPAR is PPAR ⁇ . In one embodiment, the PPAR is PPAR ⁇ and PPAR ⁇ .
• the PPAR ⁇ protein may have, for example, the amino acid sequence set forth in SEQ ID NO: 4.
• the PPAR ⁇ protein may have, for example, amino acids encoded by a nucleic acid having a base sequence set forth in SEQ ID NO: 3.
• the PPAR ⁇ protein may be a mutant of a protein having the amino acid sequence set forth in SEQ ID NO: 4 or the amino acid sequence encoded by a nucleic acid having a base sequence set forth in SEQ ID NO: 3.
• the definition of "mutant" is described later in this specification.
• the "mutant” may have an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from the amino acid sequence set forth in SEQ ID NO: 4.
• the "mutant” may have an amino acid sequence encoded by a nucleic acid having at least 70% sequence identity to the base sequence set forth in SEQ ID NO: 3, or an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from the base sequence set forth in SEQ ID NO: 3.
• the "mutant" may have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 3 or its complementary sequence.
• the “mutant” maintains the biological activity of the PPAR ⁇ protein having the amino acid sequence set forth in SEQ ID NO: 4.
• the biological activity of the PPAR ⁇ protein means, for example, the activity of binding to a ligand.
• the ligand may include both agonists and antagonists. For example, it may include those used in the examples of the present specification.
• the PPAR ⁇ protein may have, for example, the amino acid sequence set forth in SEQ ID NO: 6.
• the PPAR ⁇ protein may have, for example, amino acids encoded by a nucleic acid having a base sequence set forth in SEQ ID NO: 5.
• the PPAR ⁇ protein may be a mutant of a protein having the amino acid sequence set forth in SEQ ID NO: 6 or the amino acid encoded by a nucleic acid having a base sequence set forth in SEQ ID NO: 5.
• the definition of "mutant" is described later in this specification.
• the "mutant” may have an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from the amino acid sequence set forth in SEQ ID NO: 6.
• the “mutant” may have an amino acid sequence encoded by a nucleic acid having at least 70% sequence identity to the base sequence set forth in SEQ ID NO: 5, or an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from the base sequence set forth in SEQ ID NO: 5.
• the "mutant" may have amino acids encoded by a nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 5 or its complementary sequence.
• the “mutant” maintains the biological activity of the PPAR ⁇ protein having the amino acid sequence set forth in SEQ ID NO:6.
• the biological activity of the PPAR ⁇ protein means, for example, the activity of binding to a ligand.
• the ligand may include both agonists and antagonists. For example, it may include those used in the examples of the present specification.
• the fusion protein contains the "ligand binding domain" of the peroxisome proliferator-activated receptor.
• the "ligand binding domain" of PPAR is present on the C-terminal side of the PPAR protein (almost half of the total length).
• amino acid residues 165-441 of SEQ ID NO: 2 amino acid residues 199-468 of SEQ ID NO: 4, and amino acid residues 205-475 of SEQ ID NO: 6 correspond to the ligand binding domain of PPAR.
• the fusion protein may contain a portion other than the ligand binding domain of PPAR.
• Fluorescent protein refers to a group of proteins that emit fluorescence when irradiated with visible light such as ultraviolet light or light in the same range.
• Known fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP).
• a “cyan fluorescent protein (CFP)” is a protein that emits cyan fluorescence.
• Non-limiting examples of cyan fluorescent proteins include those having an amino acid sequence as set forth in SEQ ID NO: 8.
• Examples of cyan fluorescent proteins include those having amino acids encoded by a nucleic acid having a base sequence as set forth in SEQ ID NO: 7.
• a cyan fluorescent protein may be a mutant of a protein having an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence encoded by a nucleic acid having a base sequence as set forth in SEQ ID NO: 7. The definition of "mutant" is described later in this specification.
• a “mutant” may have an amino acid sequence having at least 70% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from the amino acid sequence as set forth in SEQ ID NO: 8.
