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WO2025174004A1 - Production d'un biocapteur protéique à base de protéase pouvant quantifier spécifiquement le sulfoxyde de méthionine présent dans une protéine cible, et son utilisation - Google Patents

Production d'un biocapteur protéique à base de protéase pouvant quantifier spécifiquement le sulfoxyde de méthionine présent dans une protéine cible, et son utilisation

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Publication number
WO2025174004A1
WO2025174004A1 PCT/KR2025/001907 KR2025001907W WO2025174004A1 WO 2025174004 A1 WO2025174004 A1 WO 2025174004A1 KR 2025001907 W KR2025001907 W KR 2025001907W WO 2025174004 A1 WO2025174004 A1 WO 2025174004A1
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Prior art keywords
protein
fluorescent
methionine
sulfoxide
biosensor
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Korean (ko)
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이병천
황혁주
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to the production of a protease-based protein biosensor capable of specifically quantifying methionine sulfoxide present in a target protein and its use, and more particularly, to a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein composed of a fluorescent protein; Msr (Methionine sulfoxide reductase) protein; an enterokinase cleavage site; a thioredoxin protein; and protein G, a biosensor including the same, and a method for quantifying methionine sulfoxide in a target protein using the same.
  • Msr Methionine sulfoxide reductase
  • ROS reactive oxygen species
  • methionine is an amino acid that is relatively easily oxidized compared to other amino acids because it has a sulfur atom in its side chain. For this reason, methionine always plays a leading role at the forefront of the battle against reactive oxygen species, and since it is not free from protein modification and loss of function due to reactive oxygen species, research has been developed to indirectly confirm the amount of reactive oxygen species by measuring the degree of oxidation of methionine residues.
  • Non-patent Document 0001 When MsrA/MsrB reacts with a protein containing an oxidized methionine residue, the thioredoxin on the other end forms a disulfide bond through the reduction process of MsrA/MsrB. At this time, the distance between MsrA/MsrB and thioredoxin becomes closer, which causes a structural change in cpYFP, resulting in a change in the fluorescence value (Non-patent Document 0001).
  • Non-patent Document 0002 In another study, by attaching an immunoglobulin protein, protein G, together with a linker behind the thioredoxin portion of the fluorescent protein described above, and then attaching an antibody to the target protein, protein G binds to the antibody and recognizes it, thereby producing a method for quantitatively measuring methionine oxidation of a specific protein (Non-patent Document 0002).
  • enterokinase is a proteolytic enzyme produced by cells in the duodenum that recognizes and cleaves specific amino acid sequences. It is involved in digestion in humans, cattle, and other animals, and cleaves amino acid bonds following the Asp-Asp-Asp-Asp-Lys sequence. Because of this sequence-specific cleavage, it is used to cleave various fusion proteins.
  • Non-patent literature 1 Lionel Tarrago et al., Nature Chemical Biology, 11:332-338, 2015
  • Non-patent literature 2 Hae Min Lee et al., ACS Sens, 7(1):131-141, 2022
  • Non-patent literature 3 Guohong Zhang et al., Biochem Biophys Res Commun, 227(3):707-11, 1996
  • the purpose of the present invention is to provide a recombinant protein for a fluorescent biosensor for quantifying methionine-sulfoxide present in a target protein.
  • Another object of the present invention is to provide a biosensor comprising the recombinant protein and a method for quantifying methionine-sulfoxide in a target protein using the same.
  • the present invention provides a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein comprising a fluorescent protein; a methionine sulfoxide reductase (Msr) protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • a fluorescent protein comprising a fluorescent protein; a methionine sulfoxide reductase (Msr) protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • Msr methionine sulfoxide reductase
  • the methionine-sulfoxide is methionine-S-sulfoxide or methionine-R-sulfoxide
  • the recombinant protein for the fluorescent biosensor for the above methionine-S-sulfoxide quantification is composed of a fluorescent protein; an MsrA protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • the recombinant protein for the fluorescent biosensor for quantifying the above methionine-R-sulfoxide may be composed of a fluorescent protein; an Msr B protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • the MsrA protein may be represented by the amino acid sequence of SEQ ID NO: 2
  • the MsrB protein may be represented by the amino acid sequence of SEQ ID NO: 17.
  • the above thioredoxin 1 (Trx1) protein may be represented by the amino acid sequence of SEQ ID NO: 5
  • the above thioredoxin 3 (Trx 3) protein may be represented by the amino acid sequence of SEQ ID NO: 19.
