Production and use of protease-based protein biosensors capable of specifically quantifying methionine sulfoxide present in target proteins

Abstract

This invention relates to the manufacture and use of a protease-based protein biosensor capable of specifically quantifying methionine sulfoxide present in a target protein, and more specifically, to: a recombinant protein for quantifying methionine sulfoxide present in a target protein, the recombinant protein comprising a fluorescent protein, a methionine sulfoxide reductase (Msr) protein, an enterokinase cleavage site, a thioredoxin, and protein G; a biosensor comprising the recombinant protein; and a method for using the recombinant protein to quantify methionine sulfoxide in a target protein. The biosensor according to the invention can accurately measure the degree of oxidation of methionine residues in a specific protein, rather than the degree of oxidation of all proteins, by detecting specific fluorescence changes, and therefore can be used as a fluorescent biosensor for diagnosing oxidative stress diseases. Furthermore, the sensor can identify the degree of aging by measuring the degree of oxidation, and thus can be effectively used for diagnosing aging and related diseases.

Description

Technical Field

[0001] This invention relates to the development and use of protease-based protein biosensors capable of specifically quantifying methionine sulfoxide present in target proteins, and more specifically, to recombinant proteins for quantifying methionine sulfoxide present in target proteins, said recombinant proteins comprising a fluorescent protein, Msr (methionine sulfoxide reductase) protein, an enterokinase cleavage site, thioredoxin, and protein G, to biosensors comprising said recombinant proteins, and to methods for using said recombinant proteins to quantify methionine sulfoxide in target proteins. Background Technology

[0002] Organisms are constantly exposed to the dangers of reactive oxygen species (ROS), generated through internal metabolic processes or external environmental stress. The problem with these ROS is that they damage macromolecules within the body, thereby triggering disease or accelerating aging. Representative examples of such macromolecules include DNA, lipids, and proteins, which are directly damaged by ROS. The level of ROS in the body is crucial information not only in aging but also in the diagnosis and monitoring of various diseases, such as cancer and cardiovascular disease.

[0003] Therefore, research was conducted on techniques for measuring the amount of reactive oxygen species (ROS) in vivo, and methods using fluorescent dyes (e.g., DCFDA and DHE staining) were developed. However, because ROS are generated instantaneously and react immediately with other macromolecules in vivo, there are limitations to directly measuring the amount of ROS in general biological samples other than living cells.

[0004] As another approach, methods have been developed to measure the accumulation of substances oxidized by reactive oxygen species (ROS). Methionine, in particular, is a relatively easily oxidized amino acid compared to others due to the presence of sulfur atoms in its side chain. Therefore, methionine consistently plays a leading role in the fight against ROS, and because it is not immune to protein modifications and loss of function caused by ROS, research has been conducted to indirectly determine the amount of ROS by measuring the degree of oxidation of methionine residues.

[0005] Prior to this, studies aimed at measuring methionine oxidation were conducted using LC-MS / MS and GC-MS / MS, and more recently, studies using fluorescent biosensors to measure methionine oxidation have been published. These techniques are briefly described as follows: they all utilize cpYFP (circularly arranged yellow fluorescent protein) proteins whose fluorescence value varies according to structural changes. A yeast or bacterial methionine sulfoxide reductase (i.e., MsrA / MsrB) is attached to one side of this fluorescent protein, while a thioredoxin (Trx) protein is attached to the other side. When MsrA / MsrB reacts with a protein containing oxidized methionine residues, the thioredoxin on the other side forms a disulfide bond through the reduction process of MsrA / MsrB. At this point, the distance between MsrA / MsrB and the thioredoxin decreases, causing a structural change in cpYFP and thus a change in fluorescence value (Non-Patent Literature 0001). In another study, protein G (an immunoglobulin) was linked to a linker following the thioredoxin portion of the aforementioned fluorescent protein, so that after an antibody is attached to a target protein, protein G binds to and recognizes the antibody, thereby enabling quantitative measurement of methionine oxidation in a specific protein (Non-Patent Document 0002).

[0006] Meanwhile, enterokinase is a proteolytic enzyme that recognizes and cleaves specific amino acid sequences, and is produced by cells present in the duodenum. It is an enzyme involved in digestion in humans and other animals (such as cattle) and is used to cleave the amino acid bonds following the Asp-Asp-Asp-Asp-Lys sequence. Due to this sequence-specific cleavage, it is used for the cleavage of various fusion proteins.

[0007] Existing technical documents

[0008] Non-patent literature 1: Lionel Tarrago et al., Nature Chemical Biology, 11:332-338, 2015

[0009] Non-patent literature 2: Hae Min Lee et al., ACS Sens, 7(1):131-141, 2022

[0010] Non-patent literature 3: Guohong Zhang et al., Biochem Biophys Res Commun, 227(3):707-11, 1996 Summary of the Invention

[0011] [Technical Issues]

[0012] Therefore, as a result of the inventors' extensive efforts to develop a biosensor capable of quantifying methionine oxidation of target proteins with higher sensitivity than conventional biosensors, the inventors have prepared a recombinant protein for quantifying methionine sulfoxide present in target proteins in a further improved form by applying the techniques described above. The recombinant protein comprises a fluorescent protein, Msr (methionine sulfoxide reductase) protein, an enterokinase cleavage site, thioredoxin, and protein G, thereby completing the present invention.