• a “mutant” may have an amino acid sequence encoded by a nucleic acid having at least 70% sequence identity to the base sequence as set forth in SEQ ID NO: 7, or an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from the base sequence as set forth in SEQ ID NO: 7.
• the "mutant" may have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 7 or its complementary sequence.
• the “mutant” maintains the biological activity of a cyan fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 8.
• the biological activity of a cyan fluorescent protein means, for example, the activity of producing cyan fluorescence upon excitation.
• Yellow fluorescent protein is a protein that emits yellow fluorescence.
• Non-limiting examples of yellow fluorescent proteins include the amino acid sequence shown in SEQ ID NO: 10.
• Yellow fluorescent proteins include, for example, amino acids encoded by a nucleic acid having a base sequence shown in SEQ ID NO: 9.
• Yellow fluorescent proteins may be mutants of proteins having the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence encoded by a nucleic acid having a base sequence shown in SEQ ID NO: 9. The definition of "mutant” is described later in this specification.
• a “mutant” may have an amino acid sequence having at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 10.
• a “mutant” may have an amino acid sequence encoded by a nucleic acid having at least 70% sequence identity to the base sequence shown in SEQ ID NO: 9, or an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from the base sequence shown in SEQ ID NO: 9.
• the "mutant" may have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 9 or its complementary sequence.
• the “mutant” maintains the biological activity of a yellow fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 10.
• the biological activity of a yellow fluorescent protein means, for example, the activity of producing yellow fluorescence upon excitation.
• Fluorescence resonance energy transfer is a phenomenon in which excitation energy is transferred directly between two adjacent dye molecules through electronic resonance, without becoming electromagnetic waves.
• donor energy absorbed by one molecule
• acceptor acceptor
• fluorescence is emitted from the acceptor.
• One method of observing FRET is to excite the donor with light equivalent to the donor's absorption spectrum and detect changes in the fluorescence intensity emitted from the donor and acceptor. Another method is to measure changes in the donor's fluorescence lifetime.
• FRET Fluorescence Activated FRET
• Both PPAR receptors and retinoic acid receptors belong to the nuclear receptor superfamily, but the amino acid sequences of their ligand-binding regions are significantly different.
• the ligand binding domain of the retinoic acid receptor used in mGEPRA-B consists of 246 amino acids, while that of PPAR ⁇ consists of 270 amino acids, that of PPAR ⁇ consists of 277 amino acids, and that of PPAR ⁇ consists of 271 amino acids.
• the fusion protein contains a cyan fluorescent protein, a ligand-binding domain of a peroxisome proliferator-activated receptor, and a yellow fluorescent protein.
• a cyan fluorescent protein Upon ligand binding to the ligand-binding domain of PPAR in the fusion protein, the structure of the ligand-binding domain changes, and the relative positions of the fluorescent proteins at both ends change. As a result, the efficiency of FRET from CFP to YFP changes, and the observed fluorescence spectrum changes. This makes it possible to use the fusion protein as a probe for detecting ligands for PPAR.
• Figure 1 A schematic diagram of PPAR ligand detection using the fusion protein is shown in Figure 1.
• Non-limiting descriptions of “mutants” of cyan fluorescent protein, peroxisome proliferator-activated receptor, and yellow fluorescent protein are provided.
• “Mutants” may have an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the specific amino acid sequences described above. “Mutants” may have an amino acid sequence encoded by a nucleic acid that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the specific base sequences described above.
• one sequence typically serves as a reference sequence to which the other sequence is compared.
• the reference sequence and the comparison sequence may be input into a computer, and sequence algorithm program parameters are selected, if desired.
• a percent sequence identity is then generated for the comparison sequence relative to the reference sequence based on the selected parameters.
• One example of an algorithm that may be suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (Nuc Acids Res 25:3389-3402, 1977) and Altschul et al., (J Mol Biol 215:403-410, 1990), respectively.
