Fusion protein, S-adenosine-L-methionine biosensor and application of S-adenosine-L-methionine biosensor

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a fusion protein, an S-adenosine-L-methionine biosensor and application of the S-adenosine-L-methionine biosensor. Specifically, the S-adenosine-L-methionine biosensor at least comprises a fusion protein composed of an S-adenosine-L-methionine specific signal recognition element and a cyclic rearrangement fluorescent protein. Experiments prove that the biosensor developed by the invention has the advantages of high response amplitude, strong fluorescence brightness, moderate affinity and the like, and can monitor the dynamic state of S-adenosine-L-methionine in living bacteria and living mammalian cells in real time in a high-specificity, high-efficiency, high-temporal-spatial-resolution and low-cost manner; high-flux and convenient detection of the activity of the S-adenosine-L-methionine-dependent methyltransferase can be realized, so that the S-adenosine-L-methionine-dependent methyltransferase has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a fusion protein, S -Adenosine-L-methionine biosensor and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] S -Adenosine-L-methionine ( S L-Adenosine (SAM) is an active methionine produced from adenosine provided by adenosine triphosphate (ATP), and is widely found in all biological groups, including microorganisms, plants, and animals. As an important methyl donor, SAM is considered one of the most chemically reactive enzyme substrates besides ATP. For example, under the catalysis of various DNA, RNA, or histone methyltransferases, SAM can transfer methyl groups to DNA, RNA, or histones, thereby regulating gene expression at the genomic, epigenomic, or transcriptomic levels. Furthermore, SAM can also provide active methyl groups for various small-molecule secondary metabolites under the action of different natural product methyltransferases, thus participating in the synthesis of bioactive molecules, biodiesel production, and fine chemical production. Given the broad biological functions of SAM and its related methyl transfer processes in medicine, chemical engineering, and other fields, establishing in vivo and in vitro real-time, in-situ detection technologies for SAM to achieve efficient monitoring of SAM-dependent methyl transfer reactions is of great significance for revealing the physiological and pathological functions of SAM and for conducting high-throughput screening of SAM-dependent methyltransferases and their inhibitors.

[0004] Currently, the detection of SAM mainly relies on expensive and complex techniques such as chromatography and mass spectrometry, making it difficult to achieve real-time, in-situ detection of SAM in living cells. Although recent studies have reported SAM biosensors based on nucleic acid aptamers for imaging the metabolic processes of SAM in living cells, these sensors lack specificity for SAM and for its structural analogues, such as... S -Adenosine-L-homocysteine ​​( SSAM-dependent methyltransferase activity also showed significant responses to α-adenosine (SAH) and adenosine. On the other hand, existing methods for detecting SAM-dependent methyltransferase activity, such as multi-enzyme coupling assays, proximity scintillation assays (SPA), and ALphaLISA homogeneous immunoassay, typically monitor substrate methylation or quantify SAH generation. However, these techniques are cumbersome and time-consuming, hindering high-throughput screening. Therefore, there is an urgent need to develop a highly specific, efficient, spatiotemporally resolved, and low-cost SAM detection method, and to build a high-throughput, convenient technical system for monitoring SAM-dependent methyltransferase activity based on this method, to meet the needs for in vitro and in vivo detection of SAM and related methyl transfer processes. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided a fusion protein... S -Adenosine-L-methionine (SAM) biosensor and its applications. Specifically, the SAM biosensor comprises at least a fusion protein consisting of a SAM-specific signal recognition element and a cyclic rearranged fluorescent protein. Experiments have demonstrated that the biosensor developed in this invention possesses advantages such as high response amplitude, strong fluorescence intensity, and moderate affinity. It can monitor the dynamics of SAM in live bacteria and live mammalian cells in real time in a highly specific, efficient, high spatiotemporal resolution, and low-cost manner, and can achieve high-throughput and convenient detection of SAM-dependent methyltransferase activity. Based on the above research results, this invention is thus completed.

[0006] Specifically, to achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a fusion protein, said fusion protein comprising at least S -Adenosine-L-methionine specific signal recognition element and fluorescent protein; wherein, the S The adenosine-L-methionine-specific signal recognition element is the SAM-binding protein PH1780 derived from Thermococcus Horikoshi; the fluorescent protein can be a cyclic rearranged fluorescent protein.

[0007] In a second aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned fusion protein.

[0008] A third aspect of the present invention provides a carrier containing the above-described nucleic acid molecules.

[0009] A fourth aspect of the present invention provides a host cell containing the above-described nucleic acid molecules, the above-described vector, or capable of expressing the above-described fusion protein.

[0010] A fifth aspect of the present invention provides the above-mentioned fusion protein, nucleic acid molecule, vector and / or host cell for detection S -Adenosine-L-methionine or preparation S Applications of α-adenosine-L-methionine biosensors.

[0011] A sixth aspect of the present invention provides S -Adenosine-L-methionine biosensor, the biosensor comprising at least the above-mentioned fusion protein.

[0012] A seventh aspect of the present invention provides an in vitro detection method. S A method for detecting adenosine-L-methionine, the method comprising at least: contacting the sample to be tested with the biosensor, and analyzing the concentration of adenosine-L-methionine in the sample based on changes in the fluorescence signal of the biosensor. S - The concentration or presence or absence of adenosine-L-methionine.

[0013] An eighth aspect of the present invention provides an intracellular detection method. S A method for expressing -adenosine-L-methionine, the method comprising at least: inducing the expression of the biosensor in cells, and analyzing intracellular fluorescence signals based on changes in the biosensor's fluorescence signal. S - The concentration or presence or absence of adenosine-L-methionine.

[0014] A ninth aspect of the present invention provides a method for detecting methyltransferase activity or for high-throughput screening of methyltransferase-related materials, the method comprising: incubating any one or more of the methyltransferase to be tested, a substrate, SAM, SAH, and methyltransferase-related materials with the biosensor; and analyzing the methyltransferase activity or evaluating the efficacy of the methyltransferase-related materials based on the rate of change of the fluorescence signal of the SAM biosensor.

[0015] The methyltransferase-related materials may be methyltransferase activators (including highly active methyltransferase mutants) or methyltransferase inhibitors.

[0016] In this invention, the methyltransferase is selected from Burkholderia paraknegaceae (…). ParaburkhoLderia xenovorans The halogenated methyltransferase BxHMT from corn smut ( UstiLago maydis ) halogenated methyltransferase UMA, from drought-sensitive tomato M82 ( SoLanum Lycopersicum Catechols in cv. M82) O -Methyltransferase SLCOMT, from Penali tomato ( SoLanum penneLLii Catechins O -Methyltransferase SpCOMT, from *Internal Rhizobium mulberry* ( Mesorhizobium muLeiense CatechinsO α-methyltransferase MesMOMT, from *Xanthomonas aeruginosa* ( Myxococcus xanthus Catechins O -Methyltransferase MxSafC.