  • the recombinant protein for a fluorescent biosensor for quantifying methionine-S-sulfoxide may be composed of a fluorescent protein; an MsrA protein represented by the amino acid sequence of SEQ ID NO: 2; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin 1 protein represented by the amino acid sequence of SEQ ID NO: 5; and a protein G represented by the amino acid sequence of SEQ ID NO: 7.
  • the present invention provides a polynucleotide encoding a recombinant protein for a fluorescent biosensor for quantifying methionine-sulfoxide present in the target protein.
  • the present invention provides a fluorescent biosensor for quantifying methionine-sulfoxide present in a target protein, comprising a recombinant protein for a fluorescent biosensor for quantifying methionine-sulfoxide present in the target protein.
  • the present invention provides a composition for quantitative detection of methionine-sulfoxide present in a target protein, comprising a recombinant protein for a fluorescent biosensor for quantitative detection of methionine-sulfoxide present in the target protein.
  • the present invention comprises the steps of (a) treating a sample containing a target protein with a primary antibody against the target protein to bind the antibody to the target protein;
  • a method for quantitatively analyzing methionine sulfoxide present in a target protein comprising the step of measuring a fluorescence spectrum value for a recombinant protein for the remaining fluorescent biosensor.
  • the higher the methionine-sulfoxide content in the target protein the higher the fluorescence spectrum value.
  • the method comprises a step of comparing a fluorescence spectrum value for the recombinant protein for the fluorescent biosensor with a fluorescence spectrum value for a normal control group, wherein the fluorescence spectrum value can be calculated as a fluorescence value through the following chemical formulas 1 to 3.
  • the present invention comprises the steps of (a) treating a biological sample separated from a test subject with a primary antibody for a target protein to bind the antibody to the target protein;
  • a method for providing information for diagnosing oxidative stress-related diseases through quantitative analysis of methionine-sulfoxide present in a target protein including a step of measuring the fluorescence spectrum value for the remaining recombinant protein for a fluorescent biosensor and then measuring the fluorescence spectrum value for a normal control group.
  • the sample may be a cell, tissue, blood, plasma, serum, saliva or urine.
  • the fluorescence spectrum value is higher than that of the normal control group, it may provide information that it is an oxidative stress-related disease.
  • the oxidative stress-related disease may be selected from the group consisting of cancer, stroke, myocardial infarction, angina pectoris, arteriosclerosis, infertility, hepatitis, osteoarthritis, acute coronary syndrome, cataract, aging, lipid metabolic disease, heart failure, hypertensive heart disease, arrhythmia, and aging.
  • the present invention comprises the steps of (a) inducing oxidative stress in a sample containing a target protein containing a methionine residue, and then treating the candidate drug;
  • a method for screening a therapeutic agent for an oxidative stress-related disease comprising the step of measuring a fluorescence spectrum value for the remaining recombinant protein for a fluorescent biosensor and then comparing the fluorescence spectrum value with that for a control group that was not treated with the candidate drug.
  • the candidate drug can be selected as a treatment agent for an oxidative stress-related disease.
  • the biosensor according to the present invention can accurately and quantitatively measure the degree of oxidation of methionine residues in a specific protein rather than the entire protein through detection of specific fluorescence changes, and thus can be used as a fluorescent biosensor for diagnosing oxidative stress diseases.
  • the degree of aging can be determined by measuring the degree of oxidation, and thus can be usefully utilized for diagnosing aging and related diseases.
  • Figure 1 is a schematic diagram showing the genetic sequence composition of a recombinant protein for a protease-based fluorescent biosensor capable of quantitatively measuring methionine sulfoxide of a target protein, and a schematic diagram of an expression vector including the genetic sequence thereof.
  • FIG. 2 is a schematic diagram showing the operating principle of the fluorescent biosensor of the present invention.
  • Figure 2a is a schematic diagram showing a fluorescent biosensor in a fully reduced state binding to a target protein and its corresponding antibody.
  • Figure 2b is a schematic diagram showing the process when a fluorescent biosensor reduces methionine-S-sulfoxide (MetSO) of a target protein to methionine (Met), thereby creating a disulfide bond between MsrA and Trx1, and then cutting the cleavage site through enterokinase.
  • MetalSO methionine-S-sulfoxide
  • Figure 2c is a schematic diagram showing the experimental process when applying the characteristics of the fluorescent biosensor of the present invention to an ELISA experiment.