• BLAST and BLAST 2.0 are well known in the art and may be used to determine the percent sequence identity of any nucleic acid or protein, such as those described herein.
• nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or contain a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website found at, e.g., ncbi.nlm.nih.gov/BLAST/).
• Sequences with such a high degree of sequence identity may be referred to as "substantially identical.” This definition also refers to or applies to the complementarity of a particular sequence. This definition may also include sequences that have deletions, additions, and/or substitutions.
• a “variant” may have an amino acid sequence in which one or more amino acids have been deleted, substituted, or added from a specific amino acid sequence.
• a “variant” may have an amino acid sequence encoded by a nucleic acid having a base sequence in which one or more bases have been deleted, substituted, or added from a specific base sequence.
• the number of amino acid residues deleted, substituted or added to a particular amino acid sequence is not particularly limited.
• the number of amino acid residues deleted, substituted or added is 1 to 150, 1 to 120, 1 to 100, 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 28, 1 to 25, 1 to 22, 1 to 20, 1 to 18, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.
• the number of amino acid residues deleted, substituted or added is within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amino acid residues in a particular amino acid sequence. In one embodiment, without limitation, the number of amino acid residues deleted, substituted or added is within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amino acid residues in the amino acid sequence corresponding to the ligand binding region of the peroxisome proliferator-activated receptor.
• the number of bases deleted, substituted, or added from a particular base sequence is not particularly limited. Without limitation, the number of bases deleted, substituted, or added corresponds to the number of amino acid residues deleted, substituted, or added in the encoded amino acid sequence. The number of amino acid residues deleted, substituted, or added in the amino acid sequence is as described in the previous paragraph.
• a “substitution” is a substitution in which the amino acid residue encoded by the substitution in the base sequence is not changed (a "silent substitution”); Alternatively, in one embodiment, the “substitution” is a substitution in which the substitution of the encoded amino acid residue resulting from the substitution of the base sequence is a "conservative substitution".
• a conservative substitution is a substitution of a specific amino acid residue with an amino acid residue having similar physicochemical characteristics, but any substitution may be made as long as it does not substantially change the structural characteristics of the original sequence, for example, any substitution may be made as long as the substituted amino acid does not destroy the helix present in the original sequence or destroy other types of secondary structures that characterize the original sequence.
• conservative substitutions of amino acid residues are categorized by substitutable residues and exemplified, but the substitutable amino acid residues are not limited to those listed below.
• Group A leucine, isoleucine, valine, alanine, methionine, glycine, cysteine, proline
• Group B aspartic acid, glutamic acid Group C: asparagine, glutamine Group D: lysine, arginine Group E: serine, threonine Group F: phenylalanine, tyrosine, tryptophan, histidine
• one member of the above types can be exchanged for another type of member.
• the amino acids of the above groups B, D, and E may be replaced with amino acids of other groups to eliminate inadvertent glycosylation.
• cysteine may be deleted or replaced with another amino acid to prevent folding into a protein in a tertiary structure.
• amino acids may be substituted in consideration of the hydropathic index of amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, p. 105-132, 1982), which is an index of hydrophobicity/hydrophilicity of amino acids, so as to maintain a balance between hydrophilicity and hydrophobicity, or to increase hydrophilicity for ease of synthesis.
• a “variant” may have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a particular base sequence or its complementary sequence.
• under stringent conditions means hybridization under moderately or highly stringent conditions.
• moderately stringent conditions can be easily determined by a person skilled in the art based on, for example, the length of DNA. The basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001.
• moderately stringent conditions include hybridization conditions of 1xSSC to 6xSSC and 42°C to 55°C, more preferably 1xSSC to 3xSSC and 45°C to 50°C, and most preferably 2xSSC and 50°C.