[0017] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects: (1) The SAM biosensor (named SAMsor) provided by the above technical solution is constructed by inserting the cyclic rearranged hypersheeted yellow fluorescent protein cpSFYFP into an appropriate position inside the SAM-binding protein PH1780 derived from Thermococcus Horikoshi. The conformational change caused by the binding of SAM to PH1780 can lead to conformational changes in the polypeptide backbone and amino acid side chains at the insertion site of the cyclic rearranged hypersheeted yellow fluorescent protein, which further significantly changes the fluorescence intensity of the cyclic rearranged hypersheeted yellow fluorescent protein. This change in fluorescence intensity can be used as an indicator for the detection of SAM concentration.

[0018] (2) The SAM biosensor provided by the above technical solution has a fluorescence response amplitude of 3536.02 ± 351.86% to SAM, and an apparent dissociation constant ( K d The molecular weight limit (MW) is 204.02 ± 20.90 μM, and the detection range is 1.81 μM-3 mM. It exhibits high specificity for SAM, temperature stability, and reversible binding.

[0019] (3) The SAM biosensor provided by the above technical solution can be expressed in the cells of live bacteria and live mammalian cells, thereby enabling real-time and in-situ detection of SAM in different bacteria and different mammalian cells, which is of great significance for the development of SAM metabolic mechanism and functional diversity research.

[0020] (4) The SAM biosensor provided by the above technical solution can achieve efficient and convenient detection of methyltransferase activity. Simply mix the purified SAMsor protein with the methyltransferase to be tested and the corresponding substrate, and combine it with a fluorescence microplate reader to detect the changes in SAMsor fluorescence signal in real time, so as to quickly achieve high-throughput detection of methyltransferase activity. Furthermore, by combining the SAMsor-based methyltransferase activity detection technology with a random mutant library of methyltransferases or a library of commercially available bioactive compounds, high-throughput screening of highly active methyltransferase mutants or their inhibitors can be achieved through a simple fluorescence microplate reader. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a schematic diagram illustrating the domain composition and structural analysis of PH1780 in Embodiment 1 of the present invention.

[0023] Figure 2 This is a ligand analysis of PH1780 in Example 1 of the present invention.

[0024] Figure 3 This is a schematic diagram of the structure of different SAM biosensor variants formed by the cyclic rearranged superfolded yellow fluorescent protein constructed in Example 1 of the present invention at different insertion sites inside PH1780, and a comparison of the response amplitudes to SAM and MTA.

[0025] Figure 4 The dose-response curves of SAMsor-0.85, a SAM biosensor variant constructed in Example 1 of this invention, for SAM and MTA are shown.

[0026] Figure 5 This study focuses on the specificity analysis of the SAM biosensor variant SAMsor-0.85 constructed in Example 1 of this invention.

[0027] Figure 6 The dose-response curves of SAM and MTA are shown for the MTA biosensor variant MTAor-0.64 constructed in Example 1 of this invention.

[0028] Figure 7 This study focuses on the specificity analysis of the MTA biosensor variant MTAor-0.64 constructed in Example 1 of this invention.

[0029] Figure 8 This is a comparison of the response magnitudes of 279 random mutants of SAMsor-0.85 Linker selected in Example 1 of the present invention to SAM.

[0030] Figure 9 This is a dose-response curve analysis of the SAM biosensor variant SAMsor screened in Example 1 of the present invention.

[0031] Figure 10 In Embodiment 2 of the present invention E. coLi Gradient response and specificity analysis of SAMsor expressed in BL21(DE3) to SAM.

[0032] Figure 11 This is implemented using SAMsor in Embodiment 2 of the present invention. E. coLi Functional identification of SAM synthesis and catabolism enzymes in BL21(DE3).

[0033] Figure 12This is an example of the gradient response and specificity analysis of SAMsor expressed in HEK293FT cells to SAM in Example 3 of the present invention. Figure A shows the specificity analysis of SAMsor expressed in HEK293FT cells; Figure B shows the gradient response of SAMsor expressed in HEK293FT cells to SAM.

[0034] Figure 13 This invention provides an example of identifying the function of SAM synthase in HEK293FT cells using SAMsor in Example 3 of this invention.

[0035] Figure 14 This is an example of SAMsor-based assay for halomethyltransferase activity in Example 4 of the present invention. Figure A shows the time progression curve of SAM generation in the halomethyltransferase catalytic system determined by SAMsor; Figure B shows a comparison of the activities of different halomethyltransferases determined by SAMsor.

[0036] Figure 15 The SAMsor-based catechin in Example 4 of this invention O α-Methyltransferase activity assay. Figure A shows the determination of catechol- methyltransferase activity by SAMsor. O Figure B shows the time progression curve of SAM consumption in the methyltransferase catalytic system; Figure B shows the different catechol-related parameters determined by SAMsor. O Comparison of methyltransferase activities. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0039] In a typical embodiment of the present invention, a fusion protein is provided, the fusion protein comprising at least a SAM-specific signal recognition element and a cyclic rearranged fluorescent protein.

[0040] The SAM-specific signal recognition element can be derived from *Thermococcus Horikoshi* (…). Pyrococcus horikoshiiThe SAM-binding protein PH1780 (NCBI number WP_048053476.1, PDB number 2YZQ) is used. Of course, based on the concept of this invention, other known SAM-specific signal recognition elements or homologous proteins of SAM-binding proteins are also applicable to the technical solutions of this application, and therefore should fall within the protection scope of this invention.

[0041] In this invention, the fluorescent protein is preferably a circularly permuted fluorescent protein (cpFP), specifically a class of reporter gene-encoded proteins for visualization, including circularly permuted cyan fluorescent protein (cpTFP), circularly permuted green fluorescent protein (cpGFP), circularly permuted yellow fluorescent protein (cpYFP), and circularly permuted red fluorescent protein (cpRFP). In one specific embodiment of this invention, the circularly permuted fluorescent protein used is a cpYFP variant containing four mutation sites (S30R, Y39N, N105T, Y145F) (circularly permuted hyperfolded yellow fluorescent protein cpSFYFP, which has the characteristic of strong fluorescence intensity). When SAM is present, the conformational change induced by the binding of SAM to the signal recognition element PH1780 can lead to a conformational change in cpSFYFP, thereby greatly changing the fluorescence properties of the sensor, thus realizing the detection of SAM.