  • Figure 3 is a graph showing the spectroscopic characteristics of the recombinant protein for the fluorescent biosensor of the present invention expressed in E. coli, including the excitation spectrum, emission spectrum, and standard curve for the fluorescence value according to the concentration of the fluorescent biosensor.
  • Figure 3a shows the excitation spectrum results for the wavelength range from 420 nm to 600 nm when 507 nm is fixed as the emission wavelength for the oxidized (blue line) and reduced (pink line) fluorescent biosensor.
  • Figure 3b is an emission spectrum result for the wavelength range from 420 nm to 600 nm when 535 nm light is fixed as the excitation wavelength.
  • Figure 3c is a standard curve for the fluorescence value for the concentration of the fluorescent biosensor, which is a curve drawn using the value of the emission wavelength of 535 nm when light of a wavelength of 485 nm is given as the excitation wavelength.
  • Figure 4 shows the SDS-PAGE results confirming the operation of the cut portion of the fluorescent biosensor.
  • the fluorescent biosensor is approximately 75 kDa in size and is divided into 50 kDa and 25 kDa fragments when cut.
  • Figure 5 is data measuring the degree of methionine oxidation of a target protein when oxidation of the target protein was induced with hydrogen peroxide.
  • Figure 5a is an SDS-PAGE result that confirms the difference in migration according to the degree of oxidation of methionine after treating methionine-rich protein IDLO (Hypothetical protein, Accession number : YP_155605) with hydrogen peroxide at various concentrations with a size of 17 kDa.
  • IDLO methionine-rich protein
  • Figure 5b shows the results of confirming the difference in the degree of oxidation of methionine through changes in the fluorescence value of a fluorescent biosensor after treating IDLO protein with hydrogen peroxide at different concentrations.
  • the present invention relates to a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein, which is composed of a fluorescent protein; a methionine sulfoxide reductase (Msr) protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • Msr methionine sulfoxide reductase
  • the fluorescent protein may be selected from the group consisting of green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), and enhanced cyan fluorescent protein (ECFP).
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • RFP red fluorescent protein
  • ERFP enhanced red fluorescent protein
  • BFP blue fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • YFP yellow fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • CFP cyan fluorescent protein
  • ECFP enhanced cyan fluorescent protein
  • EGFP protein is a protein that has a fluorescence that is approximately 35 times brighter by mutating the chromophore of the GFP protein, and is widely used as a reporter protein because it has greater sensitivity than GFP.
  • Methionine is an amino acid that contains a sulfur element and is easily oxidized by reactive oxygen species to form methionine sulfoxide. At this time, two stereoisomers, methionine-R-sulfoxide and methionine-S-sulfoxide, can be produced.
  • Msr methioninesulfoxide reductase proteins that reduce methionine sulfoxide back to methionine in response to this oxidative stress.
  • MsrA reduces methionine-S-sulfoxide within proteins or in a free state
  • MsrB reduces methionine-R-sulfoxide within proteins.
  • the recombinant protein of the present invention can be produced by selecting Msr protein or Trx protein depending on the type of methionine-sulfoxide.
  • the recombinant protein for a fluorescent biosensor for quantifying methionine-S-sulfoxide may be composed of a fluorescent protein; an MsrA protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G.
  • the recombinant protein for a fluorescent biosensor for quantifying methionine-R-sulfoxide can be composed of a fluorescent protein; Msr B protein; an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; a thioredoxin protein; and protein G, and can quantify methionine-R-sulfoxide present in a target protein.
  • the MsrA protein may be represented by the amino acid sequence of SEQ ID NO: 2
  • the MsrB protein may be represented by the amino acid sequence of SEQ ID NO: 17.
  • the thioredoxin (Trx) protein may be a thioredoxin 1 (Trx1) protein or a thioredoxin 3 (Trx 3) protein
  • Trx1 protein may be represented by the amino acid sequence of SEQ ID NO: 5
  • Trx 3 protein may be represented by the amino acid sequence of SEQ ID NO: 19.
  • protein G may be represented by the amino acid sequence of SEQ ID NO: 7.
  • yeast-derived MsrA, a linker, and an enterokinase cleavage site were linked to the C-terminus of enhanced green fluorescent protein (EGFP), and then yeast-derived thioredoxin 1 (Trx1) and streptococcus-derived protein G were sequentially linked (Fig. 1).
