New α-ketoglutaric acid optical probe, and preparation method therefor and use thereof

Abstract

The present invention relates to a new α-ketoglutaric acid optical probe, and a preparation method therefor and the use thereof. Specifically, provided is an α-ketoglutaric acid optical probe which contains an α-ketoglutaric acid sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the α-ketoglutaric acid sensitive polypeptide.

Description

Novel α-ketoglutarate optical probe, its preparation method and application

[0001] This application claims priority to application number CN202410916491.8, filed on July 9, 2024, entitled "Novel α-ketoglutarate optical probe and its preparation method and application", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of optical probe technology, and in particular to a novel α-ketoglutarate optical probe, its preparation method, and its application. Background Technology

[0003] α-Ketoglutarate is one of the most important metabolites in the tricarboxylic acid cycle and is essential for the oxidation of glucose, fatty acids, and amino acids. Currently, the main methods for detecting α-ketoglutarate include GC-MS, LC-MS, HPLC, NMR, and enzyme-linked reactions. In addition, in recent years, several protein probes have been developed for the detection of α-ketoglutarate, including the FRET probe OGsor. This series of probes has a maximum response of 95% to α-ketoglutarate and a dynamic range between 100 μM and 10 mM. The mOGsor probe, constructed in 2014, exhibits greater variability and faster kinetics than OGsor, but its response remains relatively small and it has only been applied to *E. coli*. The FRET probe, constructed in 2017, has Kd values ​​of 3 μM and 91 μM, and has only been applied to bacterial detection. Conventional chemical analysis methods involve direct or indirect determination of total α-ketoglutarate in cells, but the procedures are complex, and they cannot meet the requirements for real-time dynamic in vivo monitoring of α-ketoglutarate levels in living cells or even organelles. While existing protein probes have addressed some of the issues, they still suffer from unsuitable affinity, limited response amplitude, and inability to be applied in mammalian cells. Therefore, there is an urgent need to develop new detection methods to achieve simple, rapid, highly specific, real-time, localized, quantitative, and high-throughput detection of α-ketoglutarate both intracellularly and in vitro. Summary of the Invention

[0004] The purpose of this invention is to provide a probe and method for real-time, high-throughput, and quantitative detection of α-ketoglutarate inside and outside cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The first invention provides a variant of α-ketoglutarate-binding protein, wherein:

[0007] (a) Having the sequence shown in SEQ ID NO: 1 and having mutations at one, two, three, four, five or more sites selected from the following: E44, R45, Y46, Y51, I52, V53, D54, said mutations including amino acid modifications, substitutions or deletions.

[0008] (b) is a sequence that has at least 70% sequence identity with the sequence of (a) and has the mutation described in (1) and retains the ability to bind to α-ketoglutarate.

[0009] In one or more embodiments, the α-ketoglutarate-binding protein variant mutation includes mutations at any one, two, three, four, five or more of the following sites: E44, R45, Y46, Y51, I52, V53, D54.

[0010] In one or more embodiments, the mutation includes mutations at sites selected from any of the following groups: (1) E44, (2) R45 and Y46, (3) Y51 and I52, (4) V53 and D54.

[0011] In one or more embodiments, the E44 mutation is I; the R45 mutation is V; the Y46 mutation is S; the Y51 mutation is L; the I52 mutation is W; the V53 mutation is G, A, N, H, L, Q, S or M; and the D54 mutation is H, F, N, W, K or V.

[0012] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) E44I, (2) R45V, (3) Y46S, (4) Y51L, (5) I52W, (6) V53G, (7) V53A, (8) V53N, (9) V53H, (10) V53L, (11) V53Q, (12) V53S, (13) V53M, (14) D54H, (15) D54F, (16) D54N, (17) D54W, (18) D54K, (19) D54V, (20) R45V and Y46S, (21) Y51L and I52W, (22) V53G and D54H, (23) V53A and D54F, (24) V53G and D54F, (25) V53N and D54H, (26) V53H and D54N, (27) V53L and D54W, (28) V53Q and D54K, (29) V53L and D54H, (30) V53G and D54V, (31) V53S and D54N, (32) V53Q and D54N, (33) V53L and D54F, (34) V53M and D54N.

[0013] A second aspect of the present invention provides an optical probe comprising an α-ketoglutarate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located at one or more sites selected from the following sites of the α-ketoglutarate-sensitive polypeptide: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 4 7, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53, wherein the α-ketoglutarate-sensitive polypeptide is an α-ketoglutarate-binding protein or a functional variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof, and the α-ketoglutarate-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

[0014] In one or more embodiments, the α-ketoglutarate-sensitive polypeptide comprises an α-ketoglutarate-binding protein or a functional variant thereof. In one or more embodiments, the α-ketoglutarate-sensitive polypeptide is derived from the GlnK1 protein of *Methanococcus japonicus*.