[0013] Therefore, the objective of this invention is to provide a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in target proteins.

[0014] Another objective of the present invention is to provide a biosensor comprising the recombinant protein, and a method for using the recombinant protein to quantify methionine sulfoxide in a target protein.

[0015] [Technical means]

[0016] To achieve the above-mentioned objectives, the present invention provides a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein, the recombinant protein comprising a fluorescent protein, Msr (methionine sulfoxide reductase) protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

[0017] According to a preferred embodiment of the present invention, methionine sulfoxide is methionine-S-sulfoxide or methionine-R-sulfoxide.

[0018] The recombinant protein for a fluorescent biosensor used to quantify methionine-S-sulfoxide comprises a fluorescent protein, MsrA protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

[0019] The recombinant protein used for quantifying methionine-R-sulfoxide fluorescent biosensors may include fluorescent protein, MsrB protein, enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

[0020] According to another preferred embodiment of the present invention, the MsrA protein can be represented by the amino acid sequence of SEQ ID NO: 2, while the MsrB protein can be represented by the amino acid sequence of SEQ ID NO: 17.

[0021] According to another preferred embodiment of the invention, 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).

[0022] According to another preferred embodiment of the present invention, thioredoxin (Trx) can be thioredoxin 1 (Trx1) or thioredoxin 3 (Trx3).

[0023] Thioredoxin 1 (Trx1) can be represented by the amino acid sequence of SEQ ID NO: 5, while thioredoxin 3 (Trx3) can be represented by the amino acid sequence of SEQ ID NO: 19.

[0024] According to another preferred embodiment of the present invention, the recombinant protein for quantifying methionine-S-sulfoxide fluorescent biosensor may comprise a fluorescent protein, 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, thioredoxin 1 represented by the amino acid sequence of SEQ ID NO: 5, and protein G represented by the amino acid sequence of SEQ ID NO: 7.

[0025] In addition, the present invention provides a polynucleotide that encodes a recombinant protein for a fluorescent biosensor used to quantify methionine sulfoxide present in a target protein.

[0026] Furthermore, the present invention provides a recombinant vector containing the polynucleotide, and a recombinant strain transformed with the recombinant vector.

[0027] To achieve another objective, the present invention provides a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein, the fluorescent biosensor comprising a recombinant protein for quantifying methionine sulfoxide present in a target protein.

[0028] Furthermore, the present invention provides a composition for the quantitative detection of methionine sulfoxide present in a target protein, the composition comprising a recombinant protein of a fluorescent biosensor for the quantitative detection of methionine sulfoxide present in a target protein.

[0029] Furthermore, the present invention provides a method for quantitatively analyzing methionine sulfoxide present in a target protein, the method comprising: (a) treating a sample containing the target protein with a primary antibody against the target protein to cause the antibody to bind to the target protein;

[0030] (b) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0031] (c) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0032] (d) Measure the fluorescence spectra of the remaining recombinant proteins used for the fluorescent biosensor.

[0033] According to a preferred embodiment of the present invention, the higher the content of methionine sulfoxide in the target protein, the higher the value of the fluorescence spectrum.

[0034] According to another preferred embodiment of the present invention, the method includes the step of comparing the fluorescence spectral value of the recombinant protein used for the fluorescent biosensor with the fluorescence spectral value of a normal control group, wherein the fluorescence spectral value can be calculated as a fluorescence value using the following formulas 1 to 3:

[0035] [Formula 1]

[0036] Fluorescence value = Ex:535 nm / Em:507 nm = value at 535 nm of the excitation spectrum (when the emission wavelength is fixed at 507 nm);

[0037] [Formula 2]

[0038] Fluorescence value = Ex:485 nm / Em:535 nm = value at 485 nm of the excitation spectrum (when the emission wavelength is fixed at 535 nm);

[0039] [Formula 3]

[0040] Fluorescence value = Ex:280 nm / Em:510 nm = value at 280 nm of the excitation spectrum (when the emission wavelength is fixed at 510 nm).

[0041] Furthermore, the present invention provides a method for providing information for the diagnosis of oxidative stress-related diseases by quantitative analysis of methionine sulfoxide present in target proteins, the method comprising: (a) treating a biological sample isolated from a subject with a primary antibody against the target protein to enable the antibody to bind to the target protein;

[0042] (b) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0043] (c) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0044] (d) Measure the fluorescence spectrum of the remaining recombinant protein used for the fluorescent biosensor and then compare it with the fluorescence spectrum of the normal control group.

[0045] According to a preferred embodiment of the present invention, the sample may be cells, tissues, blood, plasma, serum, saliva, or urine.

[0046] According to another preferred embodiment of the invention, if the fluorescence spectral value is higher than that of the normal control group, it can provide information indicating oxidative stress-related diseases.