[0042] The fusion protein can be obtained by linking the cyclic rearranged fluorescent protein to the N-terminus or C-terminus of the SAM-specific signal recognition element, or by inserting the cyclic rearranged fluorescent protein into the SAM-binding protein PH1780; the fusion protein is obtained by using the cyclic rearranged hyperfolded yellow fluorescent protein cpSFYFP with an N-terminal linker of "serine-alanine-glycine" and a C-terminal linker of "glycine-glycine-cysteine".The following amino acid sites are inserted into PH1780: 9I / 10M, 10M / 11T, 14P / 15V, 15V / 16T, 20P / 21A, 21A / 22T, 34K / 35V, 35V / 36R, 42N / 43K, 43K / 44E, 44E / 45G, 45G / 46K, 46K / 47L, 54K / 55R, 55R / 56I, 57L / 58V, 58V / 59N, 60P / 61D, 61D / 62E, 63E / 64Q, 64Q / 65L, 66A / 67M, 67M / 68L, 69V / 70K, 70K / 71R, 73V / 74P, 76V / 77K, 79N / 80D, 80D / 81T, 92Y / 93D, 93D / 94Y, 103K / 104G, 104G / 105K, 111T / 112V, 120F / 121A, 121A / 122K, 122K / 123S, 123S / 124E, 126Y / 127K, 127K / 128G, 128G / 129V, 129V / 130E, 130E / 131I, 131I / 132E, 132E / 133P, 133P / 134Y, 134Y / 135Y, 13 5Y / 136Q, 136Q / 137R, 137R / 138Y, 138Y / 139V, 139V / 140S, 140S / 141I, 144E / 145G, 145G / 146T, 158S / 159N, 159N / 160S, 167D / 168S , 168S / 169E, 170G / 171N, 171N / 172L, 177D / 178E, 178E / 179T, 182L / 183R, 183R / 184D, 188V / 189R, 189R / 190I, 192K / 193S, 194T / 1 95E, 195E / 196L, 197A / 198A, 198A / 199S, 199S / 200S, 200S / 201E, 201E / 202E, 203E / 204W, 204W / 205I, 206L / 207E, 207E / 208S, 222 P / 223N, 223N / 224K, 229I / 230M, 231T / 232R, 233D / 234V, 234V / 235I, 237A / 238T, 253Y / 254S, 255I / 256E, 263G / 264E, 281V / 282K. Preferably, the fusion protein is obtained by inserting the cyclic rearranged superfolded yellow fluorescent protein cpSFYFP with an N-terminal linker of "leucine-threonine" and a C-terminal linker of "serine-proline-arginine" between amino acids 1-234 and 235-285 of pH 1780.

[0043] Specifically, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

[0044] In (a2), the substitution, deletion and / or addition of one or more amino acid residues generally refers to the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0045] The proteins shown in (a1)–(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0046] In one specific embodiment of the present invention, the fusion protein further comprises a signal peptide and / or a tag protein.

[0047] In one or more specific embodiments of the present invention, a nucleic acid molecule is provided, which is capable of encoding the above-mentioned fusion protein.

[0048] Specifically, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode a fusion protein with the same function.

[0049] It should be noted that the term "identity" refers to sequence similarity to an amino acid / nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0050] The aforementioned 40% or more of identity can be 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more of identity.

[0051] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., without specific limitations.

[0052] In one or more specific embodiments of the present invention, a carrier is provided, the carrier comprising at least the above-mentioned nucleic acid molecules.

[0053] The vector can be any one or more of a viral vector, plasmid, bacteriophage, phage particle, granule, or artificial chromosome; the viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector; the artificial chromosome includes bacterial artificial chromosome, phage P1-derived vector, yeast artificial chromosome, or mammalian artificial chromosome; more preferably, it is a plasmid; the plasmid includes, but is not limited to, pETDuet-1 and pcDNA3.1. (+) .

[0054] In one or more specific embodiments of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector, or is capable of expressing the above-mentioned fusion protein.

[0055] The host cells include bacterial cells, fungal cells, and animal cells; The bacteria can be any one or more of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0056] In one or more specific embodiments of the present invention, the bacteria include, but are not limited to, Escherichia coli (e.g., BL21(DE3)), Agrobacterium tumefaciens (e.g., GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0057] The fungal cells include yeast.

[0058] The animal cells may be mammalian cells, and more specifically, the mammalian cells include, but are not limited to, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293FT cells.

[0059] In one or more specific embodiments of the present invention, the above-mentioned fusion protein, nucleic acid molecule, vector and / or host cell are provided for detection S -Adenosine-L-methionine or preparation S Applications of α-adenosine-L-methionine biosensors.

[0060] In one or more specific embodiments of the present invention, a method is provided. S -Adenosine-L-methionine biosensor, the biosensor comprising at least the above-mentioned fusion protein.

[0061] That is, S The adenosine-L-methionine biosensor SAMsor is a biosensor constructed by inserting cpSFYFP with an N-terminal linker of "leucine-threonine" and a C-terminal linker of "serine-proline-arginine" into the spaces between amino acids 1-234 and 235-285 of PH1780 (its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2).

[0062] Furthermore, the biosensor may also include other components for... S Reagents, apparatus and / or equipment for the detection of α-adenosine-L-methionine.

[0063] For example, in this invention, the reagent may contain a detection buffer (such as a fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0064] In practical applications, the aforementioned biosensors can exist in the form of detection kits, especially for use in (in vitro) environments. S -Adenosine-L-methionine detection.

[0065] However, when biosensors are used inside the body (such as inside cells) S The detection of α-adenosine-L-methionine can directly induce the expression of the aforementioned biosensor within cells, thereby enabling its detection of intracellular... S The presence or concentration of adenosine-L-methionine.

[0066] Therefore, in one or more specific embodiments of the present invention, an in vitro detection method is provided. S A method for processing -adenosine-L-methionine, the method comprising at least: contacting the sample to be tested with the biosensor, and according to... S The fluorescence signal changes of the α-adenosine-L-methionine biosensor are analyzed to assess the presence of α-adenosine-L-methionine in the sample. S - The concentration or presence or absence of adenosine-L-methionine.

[0067] The sample to be tested contains or is suspected of containing S The sample can be a biological sample or an environmental sample. The biological sample includes, but is not limited to, bacterial culture medium, bacterial lysate, cell culture medium, cell lysate, animal blood (such as whole blood, serum, dried blood spot samples, etc.), animal urine, and animal tissue fluid. The animal may be a mammal, with humans being preferred.

[0068] The environmental samples include, but are not limited to, water bodies (such as surface water and wastewater), soil leachate, food processing liquids, and fermentation liquids. Optionally, the samples may be pretreated, such as by dilution, filtration, centrifugation to remove impurities, and adjustment of pH or ionic strength, to adapt them to the detection environment of the biosensor.