[0015] In the optical probe of the present invention, the α-ketoglutarate-sensitive polypeptide has: (i) the sequence shown in SEQ ID NO:1, or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity; (ii) a sequence of an α-ketoglutarate-binding protein variant as described in any embodiment herein; or (iii) a sequence having at least 70% sequence identity with the sequence described in (ii) and having the mutation described in (ii) and retaining sensitivity to α-ketoglutarate.

[0016] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one or more embodiments, the fluorescent protein is selected from yellow fluorescent proteins (such as cpYFP shown in SEQ ID NO:2), orange fluorescent proteins (such as cpmOrange shown in SEQ ID NO:3), red fluorescent proteins (such as mKate shown in SEQ ID NO:4 or 8, or mcherry shown in SEQ ID NO:5), green fluorescent proteins (such as cpGFP shown in SEQ ID NO:6), blue fluorescent proteins (such as cpBFP shown in SEQ ID NO:7), and apple red fluorescent proteins (such as cpmApple shown in SEQ ID NO:9). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has the sequence shown in any of SEQ ID NO:2-9.

[0017] In one or more embodiments, the optically active polypeptide has: (a) any of the sequences shown in SEQ ID NO:2-9, (b) the sequence shown in SEQ ID NO:2 and a mutation at the Y1 site, the mutation including modification, substitution or deletion of an amino acid; preferably, the mutation is selected from any one or more of the following: Y1V and Y1E, or (c) a variant sequence that has at least 70% sequence identity with (a) or (b) and retains the function of the fluorescent protein.

[0018] In one or more embodiments, the optically active polypeptide is located at one or more sites of the α-ketoglutarate-sensitive polypeptide selected from the following: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53.

[0019] In one or more embodiments, the optically active polypeptide, as shown in SEQ ID NO:2, is located at one or more sites selected from the following sites in the amino acid sequence of the α-ketoglutarate-binding protein: 43 / 47, 43 / 50, 43 / 51, 44 / 45, 44 / 52, 44 / 53, 45 / 46, 45 / 50, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 48 / 49, 48 / 53.

[0020] In one or more embodiments, the optically active polypeptide is as shown in SEQ ID NO:6, the α-ketoglutarate binding protein is as shown in SEQ ID NO:1, and the optically active polypeptide is located at one or more sites selected from the following amino acid sequences of the α-ketoglutarate binding protein: 43 / 50, 43 / 51, 44 / 53, 45 / 46, 45 / 50, 45 / 51, 46 / 47, 46 / 52, 46 / 53, 47 / 49, 47 / 50.

[0021] In one or more embodiments, the optically active polypeptide is as shown in SEQ ID NO:7, the α-ketoglutarate binding protein is as shown in SEQ ID NO:1, and the optically active polypeptide is located at one or more sites selected from the following amino acid sequences of the α-ketoglutarate binding protein: 43 / 47, 43 / 50, 43 / 51, 44 / 45, 44 / 52, 44 / 53, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 49, 47 / 50, 47 / 51.

[0022] In one or more embodiments, the optically active polypeptide is as shown in SEQ ID NO:9, and the α-ketoglutarate-binding protein is as shown in SEQ ID NO:1, wherein the optically active polypeptide is located at one or more sites selected from the following amino acid sequences of the α-ketoglutarate-binding protein: 43 / 50, 43 / 51, 43 / 53, 44 / 45, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 49, and 50 / 51.

[0023] In one or more embodiments, the optically active polypeptide is located at position 44 / 53 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53G and D54H of the α-ketoglutarate-sensitive polypeptide, (2) V53A and D54F of the α-ketoglutarate-sensitive polypeptide, (3) V53G and D54F of the α-ketoglutarate-sensitive polypeptide, (4) V53N and D54H of the α-ketoglutarate-sensitive polypeptide, (5) V53H of the α-ketoglutarate-sensitive polypeptide. (6) V53L and D54W of α-ketoglutarate-sensitive peptides, (7) V53Q and D54K of α-ketoglutarate-sensitive peptides, (8) V53L and D54H of α-ketoglutarate-sensitive peptides, (9) V53G and D54V of α-ketoglutarate-sensitive peptides, (10) V53S and D54N of α-ketoglutarate-sensitive peptides, (11) V53Q and D54N of α-ketoglutarate-sensitive peptides.