[0047] According to another preferred embodiment of the invention, oxidative stress-related diseases may be selected from the group consisting of: cancer, stroke, myocardial infarction, angina pectoris, arteriosclerosis, infertility, hepatitis, osteoarthritis, acute coronary syndrome, cataracts, aging, lipid metabolism disorders, heart failure, hypertensive heart disease, arrhythmia, and aging.

[0048] Furthermore, the present invention provides a method for screening therapeutic agents for oxidative stress-related diseases, the method comprising: (a) inducing oxidative stress in a sample containing a target protein containing methionine residues, and then treating the sample with a candidate drug;

[0049] (b) Treat the sample with a primary antibody targeting the target protein to allow the antibody to bind to the target protein;

[0050] (c) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0051] (d) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0052] (e) Measure the fluorescence spectrum of the remaining recombinant protein for the fluorescent biosensor and then compare it with the fluorescence spectrum of the control group that was not treated with the candidate drug.

[0053] According to a preferred embodiment of the present invention, if the fluorescence spectrum value obtained after treatment with the candidate drug is lower than the fluorescence spectrum value of the control group, the candidate drug can be selected as a therapeutic agent for oxidative stress-related diseases.

[0054]

Beneficial Effects of the Invention

[0055] The biosensor according to the invention can accurately and quantitatively measure the degree of oxidation of methionine residues in a specific protein, rather than the degree of oxidation of methionine residues throughout the protein, by detecting specific fluorescence changes, and can therefore be used as a fluorescent biosensor for the diagnosis of oxidative stress diseases. Furthermore, by measuring the degree of oxidation to determine the progression of aging, it can be usefully used for the diagnosis of aging and related diseases. Attached Figure Description

[0056] Figure 1 A schematic diagram illustrating the gene sequence configuration of a protease-based recombinant protein for use in a fluorescent biosensor, and a schematic diagram of an expression vector containing the corresponding gene sequence, is provided. The fluorescent biosensor is capable of quantitatively measuring methionine sulfoxide of a target protein.

[0057] Figure 2 is a schematic diagram illustrating the working principle of the fluorescent biosensor of the present invention.

[0058] Figure 2a This diagram illustrates the binding of a fluorescent biosensor to its target protein and corresponding antibody in a fully reduced state.

[0059] Figure 2b This diagram illustrates the process by which a fluorescent biosensor reduces methionine-S-sulfoxide (MetSO) of a target protein to methionine (Met), thereby forming a disulfide bond between MsrA and Trx1, which is then cleaved at the cleavage site by enterokinase.

[0060] Figure 2c This diagram illustrates the experimental procedure for applying the characteristics of the fluorescent biosensor of the present invention to an ELISA experiment.

[0061] Figure 3 shows the excitation and emission spectra, which are the spectral characteristics of the recombinant protein of the present invention for a fluorescent biosensor expressed in Escherichia coli, and a standard curve of fluorescence values ​​based on the concentration of the fluorescent biosensor.

[0062] Figure 3a The excitation spectra of the oxidized (blue line) and reduced (pink line) fluorescent biosensors in the wavelength range of 420 nm to 600 nm are shown when the emission wavelength is fixed at 507 nm.

[0063] Figure 3b The emission spectra in the wavelength range of 420 nm to 600 nm are shown when 535 nm light is fixed as the excitation wavelength.

[0064] Figure 3cThis is a standard curve of fluorescence value relative to the concentration of the fluorescent biosensor, and is a curve plotted using the emission wavelength of 535 nm when 485 nm light is used as the excitation wavelength.

[0065] Figure 4 The SDS-PAGE results are shown, confirming the operation of the cleavage site of the fluorescent biosensor. The fluorescent biosensor is approximately 75 kDa in size and, when cleaved, splits into 50 kDa and 25 kDa portions.

[0066] Figure 5 shows data obtained by measuring the degree of methionine oxidation in the target protein when hydrogen peroxide was used to induce oxidation of the target protein.

[0067] Figure 5a The SDS-PAGE results show the migration differences of the 17 kDa methionine-rich protein IDLO (hypothetical protein, accession number: YP_155605) after treatment with different concentrations of hydrogen peroxide, based on the degree of methionine oxidation.

[0068] Figure 5b The results show the differences in the degree of methionine oxidation confirmed by changes in fluorescence values ​​of a fluorescent biosensor after treating IDLO protein with different concentrations of hydrogen peroxide. Detailed Implementation

[0069] The present invention will be described in detail below.

[0070] Recombinant protein for a fluorescent biosensor to quantify methionine sulfoxide

[0071] In one aspect, the present invention relates to a recombinant protein for a fluorescent biosensor for quantifying the presence of methionine sulfoxide in a target protein, said recombinant protein comprising a fluorescent protein, Msr (methionine sulfoxide reductase) protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

[0072] In this invention, 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).

[0073] In this invention, enhanced green fluorescent protein (EGFP) is used. EGFP is a protein that exhibits approximately 35 times brighter fluorescence by introducing a mutation into the chromophore of the GFP protein, and is widely used as a reporter protein due to its higher sensitivity than GFP.