• washing conditions include 0.5xSSC to 6xSSC and 40°C to 60°C. During hybridization and washing, generally, 0.05% to 0.2%, preferably about 0.1% SDS may be added. Highly stringent conditions can also be easily determined by those skilled in the art, for example, based on the length of the DNA. In general, highly stringent (high stringent) conditions include hybridization and/or washing at a higher temperature and/or lower salt concentration than moderately stringent conditions.
• hybridization conditions include 0.1xSSC to 2xSSC and 55°C to 65°C, more preferably 0.1xSSC to 1xSSC and 60°C to 65°C, and most preferably 0.2xSSC and 63°C.
• Washing conditions include 0.2xSSC to 2xSSC, 50°C to 68°C, and more preferably 0.2xSSC, 60°C to 65°C.
• the fusion protein contains a ligand-binding domain of a peroxisome proliferator-activated receptor between a cyan fluorescent protein and a yellow fluorescent protein.
• the order within the fusion protein is not particularly limited as long as the cyan fluorescent protein and the yellow fluorescent protein are capable of interacting with each other (FRET can occur) and are in a positional relationship in which a change in the efficiency of FRET from CFP to YFP (i.e., a change in the fluorescence spectrum or a change in the fluorescence lifetime of the donor) based on a structural change in the ligand-binding domain due to ligand binding to the ligand-binding domain of PPAR can be observed.
• the fusion protein either the cyan fluorescent protein or the yellow fluorescent protein may be present on the N-terminus.
• the fusion protein contains, from the N-terminus, a cyan fluorescent protein, a ligand-binding domain of a peroxisome proliferator-activated receptor, and a yellow fluorescent protein in that order.
• a linker peptide may be included between the cyan fluorescent protein and the ligand binding region of the peroxisome proliferator-activated receptor, and/or between the ligand binding region of the peroxisome proliferator-activated receptor and the yellow fluorescent protein.
• the sequence and length of the linker peptide are not particularly affected.
• the cyan fluorescent protein and the yellow fluorescent protein can interact with each other (FRET can occur), and that the positional relationship is such that a change in the efficiency of FRET from CFP to YFP (i.e., a change in the fluorescence spectrum or a change in the fluorescence lifetime of the donor) based on a structural change in the ligand binding region due to ligand binding to the ligand binding region of PPAR can be observed.
• the length of the linker peptide is, but is not limited to, 30 or less, 20 or less, 18 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1.
• It may also contain sequences other than the ligand-binding domain of the peroxisome proliferator-activated receptor.
• the fusion protein may contain a protein other than the cyan fluorescent protein, the ligand-binding domain of the peroxisome proliferator-activated receptor, and the yellow fluorescent protein.
• it may contain a sequence other than the ligand-binding domain of the peroxisome proliferator-activated receptor. It may contain a sequence other than the above on the N-terminal or C-terminal side of the cyan fluorescent protein or the yellow fluorescent protein.
• the fusion protein does not contain a coactivator peptide.
• a "coactivator” is generally a transcriptional coagulator that binds to an activator (transcription factor) to increase the transcription rate of a gene.
• Activators have a DNA-binding domain and bind to the promoter site of a gene or to a regulatory sequence called an enhancer.
• the activator-coactivator complex recruits the basal transcription machinery to the promoter, thereby increasing the rate of transcription and enhancing gene expression.
• L leucine
• X any amino acid
• the technology of Non-Patent Document 1 is that when an agonist binds to the ligand binding domain (LBD) of PPAR ⁇ , a conformational change occurs in the LBD of PPAR ⁇ , and the coactivator peptide binds to the LBD of PPAR ⁇ , and the agonist is identified by FRET based on this.
• Non-Patent Document 1 also states that the ligand (agonist) dissociates from the LBD of PPAR ⁇ in the presence of an antagonist.
• the present invention utilizes the FRET change that occurs directly due to a structural change in the ligand-binding region of PPAR caused by the binding of a ligand to the ligand-binding region of PPAR, and the fusion protein does not contain a coactivator peptide.