  • EGFP enhanced green fluorescent protein
  • Trx1 and streptococcus-derived protein G were sequentially linked (Fig. 1).
  • Yeast-derived MsrA reduces methionine-S-sulfoxide, an oxide of methionine present in proteins, and Cys25 and Cys176 present in MsrA are oxidized to form a disulfide bond.
  • Trx1 plays a role in reducing MsrA that has been oxidized by methionine-S-sulfoxide. At this time, Cys30 present in Trx1 attacks and reduces the disulfide bond formed in MsrA, and then a new disulfide bond is created between Cys25 of MsrA and Cys30 of Trx1. Originally, Trx1 restores the function of MsrA by breaking the disulfide bond formed between MsrA and Trx1 at Cys33, but through genetic manipulation, cysteine at position 33 was changed to serine to prevent this process from occurring.
  • the two linkers were designed to allow MsrA to stably act on the substrate, and the former was designed to work well with Trx1.
  • Protein G is an immunoglobulin-binding protein that binds to the Fab and Fc regions of mammalian antibodies of the IgG type, making it a useful protein for purifying antibodies.
  • an antibody that specifically binds to a target protein is attached, and then a biosensor is bound to the antibody using protein G, enabling it to react with the target protein.
  • EGFP protein is a fluorescent protein and serves as a reporter protein in the present invention. Methionine oxidation of a protein can be quantitatively measured by measuring the final level of EGFP fluorescence.
  • the enterokinase cleavage site between MsrA and Trx1 allows each biosensor region to be linked and expressed.
  • the enterokinase cleavage site is cleaved.
  • MsrA and Trx1 form a disulfide bond and remain connected, whereas in the unoxidized biosensor, they are cleaved, and the EGFP and MsrA in front of the cleavage site are separated.
  • the fluorescence level of the remaining EGFP was measured to quantitatively determine the methionine oxidation of the target protein (Fig. 2).
  • EGFP SEQ ID NO: 9
  • yeast-derived MsrA SEQ ID NO: 10
  • the linker SEQ ID NO: 11
  • enterokinase cleavage site SEQ ID NO: 12
  • yeast-derived Trx1 SEQ ID NO: 13
  • another linker SEQ ID NO: 14
  • protein G SEQ ID NO: 15
  • the present invention relates to a polynucleotide encoding a recombinant protein for a fluorescent biosensor for quantifying methionine-sulfoxide present in the target protein.
  • polynucleotide generally refers to a nucleic acid molecule, deoxyribonucleotide or ribonucleotide, or an analog thereof, separated into any length.
  • the polynucleotide of the present invention can be prepared by (1) in vitro amplification such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis separation; and (4) synthetically such as chemical synthesis, and preferably, the isolated polynucleotide is prepared by recombinant DNA technology.
  • PCR polymerase chain reaction
  • the present invention relates to a recombinant vector comprising the polynucleotide.
  • the "vector” used in the present invention refers to a gene construct that is an expression vector capable of expressing a target protein in a suitable host cell and includes essential regulatory elements operably linked to enable expression of a gene insert.
  • operably linked means that a gene requiring expression and its regulatory sequence are functionally linked to each other to enable gene expression
  • a "regulatory element” includes a promoter for performing transcription, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • Such vectors may be, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc., and one or more known vectors may be used as long as they can express the above-mentioned genes.
  • the vector may be pNB181 (pET22B(+) based plasmid; AmpR ampicillin selection marker; T7 promoter/lac operator/ribosome binding site (RBS)/T7 transcription terminator).
  • the "recombinant vector” used in the present invention after being transformed into a suitable host cell, can replicate independently of the host cell's genome or can be incorporated into the genome itself.
  • the "suitable host cell” may include an origin of replication, a specific base sequence from which replication is initiated, as long as the vector is replicable.
  • the present invention relates to a polynucleotide encoding a recombinant protein for the fluorescent biosensor, or a recombinant strain transformed with a recombinant vector comprising the polynucleotide.
  • the term "recombinant strain” means a cell transformed by introducing a vector having a polynucleotide encoding one or more target proteins into a host cell as a transformant
  • methods for producing a transformant by introducing an expression vector into a host cell include the calcium phosphate method or the calcium chloride/rubidium chloride method described in the literature (Sambrook, J., et al. , Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989), electroporation, electroinjection, chemical treatment methods such as PEG, and methods using a gene gun, etc.