[0069] At this time, the S -Adenosine-L-methionine may also include other substances used for... S Reagents, apparatus and / or equipment for the detection of α-adenosine-L-methionine; The reagents include detection buffers (such as fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0070] In one or more specific embodiments of the present invention, an intracellular detection method is provided. S A method for inducing -adenosine-L-methionine, the method comprising at least: inducing S The α-adenosine-L-methionine biosensor is expressed intracellularly, according to... S α-Adenosine-L-methionine biosensor fluorescence signal changes, analyzing intracellular... S - The concentration or presence or absence of adenosine-L-methionine.

[0071] The cells may be bacterial cells, fungal cells, or animal cells, and are not specifically limited thereto. In specific embodiments of the present invention, the cells include, but are not limited to, Escherichia coli BL21(DE3) cells and human embryonic kidney cells (HEK293FT).

[0072] The above methods can be used to treat in vitro or in vivo conditions. S Qualitative or quantitative detection of adenosine-L-methionine.

[0073] In another specific embodiment of the present invention, a method for detecting methyltransferase activity or high-throughput screening of methyltransferase-related materials is provided. The method includes: incubating any one or more of the methyltransferase to be tested, substrate, SAM, SAH, and methyltransferase-related materials with the biosensor; and analyzing the methyltransferase activity or evaluating the efficacy of the methyltransferase-related materials based on the rate of change of the fluorescence signal of the SAM biosensor.

[0074] The methyltransferase-related materials may be methyltransferase activators (including highly active methyltransferase mutants) or methyltransferase inhibitors.

[0075] In this invention, the methyltransferase is selected from Burkholderia paraknegaceae (…). ParaburkhoLderia xenovorans The halogenated methyltransferase BxHMT from corn smut ( UstiLago maydis) halogenated methyltransferase UMA, from drought-sensitive tomato M82 ( SoLanum Lycopersicum Catechols in cv. M82) O -Methyltransferase SLCOMT, from Penali tomato ( SoLanum penneLLii Catechins O -Methyltransferase SpCOMT, from *Internal Rhizobium mulberry* ( Mesorhizobium muLeiense Catechins O α-methyltransferase MesMOMT, from *Xanthomonas aeruginosa* ( Myxococcus xanthus Catechins O -Methyltransferase MxSafC.

[0076] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. In the following embodiments, the expression vectors pETDuet-1 and pcDNA3.1 are used. (+) All materials and reagents used were purchased from Novagen; unless otherwise specified, all other materials and reagents used were obtained commercially. Unless otherwise specified, all experimental methods used were standard methods.

[0077] Example 1: Construction and Optimization of SAM Biosensors (1) Ligand analysis of PH1780 The entire genome of *Thermococcus Horikoshi* was synthesized by General Biosystems (Anhui) Co., Ltd. Pyrococcus horikoshii The PH1780 encoding gene in ) ph1780 (NCBI No.: WP_048053476.1), via BamH I / Hind The III restriction site was cloned into the pETDuet-1 expression vector to obtain the recombinant plasmid pETDuet-PH1780, which was then transformed into the expression strain. E. coLi In BL21(DE3), strains successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful PCR verification. PH1780 is a strain consisting of four CBS domains ( c ystathionine β - s SAM-binding protein (PDB number 2YZQ) composed of ynthase domain (see appendix) Figure 1Proteins containing CBS domains typically possess multiple ligand binding capabilities. To determine whether PH1780 possesses ligands other than SAM, the coding gene for cpSFYFP was inserted into the middle of the four CBS domains of PH1780 (i.e., between tyrosine and lysine positions 126 and 127 of PH1780) with an N-terminal linker of "serine-alanine-glycine" and a C-terminal linker of "glycine-glycine-cysteine," thus obtaining the initial biosensor PH1780. 1-126 -cpSFYFP-PH1780 127-285 .

[0078] After exogenous expression and purification, PH1780 1-126 -cpSFYFP-PH1780 127-285 It reacts with SAM and other common ligands of proteins containing the CBS domain, such as the deamination propyl product of SAM. S -Methyl-5'-thioadenosine ( S -methyL-5'-thioadenosine, MTA), ATP, ADP, AMP, NAD + NADH, Mg 2+ Zn 2+ Mix the ingredients. Measure the pH at 1780 using a fluorescence microplate reader. 1-126 -cpSFYFP-PH1780 127-285 Changes in fluorescence ratios (F) before and after the addition of different compounds 488 nm / F 405 nm The specificity of PH1780 was analyzed by measuring the ratio of fluorescence emission intensity at 528 nm under 488 nm excitation to that under 405 nm excitation. Results are attached. Figure 2 As shown, only SAM and MTA can trigger PH1780. 1-126 -cpSFYFP-PH1780 127-285 The change in fluorescence ratio indicates that PH1780 is a protein that can bind to SAM and MTA.

[0079] Among them, PH1780 1-126 -cpSFYFP-PH1780 127-285The construction method is as follows: Using the recombinant plasmid pETDuet-PH1780 as a template, a linearized plasmid backbone was obtained by reverse PCR amplification between tyrosine residue 126 and lysine residue 127 of PH1780; using the previously constructed pET28a-cpSFYFP as a template, the DNA fragment of cpSFYFP was obtained by PCR amplification. The plasmid backbone and the cpSFYFP fragment were ligated using a DNA assembly method based on T5 exonuclease. Specifically, 5 μL of the recombinant target gene fragment and the linearized plasmid were added to a 15 μL ligation system, with a molar ratio of target gene fragment to linearized plasmid of 4:1. The mixture was incubated at 30 ºC for 40 minutes, followed by cooling on ice for 10 minutes to obtain the recombinant plasmid pETDuet-PH1780. 1-126 -cpSFYFP-PH1780 127-285 Transform it into the expression strain E. coLi In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful PCR verification of the bacterial culture.

[0080] Among them, PH1780 1-126 -cpSFYFP-PH1780 127-285 The purification method is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] 1-126 -cpSFYFP-PH1780 127-285 of E. coLi BL21(DE3) strain was inoculated at a ratio of 1.5% in 1 L of LB medium containing ampicillin and cultured at 37 ºC and 180 rpm until OD. 600 nm Approximately 0.6 mg of the solution was added with 1 mM IPTG, and protein expression was induced overnight at 23 °C and 160 rpm. The bacterial cells were collected by centrifugation, washed twice with binding buffer, and resuspended at OD200. 600 nm The bacterial cells were lysed with 1 mM PMSF and 10% glycerol at a concentration of 20 mg / L. The mixture was then centrifuged at 12,000 rpm and 4 ºC for 50 min to remove cell debris, yielding a crude extract. This crude extract was filtered through a 0.22 μm filter and purified using a 5 mL nickel column. Elution with different concentrations of elution buffer yielded purified PH1780. 1-126 -cpSFYFP-PH1780 127-285 protein.