• coactivator peptide includes, but is not limited to, known PPAR coactivators and known nucleic acid receptor coactivators.
• the fusion protein contains, in this order from the N-terminus, a cyan fluorescent protein, a ligand-binding domain of a peroxisome proliferator-activated receptor, and a yellow fluorescent protein.
• a part of the N-terminus of the amino acid sequence of the yellow fluorescent protein may be deleted.
• 1-20 amino acid residues, 1-15 amino acid residues, 1-10 amino acid residues, 1-8 amino acid residues, 1-5 amino acid residues, 1-3 amino acid residues, or 1-2 amino acid residues may be deleted from the N-terminus of the yellow fluorescent protein.
• 1-5 amino acid residues are deleted from the N-terminus of the yellow fluorescent protein. In one embodiment, 1-3 amino acid residues are deleted from the N-terminus of the yellow fluorescent protein. In one embodiment, 3 amino acid residues are deleted from the N-terminus of the yellow fluorescent protein. In one embodiment, 1 or 3 amino acid residues are deleted from the N-terminus of the yellow fluorescent protein. In one embodiment, the PPAR is PPAR ⁇ or PPAR ⁇ , and one or three amino acid residues are deleted from the N-terminus of the yellow fluorescent protein. In one embodiment, the PPAR is PPAR ⁇ , and one or three amino acid residues are deleted from the N-terminus of the yellow fluorescent protein.
• no amino acid residues are deleted from the N-terminus of the yellow fluorescent protein.
• the PPAR is PPAR ⁇ and no amino acid residues are deleted from the N-terminus of the yellow fluorescent protein.
• constructs in which one amino acid residue and three amino acid residues were deleted from the N-terminus of the yellow fluorescent protein for PPAR ⁇ showed significantly larger signal changes.
• the rate of signal change was large even though no amino acid residues were deleted from the N-terminus of the yellow fluorescent protein.
• the fusion protein may or may not contain a tag for protein expression and identification.
• the fusion protein does not contain a FATT tag.
• a protein tag or polypeptide tag is a portion genetically bound to a protein to mark (tag) a specific protein molecule, and generally may be a short tag or a portion of a variety of proteins.
• a FLAG tag which is one type of protein tag, generally contains the amino acid sequence DYKDDDDV (SEQ ID NO: 15) or DYKDDDDK (SEQ ID NO: 16) from the N-terminus.
• the Flag tag can be widely used, for example, for isolating recombinant proteins by overexpression from proteins expressed in host cells, or for affinity chromatography to isolate protein complexes from multiple subunits.
• Figure 1B of Non-Patent Document 5 shows an example of the amino acid sequence of a FATT tag. It is contained in the FATT tag.
• Non-limiting examples of the amino acid sequence of the highly acidic region contained in the FATT tag and the base sequence of the nucleic acid that encodes it are shown in SEQ ID NO: 12 and SEQ ID NO: 11, respectively.
• Non-limiting examples of the amino acid sequence of the full-length FATT tag and the base sequence of the nucleic acid that encodes it are shown in SEQ ID NO: 14 and SEQ ID NO: 13, respectively.
• the fusion protein of the present invention even when it does not contain a FATT tag, binds to a ligand in the same way as when it does contain a FATT tag and functions as a probe.
• the fusion protein does not contain a FATT tag.
• Fusion Protein The explanation and definition of "fusion protein" is as described above in "1. Fusion Protein”.
• the fusion protein changes the structure of the ligand-binding region of PPAR when a ligand binds to the ligand-binding region of the peroxisome proliferator-activated receptor.
• CFP cyan fluorescent protein
• YFP yellow fluorescent protein
• the rate of change of the FRET signal increases by 0.01 or more, 0.02 or more, 0.03 or more, 0.05 or more, 0.1 or more, 0.2 or more, 0.4 or more, or 0.5 or more.