  • the host cell may be a mammalian cell or a bacterium, and preferably may be any one selected from the group consisting of Escherichia spp. bacteria, Bacillus spp. bacteria, Corynebacterium spp. bacteria, cyanobacteria spp. bacteria, Schizosaccharomyces spp. yeast, Kluyveromyces spp. yeast, and fungi. More preferably, in the present invention, E. coli, which is a bacterium of the genus Escherichia, was used.
  • the present invention relates to a fluorescent biosensor for quantifying methionine-sulfoxide present in a target protein, comprising a recombinant protein for a fluorescent biosensor for quantifying methionine-sulfoxide present in the target protein.
  • the present invention relates to a composition for quantitative detection of methionine-sulfoxide present in a target protein, comprising a recombinant protein for a fluorescent biosensor for quantitative detection of methionine-sulfoxide present in the target protein.
  • the present invention comprises the steps of: (a) treating a sample containing a target protein with a primary antibody against the target protein to bind the antibody to the target protein;
  • the higher the methionine-sulfoxide content in the target protein the higher the fluorescence spectrum value can be, and a step of comparing the fluorescence spectrum value for the recombinant protein for the fluorescent biosensor with the fluorescence spectrum value for a normal control group is included, wherein the fluorescence spectrum value can be calculated as a fluorescence value through the following chemical formulas 1 to 3.
  • the methionine-sulfoxide content in the target protein can be quantified using the following chemical formula and standard curve.
  • the present invention comprises the steps of: (a) treating a biological sample separated from a test subject with a primary antibody against a target protein to bind the antibody to the target protein;
  • a method for providing information for diagnosing oxidative stress-related diseases through quantitative analysis of methionine-sulfoxide present in a target protein comprising the steps of measuring the fluorescence spectrum value for the remaining recombinant protein for a fluorescent biosensor and then measuring the fluorescence spectrum value for a normal control group.
  • the sample may be a cell, tissue, blood, plasma, serum, saliva or urine.
  • the fluorescence spectrum value if it is higher than that of the normal control group, it can provide information that it is an oxidative stress-related disease, and the fluorescence spectrum value can be measured by the method described in the above ⁇ Methionine-sulfoxide quantitative analysis method>.
  • the oxidative stress-related disease may be selected from the group consisting of cancer, stroke, myocardial infarction, angina pectoris, arteriosclerosis, infertility, hepatitis, osteoarthritis, acute coronary syndrome, cataract, aging, lipid metabolic disease, heart failure, hypertensive heart disease, arrhythmia, and aging.
  • the present invention comprises the steps of: (a) inducing oxidative stress in a sample comprising a target protein containing a methionine residue, and then treating the sample with a candidate drug;
  • a method for screening a therapeutic agent for an oxidative stress-related disease comprising the step of measuring a fluorescence spectrum value for the remaining recombinant protein for a fluorescent biosensor and then comparing the fluorescence spectrum value with that for a control group that was not treated with the candidate drug.
  • the candidate drug can be selected as a treatment agent for an oxidative stress-related disease, and the fluorescence spectrum value can be measured by the method described in the above ⁇ Methionine-sulfoxide quantitative analysis method>.
  • Example 1 Preparation of recombinant proteins for fluorescent biosensors
  • [pEGFP-N1 plasmid including a gene encoding EGFP (SEQ ID NO: 9)] [pUC57-amp plasmid including a gene encoding MsrA (SEQ ID NO: 10), a gene encoding linker 1 (SEQ ID NO: 11), and a gene encoding an enterokinase cleavage site (SEQ ID NO: 12)]
  • [tpMetSOG in pET His6 TEV LIC cloning vector including a gene encoding thioredoxin 1 (Trx1) (SEQ ID NO: 13), a gene encoding linker 2 (SEQ ID NO: 14), and a gene encoding protein G (SEQ ID NO: 15)] were prepared for the genetic recombination process.
  • Each plasmid was amplified using PCR (polymerase chain reaction) as a template, and three genes were obtained, including EGFP, MsrA/linker/enterokinase cleavage site, and Trx1/linker/protein G.