[0024] In one or more embodiments, the optically active polypeptide is located at position 45 / 51 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) E44I of the α-ketoglutarate-sensitive polypeptide and Y1V of the optically active polypeptide; (2) Y51L and I52W of the α-ketoglutarate-sensitive polypeptide.

[0025] In one or more embodiments, the optically active polypeptide is located at position 46 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) R45V and Y46S of the α-ketoglutarate-sensitive polypeptide and Y1E of the optically active polypeptide.

[0026] In one or more embodiments, the optically active polypeptide is located at position 46 / 53 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53L and D54F of the α-ketoglutarate-sensitive polypeptide; (2) V53M and D54N of the α-ketoglutarate-sensitive polypeptide.

[0027] In one or more embodiments, the fluorescent protein has the sequence shown in SEQ ID NO:2 or a variant having a mutation at any one or more of the following sites at its first amino acid position: 1V and 1E, and the optically active polypeptide is located at one or more of the following sites of the α-ketoglutarate-sensitive polypeptide: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47. 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53.

[0028] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of this invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanking a linker of no more than 5 amino acids, such as linkers of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: a first portion B1 of the α-ketoglutarate-sensitive polypeptide, a first linker Y1, an optically active polypeptide A, a second linker Y2, and a second portion B2 of the α-ketoglutarate-sensitive polypeptide. In one embodiment, the optical probe of this invention does not comprise linkers.

[0029] In one embodiment, the optical probe of the present invention further includes a localization sequence for positioning the probe to a specific organelle, such as a cell. Preferred organelles are subcellular organelles, more preferably the cytoplasm, nucleus, and mitochondria.

[0030] In one or more embodiments, the amino acid sequence of the optical probe is shown in any one of SEQ ID NO:10-14.

[0031] This invention also provides fusion peptides comprising the optical probe described in any embodiment herein and other peptides, said other peptides including localization sequences, tags for easy purification, or tags for immunoreaction. In some embodiments, the optical probe described herein further comprises other peptides fused thereto. These other peptides do not affect the properties of the optical probe. In some embodiments, the other peptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other peptides include localization sequences (e.g., peptides that localize the optical probe to different organelles or suborganelles), tags for easy purification, or tags for immunoreaction (e.g., immunoblotting). A linker may be present between the optical probe and other peptides in the fusion peptide described herein.

[0032] The present invention also provides a nucleic acid molecule comprising: (a) a coding sequence for an α-ketoglutarate-binding protein variant, optical probe, or fusion polypeptide as described in any embodiment herein; or (b) a complementary sequence to (a); or (c) a fragment of (a) or (b). The fragment is a primer.

[0033] The present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants encoding the nucleic acid sequences or complementary sequences thereof.

[0034] This invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes a protein variant, optical probe, or fusion peptide as described in any embodiment of this invention.

[0035] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0036] In one or more embodiments, the nucleic acid molecule is operatively linked to an expression control sequence.

[0037] In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

[0038] In another aspect, the present invention provides a host cell that: (1) contains, expresses, or secretes the optical probe or fusion polypeptide described in any embodiment of the present invention; (2) contains the nucleic acid molecule described in any embodiment of the present invention; or (3) contains the nucleic acid construct described in any embodiment of the present invention. The host cell is preferably *Escherichia coli*.

[0039] In another aspect, the present invention provides a detection kit comprising the optical probe, fusion peptide, nucleic acid molecule, nucleic acid construct, or host cell described herein. Optionally, the detection kit may also include other reagents required for detecting α-ketoglutarate using the optical probe.

[0040] In one or more embodiments, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, α-ketoglutarate standard.

[0041] Another aspect of the present invention provides a method for preparing the optical probe or fusion peptide described herein, comprising: culturing the host cell described herein, and isolating the optical probe or fusion peptide from the culture.

[0042] In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the optical probe or fusion polypeptide described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the optical probe or fusion polypeptide.

[0043] Another aspect of the present invention provides a method for detecting α-ketoglutarate in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, and detecting changes in the optically active peptide. The detection can be performed in vivo, in vitro, subcellular, or in situ. The sample may be, for example, blood.

[0044] This article also provides a method for quantifying α-ketoglutarate in a sample, comprising: contacting the optical probe or fusion peptide or host cell described herein with the sample, detecting optical changes in the optically active peptide, and quantifying α-ketoglutarate in the sample based on the optical changes in the optically active peptide.