• the rate of change of the FRET signal ( ⁇ R/R) is greater than these values, it can be determined that a ligand (agonist) has bound to the ligand-binding region of the peroxisome proliferator-activated receptor.
• the absolute value of the rate of change of the FRET signal decreases by 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.06 or more, 0.08 or more, or 0.10 or more (negative signal change).
• the absolute value of the rate of change of the FRET signal is greater than these values, it can be determined that a ligand (antagonist) has bound to the ligand-binding domain of the peroxisome proliferator-activated receptor.
• FRET can be measured using known methods, and is not particularly limited.
• a microscope system such as that used in the examples can be used.
• the probe utilizes only the conformational change in the ligand-binding region of PPAR due to the binding of a ligand to the ligand-binding region of PPAR in the fusion protein. Therefore, the ligand is not particularly limited, and not only agonists but also antagonists of PPAR can be detected.
• the ligand includes an agonist or antagonist of a peroxisome proliferator-activated receptor. In one embodiment, the ligand is an agonist of a peroxisome proliferator-activated receptor.
• PPAR ligands include, but are not limited to, the following known agonists and antagonists:
• PPAR ⁇ BMS-687453 (Selleck, cat. no: S0170), fenofibrate, bezafibrate, clofibrate PPAR ⁇ : GW501516 (Sigma, cat. no: SML1491)
• PPAR ⁇ Pioglitazone (Selleck, cat. no: S2590), Troglitazone
• antagonists PPAR ⁇ GW6471 (Selleck, cat. no: S2798)
• PPAR ⁇ GSK3787 (Selleck, cat.no: S8025), GSK0660 (Selleck, cat.no: S5817)
• PPAR ⁇ T0070907 (Selleck, cat. no: S2871), GW9662 (Selleck, cat. no: S2915)
• the PPAR ligands in this specification also include candidate compounds that are unknown PPAR ligands.
• the present invention also relates to a kit for detecting a ligand of a peroxisome proliferator-activated receptor, comprising the fusion protein of the present invention.
• the kit may also comprise, for example, an agonist for each PPAR isoform, or an antagonist for each PPAR isoform, as a positive control.
• the present invention also relates to a tester for detecting a ligand for a peroxisome proliferator-activated receptor, comprising the fusion protein of the present invention.
• the present invention further relates to the use of the fusion protein, kit or tester of the present invention for a method for detecting a ligand of a peroxisome proliferator-activated receptor.
• the present invention further relates to a fusion protein or a kit of the present invention for use in a method for detecting a ligand for a peroxisome proliferator-activated receptor.
• the present invention relates to a method for detecting a ligand for a peroxisome proliferator-activated receptor.
• the method comprises: contacting the fusion protein of the present invention with a candidate compound for the ligand; If the fluorescent signal from the cyan fluorescent protein changes, the candidate compound is determined to be a ligand for the peroxisome proliferator-activated receptor. This includes:
• the method comprises: Contacting the fusion protein of the present invention with a biological sample; If the fluorescent signal from the cyan fluorescent protein changes, it is determined that a ligand for the peroxisome proliferator-activated receptor is present in the biological sample. This includes:
• Fusion protein The explanations and definitions for "fusion protein,” “ligand,” “candidate ligand,” “cyan fluorescent protein,” etc. are as described above in “1. Fusion protein” and “2. Probe for detecting ligands of peroxisome proliferator-activated receptors.”
• the structure of the ligand-binding domain of the PPAR changes. This changes the FRET signal from the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), allowing the PPAR signal to be identified and detected.
• CFP cyan fluorescent protein
• YFP yellow fluorescent protein
• the rate of change of the FRET signal increases by 0.01 or more, 0.02 or more, 0.03 or more, 0.05 or more, 0.1 or more, 0.2 or more, 0.4 or more, or 0.5 or more.