[0081] Among them, PH1780 1-126 -cpSFYFP-PH1780 127-285 The fluorescence detection method is as follows: The purified pH 1780 is diluted with detection buffer (50 mM Tris-HCl, pH 7.4).1-126 -cpSFYFP-PH1780 127-285 Protein was diluted to 4 / 3 μM. 2 mM SAM, MTA, ATP, ADP, AMP, and NAD were prepared using assay buffer (50 mM Tris-HCl, pH 7.4). + NADH, Mg 2+ Zn 2+ Under light-protected conditions, dilute pH 1780. 1-126 -cpSFYFP-PH1780 127-285 Proteins were mixed with different compound solutions at a volume ratio of 3:1. 100 μL of the mixture was transferred to a black flat-bottomed 96-well plate, and the pH was read using a PerkinElmer Ensight fluorescent microplate reader at 1780. 1-126 -cpSFYFP-PH1780 127-285 The fluorescence intensity change was observed. Instrument parameters were set as follows: excitation wavelengths of 488 nm and 405 nm, and emission wavelength of 528 nm. The ratio of the fluorescence intensity measured under 488 nm excitation to that measured under 405 nm excitation (F0) was calculated. 488 nm / F 405 nm pH 1780 is determined by its high or low levels. 1-126 -cpSFYFP-PH1780 127-285 Its specificity.

[0082] The LB culture medium formula mentioned in the above steps is as follows: 10 g / L peptone; 5 g / L yeast extract; 10 g / L NaCl, pH 7.0; sterilized at 121 ºC for 20 minutes.

[0083] The formulation of the 15 μL ligation system in the DNA assembly method based on T5 exonuclease described in the above steps is as follows: 4 μL 5× isothermal reaction buffer (0.5 M Tris-HCl, 0.05 M MgCl2, 0.05 M dithiothreitol), 0.004 μL 10 U / μL T5 exonuclease, and 11 μL ddH2O.

[0084] The binding buffer formulation mentioned in the above steps is: 20 mM Na2HPO4, 20 mM imidazole, 500 mM NaCl, adjusted to pH 7.4; the elution buffer formulation is: 20 mM Na2HPO4, 500 mM imidazole, 500 mM NaCl, adjusted to pH 7.4.

[0085] The pETDuet-PH1780 plasmid backbone was amplified by reverse PCR. The primers were designed as follows: PH1780 126Y / 127KUpstream primer: 5'-ATAGACGTTGTCGCTGTTGAAGCCTGCAGAATATTTCTCACTTTTTGCAAAATAA-3'; PH1780 126Y / 127K Downstream primer: 5'-GGGCACAAGCTGGAGTACAACGGGGGCTGCAAGGGCGTTGAAATTGAACCGTATT-3'; The primers for PCR amplification of the cpSFYFP fragment are designed as follows: upstream primer for cpSFYFP: 5'-TCTGCAGGCTTCAACAGCGACAACG-3'; cpSFYFP downstream primer: 5'-GCAGCCCCCGTTGTACTCCAGCTTG-3'.

[0086] (2) Construction of SAM-specific biosensors The CBS domain is a conserved multi-ligand binding domain. Different ligands induce overall conformational changes in the CBS domain by binding to different sites. It is speculated that the different insertion sites of cpSFYFP within PH1780 may disrupt the binding pocket of one ligand without affecting the binding of the other.To develop a SAM-specific biosensor, in this embodiment, based on pETDuet-PH1780, the following sites were selected for insertion into cpSFYFP: 9I / 10M, 10M / 11T, 14P / 15V, 15V / 16T, 20P / 21A, 21A / 22T, 34K / 35V, 35V / 36R, 42N / 43K, 43K / 44E, 44E / 45G, 45G / 46K, 46K / 47L, 54K / 55R, 55R / 56I, 57L / 58V, 58V / 59N, 60P / 61D, 61D / 62E, 63E / 64Q, 64Q / 65L, 66 A / 67M, 67M / 68L, 69V / 70K, 70K / 71R, 73V / 74P, 76V / 77K, 79N / 80D, 80D / 81T, 92Y / 93D, 93D / 94Y, 103K / 104G, 104G / 105K, 111T / 112V, 120F / 1 21A, 121A / 122K, 122K / 123S, 123S / 124E, 126Y / 127K, 127K / 128G, 128G / 129V, 129V / 130E, 130E / 131I, 131I / 132E, 132E / 133P, 133P / 134Y, 134Y / 135Y, 135Y / 136Q, 136Q / 137R, 137R / 138Y, 138Y / 139V, 139V / 140S, 140S / 141I, 144E / 145G, 145G / 146T, 158S / 159N, 159N / 160S, 167D / 168S, 168S / 169E, 170G / 171N, 171N / 172L, 177D / 178E, 178E / 179T, 182L / 183R, 183R / 184D, 188V / 189R, 189R / 190I, 192K / 193S, 194T / 195 Ninety sensor variant encoding plasmids were constructed, consisting of E, 195E / 196L, 197A / 198A, 198A / 199S, 199S / 200S, 200S / 201E, 201E / 202E, 203E / 204W, 204W / 205I, 206L / 207E, 207E / 208S, 222P / 223N, 223N / 224K, 229I / 230M, 231T / 232R, 233D / 234V, 234V / 235I, 237A / 238T, 253Y / 254S, 255I / 256E, 263G / 264E, and 281V / 282K. In the above recombinant plasmids, cpSFYFP is linked to the upper and lower fragments of PH1780 by linkers with N-terminal "serine-alanine-glycine" and C-terminal "glycine-glycine-cysteine", respectively.

[0087] The above 90 biosensor variants were exogenously expressed, isolated, and purified according to the method described in (1), and the response of each biosensor variant to 100 μM SAM or 100 μM MTA was detected one by one. (See attached...) Figure 3 As shown, the sensor variant (named SAMsor-0.85) corresponding to the insertion of cpSFYFP between the 234V / 235I amino acid sites of PH1780 has a specific response to SAM, while the sensor variant (named MTAsor-0.64) corresponding to the insertion of cpSFYFP between the 182L / 183R amino acid sites of PH1780 has a specific response to MTA.