  • the above process was performed by adding sterile water, 10X PCR buffer, dNTPs, forward/reverse primers for each template (Table 1), and nPfu polymerase in order of increasing amount to a 0.2 ml PCR tube, and then spinning down at 100 x g for about 3 seconds using a centrifuge to remove any substances adhering to the tube wall. After gently mixing the substances well, PCR was performed according to the experimental protocol. The PCR reaction conditions for each gene part were as follows: initial denaturation at 95 ° C for 2 minutes, followed by 32 cycles of denaturation at 95 ° C for 30 seconds, annealing at 54 ° C for 45 seconds, and elongation for 42 seconds, and then elongation at 72 ° C for 5 minutes.
  • Primer sequence Primer sequence (5' -> 3') Sequence number EGFP Forward AAAAAGCTAGCATGGTGAGCAAGGGCGAG Sequence number 21 Reverse GCGACGACATCTTGTACAGC Sequence number 22 MsrA Forward GCTGTACAAGATGTCGTCGC Sequence number 23 Reverse AGCAATTGCAGAGTCGAATTCG Sequence number 24 trx1 Forward ATGGTTACTCAATTCAAAACTGCCAG Sequence number 25 Reverse ACTCGAGTTCAGTAACTGTAAAGGTCTTAGTCGCA Sequence number 26
  • Trx1 1/linker/protein G portion was inserted into the MCS of the pET21a plasmid through a genetic cloning process. Then, the EGFP gene and the MsrA/linker/enterokinase cleavage site gene were sequentially linked through a genetic recombination process and inserted into the pET21a plasmid into which Trx1/linker/protein G had been previously inserted, finally connecting all genes (SEQ ID NO: 16). Finally, the base sequence of the recombinant plasmid was analyzed and confirmed, and it was named "pGACTLG.”
  • strains having a fluorescent biosensor protein were secured through a process of transforming the prepared E. coli strain with the above pGACTLG vector and then inducing expression.
  • the protein was eluted using an elution solution (50 mM Tris-HCl, 150 mM NaCl, 2 mM beta mercaptoethanol, 500 mM imidazole pH 8) with a gradient of imidazole concentration from 0 to 500 mM.
  • the presence or absence of protein was confirmed by SDS-PAGE using a 12% polyacrylamide gel, and then concentrated using a 50 ml centrifugal filter.
  • Example 2 Operation verification and fluorescence characteristic analysis of a fluorescent biosensor
  • the purified fluorescent biosensor protein is diluted to a concentration of 20 ⁇ M, then treated with DTT (dithiothreitol), a substance that reduces and breaks disulfide bonds, to a concentration of 10 mM (final concentration) and reacted at room temperature for 30 minutes. Through this process, the fluorescent biosensor is completely reduced.
  • DTT dithiothreitol
  • the reduced fluorescent biosensor was diluted to a concentration of 5 ⁇ M, and then the reduced fluorescent biosensor was treated with the oxidizing agent N-acetyl methionine sulfoxide and the reducing agent GSH, respectively, and reacted at 37°C for 30 min. After the reaction, the fluorescence spectra of the completely oxidized and reduced fluorescent biosensors were measured. The emission value was set at 507 nm, and the excitation spectrum was measured. For the emission spectrum, the excitation wavelength was measured at 535 nm, which had the highest value in the excitation spectrum (Fig. 3a and Fig. 3b).
  • the fluorescent protein diluted to 5 ⁇ M was serially diluted and the fluorescence for each dilution was measured.
  • the emission wavelength of 535 nm was measured at an excitation wavelength of 485 nm, and the concentration value of the dilution was corrected using Nanodrop for more accurate concentration measurement.
  • a standard curve according to the fluorescence value according to the concentration of the fluorescent biosensor was obtained (Fig. 3c).
  • the fluorescent biosensor was prepared in a completely reduced state using DTT at a concentration of 5 ⁇ M.
  • N-acetyl methionine sulfoxide was treated to a final concentration of 100 ⁇ M, 50 ⁇ M, 10 ⁇ M, and 5 ⁇ M, and the reaction was incubated at 37°C for 30 minutes.
  • 1 ⁇ g of enterokinase was treated and reacted for another 30 minutes.
  • SDS-PAGE was performed using SDS sample buffer, which does not affect disulfide bonds.
  • a reaction was prepared by treating glutathione at a final concentration of 5 mM.
  • the SDS-PAGE results show that as the concentration of N-acetyl methionine sulfoxide decreases, the amount of the truncated fluorescent biosensor (approximately 50 kDa in size) increases. In other words, it was confirmed that the fluorescent biosensor of the present invention was cleaved by enterokinase (Fig. 4).