• the rate of change of the FRET signal ( ⁇ R/R) is greater than these values, it can be determined that a ligand (agonist) has bound to the ligand binding region of the peroxisome proliferator-activated receptor, i.e., that the candidate compound is a ligand (agonist) of the peroxisome proliferator-activated receptor, or that a ligand (agonist) of the peroxisome proliferator-activated receptor is highly likely to be present in the biological sample.
• the rate of change of the FRET signal decreases by 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.06 or more, 0.08 or more, or 0.10 or more (negative signal change).
• the rate of change of the FRET signal ( ⁇ R/R) is greater than these values, it can be determined that a ligand (antagonist) has bound to the ligand binding region of the peroxisome proliferator-activated receptor, i.e., that the candidate compound is a ligand (antagonist) of the peroxisome proliferator-activated receptor, or that there is a high possibility that a ligand (antagonist) of the peroxisome proliferator-activated receptor is present in the biological sample.
• the ligand is an agonist or antagonist of a peroxisome proliferator-activated receptor.
• the origin of the "biological sample” is not particularly limited.
• the biological sample is derived from a vertebrate that expresses a peroxisome proliferator-activated receptor. Vertebrates include mammals, birds, reptiles, amphibians, and fish.
• the biological sample is a mammal.
• the biological sample is derived from a mammal, for example, a primate such as a human and a chimpanzee, a laboratory animal such as a rat, a mouse, and a rabbit, a livestock animal such as a pig, a cow, a horse, a sheep, and a goat, and a pet animal such as a dog and a cat.
• the biological sample is derived from a human.
• the biological sample is derived from an animal other than a human.
• Biosamples include, but are not limited to, urine, saliva, sweat, sputum, stool, blood, serum, plasma, lymph, etc.
• the method for detecting the ligand of the peroxisome proliferator-activated receptor is an in vitro method, an ex vivo method, or an in vivo method. In one embodiment, the method is an in vitro method. In one embodiment, the method of the present invention is also capable of detecting the ligand of the peroxisome proliferator-activated receptor in a cell.
• Plasmids (i) Construction of pCS2/mPPARP-G A human liver cDNA library (Takara, Human cDNA Library (Plasmid) Liver, cat. no: 9505) was amplified by PCR using a primer having an SphI site at 5' (SphI PPARg LBD, SEQ ID NO: 17) and a primer having a SacI site at 3' (PPARg LBD SacI, SEQ ID NO: 18). As a result, a cDNA encoding the ligand-binding domain of human PPAR ⁇ (SEQ ID NO: 6) was obtained.
• the PCR fragment containing the obtained human PPAR ⁇ ligand-binding domain cDNA and the retinoic acid probe (pCS2/mGEPRA-B) (base sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20) in the pCS2 vector were subjected to restriction enzyme treatment with SphI/SacI to swap the ligand-binding domain of the retinoic acid receptor with the ligand-binding domain of PPAR ⁇ .
• the m in "mGEPRA” indicates that a short FATT tag (hereinafter referred to as "sFATT”) (base sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12) has been added, improving expression in mammalian cells.
• pCS2/PPAR probe-G d0 (hereinafter referred to as "pCS2/mPPARP-G d0") was obtained, in which the ligand-binding domain cDNA of human PPAR ⁇ was incorporated into the pCS2 vector.
• cDNAs encoding five types of YPet mutants with one to five amino acids deleted from the N-terminus were generated by PCR (referred to as "d1YPet”, “d2YPet”, “d2YPet”, “d3YPet”, “d4YPet”, and "d5YPet”, respectively).
• SacI d1Ypet to SacI d5YPet were both YPet XhoI (sequence number 26).
• sequence of YPet (before mutation) incorporated into pcDNA3 is sequence number 9 (the base sequence of YFP).
• YPet was extracted from pCS2/mPPARP-G d0 by treatment with SacI/XhoI restriction enzymes and replaced with the d1YPet to d5YPet PCR fragments that had also been treated with SacI/XhoI restriction enzymes (pCS2/mPPARP-G d1 to d5).