[0074] In this invention, when methionine sulfoxide is methionine-S-sulfoxide, the Msr protein is MsrA protein, and when methionine sulfoxide is methionine-R-sulfoxide, the Msr protein can be MsrB protein.

[0075] Methionine is a sulfur-containing amino acid that is readily oxidized by reactive oxygen species to form methionine sulfoxide. This produces two stereoisomers: methionine-R-sulfoxide and methionine-S-sulfoxide. In vivo, several types of Msr (methionine sulfoxide reductase) proteins exist that respond to this oxidative stress by reducing methionine sulfoxide back to methionine. MsrA reduces methionine-S-sulfoxide in the protein or in a free state, while MsrB reduces methionine-R-sulfoxide in the protein.

[0076] Therefore, the recombinant protein of the present invention can be prepared by selecting Msr protein or Trx protein according to the type of methionine sulfoxide.

[0077] Specifically, the recombinant protein for quantifying methionine-S-sulfoxide fluorescent biosensors may comprise a fluorescent protein, MsrA protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

[0078] In addition, the recombinant protein of the fluorescent biosensor for quantifying methionine-R-sulfoxide may include a fluorescent protein, MsrB protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G, and may quantify methionine-R-sulfoxide present in the target protein.

[0079] More specifically, the MsrA protein can be represented by the amino acid sequence of SEQ ID NO: 2, while the MsrB protein can be represented by the amino acid sequence of SEQ ID NO: 17.

[0080] In this invention, thioredoxin (Trx) can be thioredoxin 1 (Trx1) or thioredoxin 3 (Trx3). Trx1 protein can be represented by the amino acid sequence of SEQ ID NO: 5, while Trx3 protein can be represented by the amino acid sequence of SEQ ID NO: 19. Furthermore, protein G can be represented by the amino acid sequence of SEQ ID NO: 7.

[0081] In a specific embodiment of the present invention, yeast-derived MsrA, a linker, and an enterokinase cleavage site are ligated to the C-terminus of EGFP, and then yeast-derived thioredoxin 1 (Trx1) and streptococcal-derived protein G (…) are sequentially ligated. Figure 1 ).

[0082] Yeast-derived MsrA reduces methionine-S-sulfoxide (methionine oxide present in proteins), while Cys25 and Cys176 present in MsrA are oxidized to form disulfide bonds.

[0083] Trx1 is used to reduce MsrA, which has been oxidized by methionine-S-sulfoxide, back to its reduced form. At this point, Cys30 present in Trx1 attacks and reduces the disulfide bonds formed in MsrA, subsequently forming a new disulfide bond between Cys25 of MsrA and Cys30 of Trx1. In the initial Trx1, Cys33 cleaves the disulfide bond formed between MsrA and Trx1 to restore the function of MsrA; however, through genetic manipulation, cysteine ​​33 is converted to serine, preventing this process from occurring.

[0084] For the linker between MsrA and Trx1 and the linker between Trx1 and protein G, both linkers are designed to enable MsrA to act stably on its substrate, and the former is designed to interact well with Trx1.

[0085] Protein G is an immunoglobulin-binding protein, and because it can bind to the Fab and Fc regions of IgG type antibodies present in mammals, it is used for antibody purification. In this invention, after attaching an antibody that specifically binds to the target protein, this protein G is used to bind a biosensor to the antibody, enabling it to react with the target protein.

[0086] EGFP protein is a fluorescent protein and functions as a reporter protein in this invention. The oxidation of methionine in the protein can be quantitatively measured by measuring the final degree of fluorescence of EGFP.

[0087] The enterokinase cleavage site located between MsrA and Trx1 allows the various parts of the biosensor to be expressed as a connected unit. When the biosensor reacts with a substrate and is then treated with enterokinase, the enterokinase cleavage site is cleaved. In the oxidized biosensor, MsrA and Trx1 form a disulfide bond and thus remain connected, while in the non-oxidized biosensor, cleavage occurs, causing the EGFP preceding the cleavage site and MsrA to separate and be released. Methionine oxidation of the target protein is quantitatively measured by measuring the fluorescence intensity of the remaining EGFP (Figure 2).

[0088] Using gene recombination technology, the above-mentioned protein gene (SEQ ID NO: 9) was linked into a long sequence and then inserted into the pET-21a and pET-28a cloning vectors. Based on the nucleotide sequence represented by SEQ ID NO: 16, EGFP (SEQ ID NO: 9) has a nucleotide sequence of 1 bp to 717 bp, yeast-derived MsrA (SEQ ID NO: 10) has a nucleotide sequence of 718 bp to 1269 bp, the linker (SEQ ID NO: 11) and enterokinase cleavage site (SEQ ID NO: 12) have a nucleotide sequence of 1270 bp to 1359 bp, yeast-derived Trx1 (SEQ ID NO: 13) has a nucleotide sequence of 1360 bp to 1668 bp, another linker (SEQ ID NO: 14) has a nucleotide sequence of 1669 bp to 1767 bp, and protein G (SEQ ID NO: 15) has a nucleotide sequence of 1768 bp to 1929 bp.