[0088] Mix SAMsor-0.85 with gradient concentrations of SAM or MTA, and then calculate the ratio of the measured fluorescence intensities (F). 488 nm / F 405 nm With SAM or MTA concentration set as the ordinate and SAM or MTA concentration as the abscissa, dose-response curves of SAMsor-0.85 to SAM or MTA were obtained using OriginPro software. The results are attached. Figure 4 As shown, SAM significantly increased the fluorescence ratio of SAMsor-0.85 in a dose-dependent manner. The maximum fluorescence ratio change of SAMsor-0.85 to SAM (i.e., response amplitude) was observed. R max The calculated value is 41.76 ± 4.60%, and the apparent dissociation constant is ( K d The calculated value was 8.31 ± 2.89 μM. Furthermore, SAMsor-0.85 exhibits high specificity for SAM, showing no significant response to MTA and other SAM derivatives such as SAH, adenosine, and L-methionine (see appendix). Figure 5 ).

[0089] In addition, MTAsor-0.64 was mixed with gradient concentrations of SAM or MTA, and the ratio of the measured fluorescence intensities (F) was calculated. 488 nm / F 405 nm The values ​​of MTA and SAM were set as the ordinate and the concentrations of MTA or SAM as the abscissa. Dose-response curves of MTA-0.64 to SAM or SAM were obtained using OriginPro software. The results are attached. Figure 6 As shown, MTA significantly reduced the fluorescence ratio of MTAsor-0.64 in a dose-dependent manner. The maximum fluorescence ratio change of MTAsor-0.64 to MTA (i.e., the response amplitude) was [not specified]. R maxThe calculated value is 30.98 ± 3.06%, and the apparent dissociation constant is ( K d The calculated value was 392.58 ± 229.83 μM. Furthermore, MTAsor-0.64 exhibited high specificity for MTA, but showed no significant response to SAM and other SAM derivatives such as SAH, adenosine, and L-methionine (see appendix). Figure 7 ).

[0090] The construction methods for the aforementioned 90 biosensor variants are as follows: Using recombinant plasmid pETDuet-PH1780 as a template, a linearized plasmid backbone was obtained by reverse PCR amplification between insertion sites within PH1780; using the inventor's previously constructed pET28a-cpSFYFP as a template, the cpSFYFP DNA fragment was obtained by PCR amplification. The plasmid backbone and the cpSFYFP fragment were then ligated using a T5 exonuclease-based DNA assembly method to obtain the recombinant plasmid pETDuet-PH1780. 1-n -cpSFYFP-PH1780 n+1-285 Transform it into the expression strain E. coLi In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful PCR verification of the bacterial culture.

[0091] The pETDuet-PH1780 plasmid backbone was amplified by reverse PCR, and the cpSFYFP fragment was amplified by PCR simultaneously. The primers were designed as follows: upstream primer for cpSFYFP: 5'-TCTGCAGGCTTCAACAGCGACAACG-3'; cpSFYFP downstream primer: 5'-GCAGCCCCCGTTGTACTCCAGCTTG-3'.

[0092] (3) System optimization of SAM-specific biosensors In this embodiment, based on SAMsor-0.85, a mutant library was constructed targeting the linker between cpSFYFP and PH1780 in SAMsor-0.85. Combined with high-throughput screening technology mediated by a fluorescence microplate reader, the detection performance of SAMsor-0.85 was systematically optimized. The recombinant plasmid pETDuet-SAMsor-0.85 (i.e., pETDuet-PH1780) was used. 1-234 -cpSFYFP-PH1780 235-285Using a template, the cpSFYFP fragment containing a random linker was amplified by PCR using degenerate primers (such as ON-mutation and OC-mutation described below). Using the recombinant plasmid pETDuet-SAMsor-0.85 as a template, reverse PCR was performed to obtain a linearized pETDuet-SAMsor-0.85 plasmid backbone with breaks at 234V and 235I sites. The recombinant cpSFYFP fragment containing a random linker was ligated to the linearized plasmid backbone using a T5 exonuclease-based DNA assembly method to obtain a recombinant plasmid encoding a sensor variant containing a random linker. The recombinant plasmid was then transformed into... E. coLi BL21(DE3) expression strain, supplemented with 100 mg L -1 The cells were cultured in LB agar plates containing ampicillin. Single colonies exhibiting bright fluorescence were picked from the plates and induced to express protein overnight in the presence of 1 mM IPTG. 15 mL of the bacterial culture was centrifuged at 4 °C and 6,000 rpm for 10 min to collect the cells. The cells were resuspended in 2.5 mL of detection buffer (50 mM Tris-HCl, pH 7.4) and transferred to 48-well deep-well plates. PMSF was added to a final concentration of 1 mM. The cells were disrupted using a Scientz-48TD multichannel ultrasonic disruptor. 1 mL of the disruption buffer was centrifuged at 4 °C and 13,000 rpm for 5 min to collect the supernatant. The supernatant was mixed with 0 μM and 500 μM SAM, respectively. The response of each random mutant to 500 μM SAM was analyzed using a fluorescence microplate reader. Random mutants with high response amplitude and fluorescence intensity were screened, and the sensor sequences were obtained through sequencing. (See attached image.) Figure 8 As shown, among the 279 randomly selected mutants, the sensor variants corresponding to the N-terminal Linker mutation of cpSFYFP to "leucine-threonine" and the C-terminal Linker mutation to "serine-proline-arginine" exhibited the highest response amplitude to SAM. Analysis of their dose-response curves to SAM showed that this sensor variant had a Δ response amplitude to SAM. R max The value increased to 3536.02 ± 351.86%. K d The value was 204.02 ± 20.90 μM (see attached image). Figure 9 It was named SAMsor and used to establish a method for the in vivo detection of SAM and the detection of SAM-dependent methyltransferase activity.

[0093] The SAMsor-0.85 mutant containing a random linker was obtained by PCR amplification. The primers were designed as follows: 0N-mutation: 5'-AATTATGACCCGTGATGTTTTCAACAGCGACAACGTCT-3'; 1N-mutation: 5'-AATTATGACCCGTGATGTTNNBTTCAACAGCGACAACGTCT-3'; 2N-mutation: 5'-AATTATGACCCGTGATGTTNNBNNBTTCAACAGCGACAACGTCT-3'; 3N-mutation: 5'-AATTATGACCCGTGATGTTNNBNNBNNBTTCAACAGCGACAACGTCT-3'; 0C-mutation: 5'-TATGCGGGGTTGCAACAATGTTGTACTCCAGCTTGTGCC-3'; 1C-mutation: 5'-TATGCGGGGTTGCAACAATVNNGTTGTACTCCAGCTTGTGCC-3'; 2C-mutation: 5'-TATGCGGGGTTGCAACAATVNNVNNGTTGTACTCCAGCTTGTGCC-3'; 3C-mutation: 5'-TATGCGGGGTTGCAACAATVNNVNNVNNGTTGTACTCCAGCTTGTGCC-3'; Reverse PCR upstream primer: 5'-AACATCACGGGTCATAATTTCGGCC-3'; Reverse PCR downstream primer: 5'-ATTGTTGCAACCCCGCATATGACCGTTCATGAAGT-3'.