• the artificially synthesized ligand binding regions of human PPAR ⁇ and human PPAR ⁇ (SEQ ID NO: 3 and SEQ ID NO: 1, respectively) (both subtypes were synthesized by GenScript) were amplified with primers having SphI at 5' and SacI at 3', respectively, and ligated with the above-mentioned pCS2/mPPARP-G d0-d5 SphI/SacI to prepare pCS2/mPPARP-A d0-d5 and pCS2/mPPARP-D d0-d5.
• the primers used for amplifying human PPAR ⁇ were SphI PPARa LBD (forward) (SEQ ID NO: 27) and PPARa LBD SacI (reverse) (SEQ ID NO: 28).
• the primers used for amplifying human PPAR ⁇ were SphI PPARd LBD (forward) (SEQ ID NO: 29) and PPARd LBD SacI (reverse) (SEQ ID NO: 30).
• sFATT-free constructs were prepared as follows. pCS2/mPPARP-A d0 and pCS2/mPPARP-G d3 were treated with restriction enzymes SphI/XhoI to extract the PPAR ⁇ ligand binding region and YPet (hereinafter, "PPAR ⁇ ligand binding region": YPet) and the PPAR ⁇ ligand binding region: d3YPet.
• pCS2/GEPRA-B (not containing the sFATT tag) was similarly treated with SphI/XhoI and ligated with the aforementioned PPAR ⁇ ligand binding region: YPet and PPAR ⁇ ligand binding region: d3YPet to obtain pCS2/PPARP-A d0 and pCS2/PPARP-G d3.
• pCS2/PPARP-D d3 was prepared by removing the retinoic acid ligand binding domain: YPet from pCS2/GEPRA-B using restriction enzymes SphI/XbaI and replacing it with the PPAR ⁇ ligand binding domain: d3YPet.
• Stimulation with ligands was performed by adding 500 ⁇ L of HBSS solution at twice the final concentration to wells containing 500 ⁇ L HBSS (ligands used are shown below).
• the ligands used are the agonists and antagonists in the table below.
• the acquired image data was analyzed using the software Fiji/ImageJ (version 1.53f51).
• the minimum value of the background image was subtracted from the CFP and FRET channel images to calculate the ratio of CFP to FRET (CFP/FRET).
• CFP/FRET ratio of CFP to FRET
• Example 1 Probe response due to N-terminal deletion of acceptor fluorescent protein
• constructs in which one amino acid residue and three amino acid residues were truncated at the N-terminus of YPet showed significantly greater signal changes. This suggests that truncating the N-terminus is one of the most effective designs for increasing the response of a probe.
• Example 2 Study of reaction to antagonists Conventional techniques for fluorescent detection of PPAR ligands target only agonists and do not react to antagonists.
• the PPAR ligand probe of the present invention utilizes only the structural change in the ligand-binding region, so in principle it can also detect antagonists (which bind to the ligand-binding region).
• the reaction of the PPAR probe group of the present invention to antagonists was examined.
• antagonists for each PPAR subtype were administered, a reaction was observed in the probes of all subtypes ( Figure 3).
• the antagonist examined this time induced a signal change with a negative rate of change. Since the agonist induced a signal with a positive rate of change, the agonist and antagonist caused signal changes of opposite polarity. It was shown that the method using the fusion protein of the present invention as a probe can also detect antagonists, and it was found to be useful in searching for PPAR ligands, both agonists and antagonists.
• Example 3 Examination of the necessity of the FATT tag for PPAR ligand probe response
• the PPAR probe group of the present invention used in Examples 1 and 2 was tagged with sFATT, a tag that is thought to improve the expression of proteins that tend to form aggregates into the soluble fraction when Escherichia coli is used as the host cell.
• the fusion protein of the present invention enables more accurate detection of PPAR ligands.
• the fusion protein of the present invention can detect PPAR antagonists, which was previously impossible, making it possible to develop new medicines and diagnostic methods.

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