[0089] In another aspect, the present invention relates to polynucleotides encoding recombinant proteins for use in a fluorescent biosensor to quantify methionine sulfoxide present in a target protein.

[0090] In this invention, the term "polynucleotide" generally refers to an isolated nucleic acid molecule, deoxyribonucleotide, or ribonucleotide of any length or an analogue thereof. In some embodiments, the polynucleotides 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) synthesis, such as chemical synthesis, and preferably, preparation of isolated polynucleotides by recombinant DNA technology.

[0091] In another respect, the present invention relates to recombinant vectors containing the polynucleotide.

[0092] As used in this invention, "vector" refers to an expression vector capable of expressing a protein of interest in a suitable host cell, and means a gene construct containing operablely linked basic regulatory elements to express an inserted gene. Here, "operably linked" means that the gene to be expressed and its regulatory sequences are functionally linked together in a manner that enables gene expression; and "regulatory elements" include promoters for implementing transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences for terminating transcription and translation. Such vectors can be plasmid vectors, granular vectors, phage vectors, viral vectors, etc., but are not limited to these, and any one or more known vectors capable of expressing the aforementioned genes can be used. As a specific example, the vector can be pNB181 (a pET22B(+) based plasmid; AmpR ampicillin selection marker; T7 promoter / lac operator / ribosome binding site (RBS) / T7 transcription terminator).

[0093] The "recombinant vector" used in this invention, after being transformed into a suitable host cell, can replicate independently of the host cell's genome or can integrate into the genome itself. Here, "suitable host cell" refers to a cell in which the vector can replicate, and may include the origin of replication, i.e., the specific nucleotide sequence where replication is initiated.

[0094] In another aspect, the present invention relates to recombinant strains transformed with polynucleotides encoding recombinant proteins for fluorescent biosensors or transformed with recombinant vectors containing said polynucleotides.

[0095] In this invention, the term "recombinant strain" refers to a transformant, i.e., a cell transformed by introducing a vector containing a polynucleotide encoding one or more proteins of interest into a host cell. Methods for preparing transformants by introducing an expression vector into a host cell include the calcium phosphate method or the calcium chloride / rubidium chloride method as described in the literature (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, 1.74, 1989), electroporation, electroinjection, chemical treatment methods (e.g., PEG), and methods using a gene gun.

[0096] The host cell can be a mammalian cell or a bacterium, and preferably can 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 this invention, Escherichia coli (E. coli bacteria) is used.

[0097] Fluorescent biosensor for quantifying methionine sulfoxide

[0098] In another aspect, the present invention relates to a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein, the fluorescent biosensor comprising a recombinant protein for quantifying methionine sulfoxide present in a target protein.

[0099] In another aspect, the present invention relates to compositions for the quantitative detection of methionine sulfoxide present in target proteins, said compositions comprising a recombinant protein of a fluorescent biosensor for the quantitative detection of methionine sulfoxide present in target proteins.

[0100] Methods for quantitative analysis of methionine sulfoxide

[0101] In another aspect, the present invention relates to a method for quantitatively analyzing methionine sulfoxide present in a target protein, the method comprising: (a) treating a sample containing the target protein with a primary antibody against the target protein to cause the antibody to bind to the target protein;

[0102] (b) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0103] (c) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0104] (d) Measure the fluorescence spectra of the remaining recombinant proteins used for the fluorescent biosensor.

[0105] In this invention, the higher the methionine sulfoxide content in the target protein, the higher the fluorescence spectral value. The method includes comparing the fluorescence spectral value of the recombinant protein used in the fluorescent biosensor with that of a normal control group, wherein the fluorescence spectral value can be calculated as a fluorescence value using the following formulas 1 to 3. Furthermore, the methionine sulfoxide content in the target protein can be quantified using the following formula and standard curve:

[0106] [Formula 1]

[0107] Fluorescence value = Ex:535 nm / Em:507 nm = value at 535 nm of the excitation spectrum (when the emission wavelength is fixed at 507 nm).

[0108] [Formula 2]

[0109] Fluorescence value = Ex:485 nm / Em:535 nm = value at 485 nm of the excitation spectrum (when the emission wavelength is fixed at 535 nm).

[0110] [Formula 3]

[0111] Fluorescence value = Ex:280 nm / Em:510 nm = value at 280 nm of the excitation spectrum (when the emission wavelength is fixed at 510 nm).

[0112] Diagnostic of oxidative stress-related diseases by quantitative analysis of methionine sulfoxide

[0113] In another aspect, the present invention relates to a method for providing information for the diagnosis of oxidative stress-related diseases by quantitative analysis of methionine sulfoxide present in target proteins, the method comprising: (a) treating a biological sample isolated from a subject with a primary antibody against the target protein to enable the antibody to bind to the target protein;

[0114] (b) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0115] (c) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0116] (d) Measure the fluorescence spectrum of the remaining recombinant protein used for the fluorescent biosensor and then compare it with the fluorescence spectrum of the normal control group.