  • IDLO oxidation level of a specific protein
  • purified IDLO protein was prepared at 1 mM and reacted with hydrogen peroxide at concentrations of 0 mM, 10 mM, 20 mM, 30 mM, and 40 mM for 2 hours at room temperature to vary the degree of oxidation. After confirming that the oxidized IDLO protein migrated differently on SDS-PAGE (Fig. 5a), the degree of oxidation was measured using the oxidized methionine quantification method using 5 ⁇ M of the fluorescent biosensor of the present invention.
  • the biosensor according to the present invention can accurately and quantitatively measure the degree of oxidation of methionine residues in a specific protein, and thus can be used as a fluorescent biosensor for diagnosing oxidative stress diseases.
  • the degree of aging can be determined by measuring the degree of oxidation, and thus can be usefully utilized for diagnosing aging and related diseases.

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Abstract

La présente invention concerne la production d'un biocapteur protéique à base de protéase pouvant quantifier spécifiquement le sulfoxyde de méthionine présent dans une protéine cible et son utilisation, et plus précisément : une protéine recombinante pour un biocapteur fluorescent permettant de quantifier le sulfoxyde de méthionine présent dans une protéine cible, la protéine recombinante comprenant une protéine fluorescente, une protéine du type méthionine sulfoxyde réductase (Msr), un site de clivage d'entérokinase, une protéine du type thiorédoxine et une protéine G ; un biocapteur la comprenant ; et un procédé de quantification de sulfoxyde de méthionine dans une protéine cible l'utilisant. Le biocapteur selon la présente invention peut mesurer avec exactitude et de manière quantitative le degré d'oxydation de résidus méthionine dans une protéine spécifique plutôt que dans toutes les protéines par le biais de la détection de changements de fluorescence spécifiques, et peut ainsi être utilisé en tant que biocapteur fluorescent pour diagnostiquer des maladies liées au stress oxydatif. De plus, le biocapteur peut reconnaître le degré de vieillissement par le biais de la mesure du degré d'oxydation, et peut ainsi être efficacement utilisé pour diagnostiquer le vieillissement et des maladies associées.
PCT/KR2025/001907 2024-02-16 2025-02-10 Production d'un biocapteur protéique à base de protéase pouvant quantifier spécifiquement le sulfoxyde de méthionine présent dans une protéine cible, et son utilisation Pending WO2025174004A1 (fr)

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EP1441030A1 (fr) * 2001-10-11 2004-07-28 Katakura Industries Co., Ltd. Procede de purification de proteine fusionnee recombinee et procede de production de proteine utilisant ledit procede de purification
JP2007508014A (ja) * 2003-10-10 2007-04-05 プロメガ　コーポレイション ルシフェラーゼバイオセンサー
KR20220043971A (ko) * 2020-09-28 2022-04-06 고려대학교 산학협력단 특정 단백질의 메티오닌 잔기의 산화정도를 정량적으로 측정할 수 있는 형광단백질 센서 및 이의 용도
KR20220064486A (ko) * 2020-11-11 2022-05-19 고려대학교 산학협력단 유리 메티오닌­ｒ­설폭사이드를 정량적으로 측정할 수 있는 형광단백질 센서 및 이의 용도
KR20230006225A (ko) * 2021-07-02 2023-01-10 강원대학교산학협력단 발현 수준 및 가용성이 증대된 인간 엔테로키나제 융합 단백질 및 그 제조방법

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EP1441030A1 (fr) * 2001-10-11 2004-07-28 Katakura Industries Co., Ltd. Procede de purification de proteine fusionnee recombinee et procede de production de proteine utilisant ledit procede de purification
JP2007508014A (ja) * 2003-10-10 2007-04-05 プロメガ　コーポレイション ルシフェラーゼバイオセンサー
KR20220043971A (ko) * 2020-09-28 2022-04-06 고려대학교 산학협력단 특정 단백질의 메티오닌 잔기의 산화정도를 정량적으로 측정할 수 있는 형광단백질 센서 및 이의 용도
KR20220064486A (ko) * 2020-11-11 2022-05-19 고려대학교 산학협력단 유리 메티오닌­ｒ­설폭사이드를 정량적으로 측정할 수 있는 형광단백질 센서 및 이의 용도
KR20230006225A (ko) * 2021-07-02 2023-01-10 강원대학교산학협력단 발현 수준 및 가용성이 증대된 인간 엔테로키나제 융합 단백질 및 그 제조방법

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