[0094] Example 2: Application of SAM biosensor in real-time detection of SAM in live bacteria (1) Functional identification of SAMsor in Escherichia coli BL21(DE3) The construction in Example 1 E. coLi BL21(DE3)-pETDuet-SAMsor strain was inoculated at a 1% inoculum in 50 mL of solution containing 100 mg L. -1 Ampicillin was cultured in LB medium at 37 °C and 180 rpm until OD. 600 nmApproximately 0.6 mg / L was added to 1 mM IPTG, and SAMsor expression was induced overnight at 23 °C and 160 rpm. An appropriate amount of bacterial culture was collected by centrifugation at 6000 rpm and 4 °C for 2 minutes. The cells were washed twice with phosphate buffer, and then resuspended in phosphate buffer supplemented with 9.5 mM EDTA, 0.1% toluene, and 1% glycerol to OD200. 600 nm The concentration was 5.0. After vortexing for 2 min, the mixture was transferred to 4 ℃ and incubated for 10 h to induce bacterial permeation. The permeated bacterial solution was diluted 10-fold with ddH2O. 99 μL of the diluted bacterial solution was mixed with 1 μL of a gradient concentration of SAM, MTA, SAH, adenosine, and L-methionine in a black flat-bottomed 96-well plate, and the fluorescence intensity was continuously read using a PerkinElmer Ensight fluorescence microplate reader. The instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, emission wavelengths of 528 nm, temperature of 30 ℃, rotation speed of 180 rpm, and detection interval of 1 minute. The results are attached. Figure 10 As shown, in E. coLi The SAMsor expressed in BL21(DE3) can respond in real time to the addition of gradient concentrations of exogenous SAM in a dose-dependent manner, and has high specificity for SAM. It does not show significant changes in fluorescence ratios for MTA, SAH, adenosine, and L-methionine.

[0095] The formulation of the above phosphate buffer (1 L) is as follows: 2.26 g KH2PO4, 4.1 g K2HPO4, 2.24 g NaH2PO4·H2O, 3.34 g Na2HPO4, pH adjusted to 7.0 with NaOH, sterilized at 121 ºC for 20 minutes.

[0096] (2) Real-time and in-situ detection of SAM in Escherichia coli BL21(DE3) using SAMsor exist E. coLi In BL21(DE3), SAM synthase (MetK) catalyzes the reaction of L-methionine with ATP to produce SAM. Expression of SAMsor... E. coLi The fluorescence ratio of BL21(DE3) did not respond to the addition of exogenous L-methionine, while overexpression of MetK under the same conditions significantly increased the fluorescence ratio of SAMsor (see appendix). Figure 11 In addition, in E. coLi In BL21(DE3), the expression of a key enzyme in L-methionine biosynthesis is repressed by the transcriptional regulator MetJ. This is consistent with MetK overexpression. metJ Gene knockout also significantly increased intracellular SAM levels. SAM synthase (RLSS) from mouse liver and SAM hydrolase (T3SH) from bacteriophage are commonly used in... E. coLiOverexpression of RLSS in BL21(DE3) was used to coordinate intracellular SAM levels. As expected, overexpression of RLSS promoted intracellular SAM accumulation under L-methionine-added conditions, while overexpression of T3SH significantly reduced intracellular SAM levels (see appendix). Figure 11 ).

[0097] Example 3: Application of SAM biosensors in real-time detection of SAM in mammalian cells (1) Functional identification of SAMsor in human cell HEK293FT The SAMsor nucleic acid sequence underwent mammalian codon optimization and whole-genome synthesis by General Biosystems (Anhui) Co., Ltd., and a Kozak sequence (5'-GCCACC-3') was added before the start codon, which was then ligated into pcDNA3.1. (+) plasmids, and stored in E. coLi Top 10 strains. Recombinant plasmid pcDNA3.1-SAMsor was extracted from this strain and transfected into HEK293FT cells. 26 h after transfection, cells were washed twice with 1× Hank's balanced salt buffer supplemented with 20 mM HEPES. Cells were then subjected to fluorescence imaging at 30-second intervals under a Zeiss 900 laser confocal microscope. Instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, and emission wavelengths from 497 to 617 nm. Cells were first treated with 80 μM digitalis saponin in the imaging medium to induce cell permeation. After 5 min, SAH, MTA, L-methionine, adenosine, and SAM were added sequentially, or gradient concentrations of SAM were added after 5 min. The response of SAMsor expressed in HEK293FT cells to SAM and different structural analogs was continuously imaged. (See attached image.) Figure 12 As shown, SAMsor can respond in real time to the addition of exogenous SAM in a dose-dependent manner, but has no significant response to SAH, MTA, L-methionine and adenosine.

[0098] (2) Real-time, in-situ detection of SAM in human HEK293FT cells using SAMsor In HEK293FT cells, SAM synthase (MATase) catalyzes the adenylation of L-methionine to produce SAM. MATase transcription is activated by vorinostat (SAHA, N-hydroxy-N'-phenyloctanoic acid), and its activity is inhibited by cyclic leucine or AG-270. (See attached image.) Figure 13 As shown, the addition of exogenous L-methionine or SAHA can increase the fluorescence ratio of SAMsor expressed in HEK293FT cells, while the addition of exogenous cyclic leucine or AG-270 can decrease the fluorescence ratio of SAMsor expressed in HEK293FT cells.

[0099] Example 4: Application of SAM biosensor in the determination of SAM-dependent methyltransferase activity Currently, the detection of SAM-dependent methyltransferase activity mainly relies on complex and cumbersome techniques such as multi-enzyme coupling, proximity scintillation assay (SPA), and homogeneous immunoassay using ALphaLISA. Based on SAMsor-mediated real-time SAM detection technology, a convenient, efficient, and high-throughput method for detecting different SAM-dependent methyltransferase activities can be established. This embodiment uses halogenated methyltransferases and catechol- O Taking the establishment of a method for detecting SAM-dependent methyltransferase activity as an example, this paper illustrates the feasibility of using SAMsor in the determination of SAM-dependent methyltransferase activity. Among these, halogenated methyltransferases derived from *Burkholderia paraknegriensis* (…) are used as examples. ParaburkhoLderia xenovorans The halogenated methyltransferase BxHMT from corn smut ( UstiLago maydis Taking the halogenated methyltransferase UMA as an example, the enzyme activity assay reaction system includes: enzyme activity assay buffer, 5 mM MeOT, 100 μM SAH, 1 μM SAMsor, and an appropriate amount of purified BxHMT protein or UMA protein. (See attached...) Figure 14 As shown, SAMsor can detect the generation of SAM during the catalysis of halomethyltransferases in real time. The specific enzyme activities of BxHMT and UMA for 100 μM SAH were 2.61 U mg and 2.61 U, respectively. -1 and 1.06 U mg -1 .