[0117] In this invention, the sample may be cells, tissues, blood, plasma, serum, saliva, or urine.

[0118] In this invention, if the fluorescence spectral value is higher than that of the normal control group, it can provide information indicating oxidative stress-related diseases, and the fluorescence spectral value can be measured by the method described in the above <Method for Quantitative Analysis of Methionine Sulfoxide>.

[0119] In this invention, oxidative stress-related diseases can be selected from the group consisting of: cancer, stroke, myocardial infarction, angina pectoris, arteriosclerosis, infertility, hepatitis, osteoarthritis, acute coronary syndrome, cataracts, aging, lipid metabolism disorders, heart failure, hypertensive heart disease, arrhythmia, and aging.

[0120] Methods for screening therapeutic agents for oxidative stress-related diseases

[0121] In another aspect, the present invention relates to a method for screening therapeutic agents for oxidative stress-related diseases, the method comprising: (a) inducing oxidative stress in a sample containing a target protein containing methionine residues, and then treating the sample with a candidate drug;

[0122] (b) Treat the sample with a primary antibody targeting the target protein to allow the antibody to bind to the target protein;

[0123] (c) Treatment with the recombinant protein of the present invention for a fluorescent biosensor, such that the primary antibody binds to the protein G portion of the recombinant protein; and

[0124] (d) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and

[0125] (e) Measure the fluorescence spectrum of the remaining recombinant protein for the fluorescent biosensor and then compare it with the fluorescence spectrum of the control group that was not treated with the candidate drug.

[0126] In this invention, if the fluorescence spectrum value obtained after treatment with the candidate drug is lower than that of the control group, the candidate drug can be selected as a therapeutic agent for oxidative stress-related diseases, and the fluorescence spectrum value can be measured by the method described in the above <Method for Quantitative Analysis of Methionine Sulfoxide>.

[0127] Example

[0128] The invention will be described in more detail below through examples.

[0129] It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention to these embodiments.

[0130] Example 1: Preparation of recombinant proteins for fluorescent biosensors

[0131] 1-1: Gene recombination encoding recombinant proteins for fluorescent biosensors

[0132] In this invention, for the gene recombination process, the following were prepared: [pEGFP-N1 plasmid containing the gene encoding EGFP (SEQ ID NO: 9)]; [pUC57-amp plasmid containing the gene encoding MsrA (SEQ ID NO: 10), the gene encoding linker 1 (SEQ ID NO: 11), and the gene encoding the enterokinase cleavage site (SEQ ID NO: 12)]; and [tpMetSOG in pET His6 TEV LIC cloning vector containing the gene encoding thioredoxin 1 (Trx1) (SEQ ID NO: 13), the gene encoding linker 2 (SEQ ID NO: 14), and the gene encoding protein G (SEQ ID NO: 15).

[0133] Each plasmid was used as a template to amplify the gene via PCR (polymerase chain reaction), resulting in three genes: EGFP, MsrA / linker / enterokinase cleavage site, and Trx1 / linker / protein G.

[0134] In the above process, sterile water, 10X PCR buffer, dNTPs, forward / reverse primers for each template (Table 1), and nPfu polymerase were added to a 0.2 mL PCR tube in decreasing order. The tube was then centrifuged at 100 × g for approximately 3 seconds to ensure no residue remained on the tube walls. After gently mixing to ensure thorough mixing, PCR was performed according to the experimental protocol. The PCR reaction conditions for each gene moiety 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 extension for 42 seconds, followed by an extension reaction at 72°C for 5 minutes.

[0135] [Table 1]

[0136] Primer sequence

[0137]

[0138] First, the Trx1 / linker / protein G portion was inserted into the MCS of the pET21a plasmid via gene cloning. Then, the EGFP gene and the MsrA / linker / enterokinase cleavage site gene were sequentially ligated via gene recombination and then inserted into the previously inserted Trx1 / linker / protein G pET21a plasmid, thus finally ligating all genes (SEQ ID NO: 16). Finally, the nucleotide sequence of the recombinant plasmid was analyzed and confirmed, and it was named "pGACTLG".

[0139] 1-2: Preparation and purification of recombinant proteins

[0140] Using the vector prepared through the gene recombination process described in Example 1-1 above, transformed *Escherichia coli* was prepared, and the recombinant protein biosensor was expressed in large quantities from this *E. coli*. Subsequently, purified fluorescent biosensor protein was prepared by affinity chromatography using a His tag. The amino acid sequence of the fluorescent biosensor protein is shown in Table 2 below.

[0141] [Table 2]

[0142] Amino acid sequence of fluorescent biosensor protein

[0143]

[0144]

[0145]

[0146] Specifically, the pGACTLG vector was transformed into the prepared Escherichia coli strain, and strains with fluorescent biosensor proteins were obtained through an induced expression process.

[0147] All purification procedures were performed using a FPLC (Rapid Protein Liquid Chromatography) system. The strain was grown in 6 L of LB broth to induce protein expression, followed by centrifugation to precipitate the cells. The precipitated bacterial cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM β-mercaptoethanol, pH 8) and sonicated at 4 °C. The lysate was centrifuged at 14,000 × g, and only the supernatant was filtered through a 0.45 μm filter and then reacted with and attached to Ni-NTA resin at a rate of 2.5 mL / min. Subsequently, the protein was eluted using an elution buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM β-mercaptoethanol, 500 mM imidazole, pH 8) with an imidazole concentration gradient from 0 mM to 500 mM. The presence of proteins was checked by SDS-PAGE using a 12% polyacrylamide gel, and then concentrated using a 50 mL centrifugal filter.

[0148] Example 2: Operational Validation and Fluorescence Characterization of a Fluorescent Biosensor

[0149] The purified fluorescent biosensor protein was diluted to a concentration of 20 μM, and then DTT (dithiothreitol) (a substance that reduces and cleaves disulfide bonds) was added to a final concentration of 10 mM. The mixture was then reacted at room temperature for 30 minutes. Through this process, the fluorescent biosensor was completely reduced.

[0150] To remove the reducing agent DTT, a desalting column was performed according to the experimental protocol. The reduced fluorescent biosensor was diluted to a concentration of 5 μM and then treated with the oxidant N-acetylsethionine sulfoxide and the reducing agent GSH, respectively, at 37 °C for 30 min. After the reaction, the fluorescence spectra of the fully oxidized and reduced fluorescent biosensors were measured. The excitation spectrum was measured with 507 nm set as the emission value. In the case of the emission spectrum, 535 nm was used for measurement, which is the excitation wavelength with the highest value in the excitation spectrum. Figure 3a and Figure 3b ).

[0151] Furthermore, to measure fluorescence values ​​based on the concentration of the fluorescent biosensor according to previously measured fluorescence spectra, the fluorescent protein was serially diluted to 5 μM, and the fluorescence of each dilution was then measured. The emission wavelength was measured at 535 nm at an excitation wavelength of 485 nm, and the concentration values ​​of the dilutions were corrected using Nanodrop for more accurate concentration measurements. Using this method, a standard curve of fluorescence values ​​based on the concentration of the fluorescent biosensor was obtained. Figure 3c ).

[0152] Example 3: Confirmation of enterokinase cleavage

[0153] To confirm the cleavage of the fluorescent biosensor of the present invention by enterokinase, a fully reduced fluorescent biosensor was prepared using DTT at a concentration of 5 μM. Specifically, N-acetylsethionine sulfoxide was added to final concentrations of 100 μM, 50 μM, 10 μM, and 5 μM, and then reacted at 37°C for 30 minutes. Then, 1 μg of enterokinase was added and reacted for another 30 minutes. Next, SDS sample buffer, which does not affect disulfide bonds, was added, and SDS-PAGE was performed. For comparison, samples treated and reacted with glutathione at a final concentration of 5 mM were prepared.

[0154] The SDS-PAGE results show that the amount of cleaved fluorescent biosensor (approximately 50 kDa) increases with decreasing N-acetylsethionine sulfoxide concentration. This confirms that the fluorescent biosensor of the present invention is cleaved by enterokinase (…). Figure 4 ).

[0155] Example 4: Confirmation of the measurement of the degree of oxidation of purified methionine-rich protein

[0156] To confirm whether the fluorescent biosensor of the present invention can measure the degree of oxidation of a specific protein, IDLO, one of the methionine-rich proteins, was purified and prepared.

[0157] First, purified IDLO protein was prepared at 1 mM and oxidized to varying degrees by treatment with hydrogen peroxide at concentrations of 0 mM, 10 mM, 20 mM, 30 mM, and 40 mM for 2 hours at room temperature. Subsequently, after confirming the different migration patterns of the oxidized IDLO protein on SDS-PAGE... Figure 5a The degree of oxidation was measured using a 5 μM fluorescent biosensor of the present invention via a quantitative method for oxidized methionine.

[0158] As a result, Figure 5b As shown, when the fluorescence biosensor of the present invention was used to measure the degree of methionine oxidation of the target protein, it was confirmed that the measured fluorescence value increased with increasing hydrogen peroxide concentration, and thus the degree of oxidation increased.

[0159] Industrial applicability

[0160] The biosensor according to the invention can accurately and quantitatively measure the degree of oxidation of methionine residues in specific proteins, and can therefore be used as a fluorescent biosensor for the diagnosis of oxidative stress diseases. Furthermore, by measuring the degree of oxidation to determine the progression of aging, it can be usefully used for the diagnosis of aging and related diseases.

Claims

1. A recombinant protein for a fluorescent biosensor used to quantify methionine sulfoxide present in a target protein, said recombinant protein comprising: Fluorescent proteins; Msr (methionine sulfoxide reductase) protein; The enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4; Thioredoxin; and Protein G.