[0100] Catechol- O -Methyltransferase from drought-sensitive tomato M82 ( SoLanum Lycopersicum Catechols in cv.M82) O -Methyltransferase SLCOMT, from Penali tomato ( SoLanum penneLLii Catechins O -Methyltransferase SpCOMT, from *Internal Rhizobium mulberry* ( Mesorhizobium muLeiense Catechins O α-methyltransferase MesMOMT, from *Xanthomonas aeruginosa* ( Myxococcus xanthus Catechins O Taking methyltransferase MxSafC as an example, the enzyme activity assay reaction system includes: enzyme activity assay buffer, 100 mM MgCl2, 300 μM SAM, 500 μM catechol, 1 μM SAMsor, and appropriate amounts of purified SLCOMT protein, SpCOMT protein, MesMOMT protein, or MxSafC protein. (See attached...) Figure 15 As shown, SAMsor can detect catechol- in real time. OSAM is consumed during the catalysis of methyltransferases. It should be noted that, consistent with literature reports, SLCOMT and SpCOMT are currently the two catechol-O-methyltransferases with the highest specific activity, significantly higher than MesMOMT and MxSafC, at 0.17 U mg. -1 and 0.15 U mg -1 .

[0101] Furthermore, the aforementioned SAMsor-based SAM-dependent methyltransferase activity detection technology can be combined with a random mutant library of methyltransferases to achieve high-throughput screening of highly active methyltransferase mutants. Specifically, assay buffer, SAMsor, substrate, and SAM or SAH are added sequentially to a solution containing different random mutants of methyltransferases, and a fluorescence microplate reader is used to screen for mutants with high SAMsor fluorescence change rates. Additionally, the aforementioned SAMsor-based SAM-dependent methyltransferase activity detection technology can be combined with a library of commercially available bioactive compounds to achieve high-throughput screening of methyltransferase inhibitors. Specifically, assay buffer, methyltransferase, SAMsor, substrate, SAM or SAH, and different bioactive compounds are added sequentially to the detection system, and a fluorescence microplate reader is used to screen for compounds that induce a decrease in the SAMsor fluorescence change rate.

[0102] The formulation of the enzyme activity assay buffer (1 L) is as follows: 6.06 g Tris, pH adjusted to 7.4 with HCl, and impurities removed by filtration through a 0.22 μm aqueous filter membrane before use.

[0103] The nucleotide / amino acid sequences involved in this invention SAMsor's amino acid sequence: (SEQ ID NO.1) SAMsor's nucleic acid sequence: The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises S -Adenosine-L-methionine specific signal recognition elements and fluorescent proteins; Among them, the S The α-adenosine-L-methionine-specific signal recognition element includes the SAM-binding protein PH1780; The fluorescent protein is specifically a cyclic rearranged fluorescent protein, further including any one or more of cyclic rearranged cyan fluorescent protein, cyclic rearranged green fluorescent protein, cyclic rearranged yellow fluorescent protein, and cyclic rearranged red fluorescent protein; Furthermore, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1; Furthermore, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode a fusion protein with the same function.

3. A carrier, characterized in that, The carrier contains at least the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2, the vector of claim 3, or is capable of expressing the fusion protein of claim 1.

5. The fusion protein of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, and / or the host cell of claim 4 in the preparation of detection assays. S -Adenosine-L-methionine or preparation S Applications of α-adenosine-L-methionine biosensors.

6. A kind S -Adenosine-L-methionine biosensor, characterized in that... The biosensor comprises at least the fusion protein of claim 1.

7. The biosensor as described in claim 6, characterized in that, The biosensor also includes other components for... S Reagents, apparatus and / or equipment for the detection of α-adenosine-L-methionine; Furthermore, the reagent contains a detection buffer.

8. An in vitro detection method S The method for -adenosine-L-methionine, characterized in that... The method includes at least: incubating the sample to be tested with the biosensor described in claim 6 or 7, according to... S The fluorescence signal changes of the α-adenosine-L-methionine biosensor are analyzed to assess the presence of α-adenosine-L-methionine in the sample. S -Concentration or presence / absence of adenosine-L-methionine; The sample to be tested can contain any... S -Adenosine-L-methionine or suspected of containing S Biological or environmental samples of adenosine-L-methionine, wherein the biological samples include, but are not limited to, subject serum, urine, cell culture medium and cell lysate; the subject may be human or non-human animal, preferably human; the environmental samples include, but are not limited to, water bodies, soil leachate, food processing liquid and fermentation liquid.

9. An intracellular detection method S The method for -adenosine-L-methionine, characterized in that... The method includes at least: inducing the method described in claim 6. S The α-adenosine-L-methionine biosensor is expressed intracellularly, according to... S α-Adenosine-L-methionine biosensor fluorescence signal changes, analyzing intracellular... S -Concentration or presence / absence of adenosine-L-methionine; Furthermore, the cells are bacterial cells, fungal cells, or animal cells.

10. A method for detecting methyltransferase activity or for high-throughput screening of methyltransferase-related materials, characterized in that, The method includes: incubating the methyltransferase to be tested, substrate, SAM, SAH, methyltransferase-related materials, any one or more of them with the biosensor described in claim 6 or 7, and analyzing the methyltransferase activity or evaluating the efficacy of the methyltransferase-related materials based on the rate of change of fluorescence signal of the SAM biosensor. The methyltransferase-related materials are methyltransferase activators or methyltransferase inhibitors; The methyltransferases include, but are not limited to, Burkholderia paraknockhausensis (… ParaburkhoLderia xenovorans The halogenated methyltransferase BxHMT from corn smut ( UstiLago maydis ) halogenated methyltransferase UMA, from drought-sensitive tomato M82 ( SoLanum Lycopersicum Catechols in cv. M82) O -Methyltransferase SLCOMT, from Penali tomato ( SoLanum penneLLii Catechins O -Methyltransferase SpCOMT, from *Internal Rhizobium mulberry* ( Mesorhizobium muLeiense Catechins O α-methyltransferase MesMOMT, from *Xanthomonas aeruginosa* ( Myxococcus xanthus Catechins O -Methyltransferase MxSafC.

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