2. The recombinant protein of the fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to claim 1, wherein, The methionine sulfoxide is either methionine-S-sulfoxide or methionine-R-sulfoxide. The recombinant protein for quantifying the methionine-S-sulfoxide fluorescent biosensor comprises a fluorescent protein, MsrA protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G, and The recombinant protein used for quantifying the methionine-R-sulfoxide fluorescent biosensor comprises a fluorescent protein, MsrB protein, an enterokinase cleavage site represented by the amino acid sequence of SEQ ID NO: 4, thioredoxin, and protein G.

3. The recombinant protein of the fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to claim 2, wherein, The MsrA protein is represented by the amino acid sequence of SEQ ID NO: 2, and the MsrB protein is represented by the amino acid sequence of SEQ ID NO:

17.

4. The recombinant protein of the fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to claim 1, wherein, The fluorescent protein is 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).

5. The recombinant protein of the fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to claim 1, wherein, The thioredoxin (Trx) is either thioredoxin 1 (Trx1) or thioredoxin 3 (Trx3). The thioredoxin 1 (Trx1) is represented by the amino acid sequence of SEQ ID NO: 5; and The thioredoxin 3 (Trx3) is represented by the amino acid sequence of SEQ ID NO:

19.

6. A polynucleotide encoding a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5.

7. A recombinant vector comprising a polynucleotide encoding a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5.

8. A recombinant strain, said recombinant strain being transformed with a polynucleotide encoding a recombinant protein for a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5, or a recombinant vector containing said polynucleotide.

9. A fluorescent biosensor for quantifying methionine sulfoxide present in a target protein, said fluorescent biosensor comprising the recombinant protein of any one of claims 1 to 5 for quantifying methionine sulfoxide present in a target protein.

10. A composition for quantitative detection of methionine sulfoxide present in a target protein, said composition comprising a recombinant protein of a fluorescent biosensor for quantitative detection of methionine sulfoxide present in a target protein according to any one of claims 1 to 5.

11. A method for quantitatively analyzing methionine sulfoxide present in a target protein, the method comprising: (a) Treat a sample containing a target protein with a primary antibody against the target protein so that the antibody binds to the target protein; (b) Treatment with a recombinant protein of a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5, such that the primary antibody binds to the protein G moiety of the recombinant protein; and (c) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and (d) Measure the fluorescence spectra of the remaining recombinant proteins used in the fluorescent biosensor. The higher the content of methionine sulfoxide in the target protein, the higher the value of the fluorescence spectrum.

12. A method for providing information for the diagnosis of oxidative stress-related diseases by quantitative analysis of methionine sulfoxides present in target proteins, the method comprising: (a) Treat a biological sample isolated from a subject with a primary antibody against the target protein to allow the antibody to bind to the target protein; (b) Treatment with a recombinant protein of a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5, such that the primary antibody binds to the protein G moiety of the recombinant protein; and (c) Treat with enterokinase and then remove the cleaved recombinant protein for the fluorescent biosensor; as well as (d) Measure the fluorescence spectrum of the remaining recombinant protein used for the fluorescent biosensor and then compare it with the fluorescence spectrum of the normal control group. If the fluorescence spectral value is higher than that of the normal control group, it provides information indicating oxidative stress-related diseases.

13. A method for screening therapeutic agents for oxidative stress-related diseases, the method comprising: (a) Inducing oxidative stress in a sample containing a target protein containing methionine residues, and then treating the sample with a candidate drug; (b) Treat the sample with a primary antibody against the target protein to allow the antibody to bind to the target protein; (c) Treatment with a recombinant protein of a fluorescent biosensor for quantifying methionine sulfoxide present in a target protein according to any one of claims 1 to 5, such that the primary antibody binds to the protein G moiety of the recombinant protein; and (d) Treatment with enterokinase, followed by removal of the cleaved recombinant protein for the fluorescent biosensor; and (e) Measure the fluorescence spectrum of the remaining recombinant protein for the fluorescent biosensor and then compare it with the fluorescence spectrum of the control group that was not treated with the candidate drug. If the fluorescence spectrum value obtained after treatment with the candidate drug is lower than the fluorescence spectrum value of the control group, then the candidate drug is selected as a therapeutic agent for oxidative stress-related diseases.

14. The method according to any one of claims 11 to 13, wherein, The fluorescence spectral value is calculated as the fluorescence value using the following formulas 1 to 3: [Formula 1] Fluorescence value = Ex:535 nm / Em:507 nm = value at 535 nm of the excitation spectrum (when the emission wavelength is fixed at 507 nm). [Formula 2] Fluorescence value = Ex:485 nm / Em:535 nm = the value at 485 nm of the excitation spectrum (when the emission wavelength is fixed at 535 nm). [Formula 3] Fluorescence value = Ex:280 nm / Em:510 nm = value at 280 nm of the excitation spectrum (when the emission wavelength is fixed at 510 nm).

15. The method according to claim 12 or 13, wherein, The oxidative stress-related diseases are selected from the group consisting of: cancer, stroke, myocardial infarction, angina pectoris, arteriosclerosis, infertility, hepatitis, osteoarthritis, acute coronary syndrome, cataracts, aging, lipid metabolism disorders, heart failure, hypertensive heart disease, arrhythmia, and aging.

Images