[0045] Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion peptide or host cell described herein with a candidate compound in a system containing α-ketoglutarate, detecting optical changes in the optically active peptide, and screening the compound based on the optical changes in the optically active peptide. The method can screen compounds in high throughput.

[0046] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a system containing α-ketoglutarate, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of α-ketoglutarate by the cells.

[0047] Another aspect of the present invention provides a method for intracellular and / or extracellular localization of the α-ketoglutarate, comprising: contacting the system containing α-ketoglutarate with the optical probe or the host cell, and detecting optical changes in the optically active polypeptide.

[0048] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0049] Another aspect of the present invention provides the application of the α-ketoglutarate optical probe, fusion peptide, or host cell described herein in the detection of α-ketoglutarate in a sample, screening of compounds, or intracellular and / or extracellular localization of α-ketoglutarate. In one or more embodiments, the localization is real-time localization.

[0050] Another aspect of the present invention provides the use of the α-ketoglutarate optical probe or fusion peptide or polynucleotide or nucleic acid construct described herein in the preparation of a kit for detecting α-ketoglutarate in a sample, screening compounds, or intracellular and / or extracellular localization of α-ketoglutarate. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0052] Figure 1 is an SDS-PAGE image of the exemplary α-ketoglutarate optical probe described in Example 1.

[0053] Figure 2 shows the fluorescence spectral properties of the exemplary α-ketoglutarate optical probe described in Example 7.

[0054] Figure 3 shows the titration curves of the exemplary α-ketoglutarate probe described in Example 7 to different concentrations of α-ketoglutarate.

[0055] Figure 4 is a bar chart showing the specificity of the exemplary α-ketoglutarate optical probe described in Example 7 for the detection of α-ketoglutarate analogs.

[0056] Figure 5 is a photograph showing the subcellular organelle localization of the exemplary α-ketoglutarate optical probe described in Example 8 in mammalian cells.

[0057] Figure 6 is a schematic diagram of the dynamic monitoring of the concentration of α-ketoglutarate in the cytoplasm of mammalian cells using the exemplary α-ketoglutarate optical probe described in Example 8.

[0058] Figure 7 is a dot plot of high-throughput compound screening at the live cell level using the exemplary α-ketoglutarate optical probe described in Example 9.

[0059] Figure 8 is a bar chart showing the quantitative analysis of α-ketoglutarate in mouse and human blood using the exemplary α-ketoglutarate optical probe described in Example 10. Detailed Implementation

[0060] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0061] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0062] As used herein, the terms “α-ketoglutarate-sensitive peptide” or “α-ketoglutarate-responsive peptide” refer to a peptide that responds to α-ketoglutarate, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. “Conformation” is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The α-ketoglutarate-sensitive peptides described herein may also include their functional variants. Functional variants of α-ketoglutarate-sensitive peptides include, but are not limited to, variants that can interact with α-ketoglutarate and thus undergo the same or similar changes as the parental α-ketoglutarate-sensitive peptide.

[0063] As used herein, the term "optical probe" refers to an α-ketoglutarate-sensitive peptide fused to an optically active peptide (e.g., a fluorescent protein), which is operatively inserted into the α-ketoglutarate-sensitive peptide (e.g., an α-ketoglutarate-binding protein). The α-ketoglutarate-binding protein can sense changes in α-ketoglutarate concentration, and its spatial conformation changes during dynamic changes in α-ketoglutarate concentration. The inventors discovered that when an optically active peptide is fused to an α-ketoglutarate-sensitive peptide (e.g., an α-ketoglutarate-binding protein), the conformational change resulting from the α-ketoglutarate-sensitive peptide's specific binding to physiological concentrations of α-ketoglutarate induces a conformational change in the optically active peptide (e.g., a fluorescent protein), thereby altering the optical properties of the optically active peptide. By plotting standard curves using fluorescence data of the fluorescent protein measured at different α-ketoglutarate concentrations, the presence and / or level of α-ketoglutarate can be detected and analyzed. The α-ketoglutarate-sensitive polypeptides described in this invention include, but are not limited to, α-ketoglutarate-binding protein GlnK1 or other peptides with more than 90% homology to it. II Protein mutants. An exemplary GlnK1 protein is shown in SEQ ID NO:1. The exemplary α-ketoglutarate-binding protein GlnK1 described in this invention is derived from *Methanococcus jannaschii*, contains the typical T-loop structure of GlnK proteins, and can bind α-ketoglutarate in the presence of Mg-ATP. The α-ketoglutarate-binding protein can sense changes in α-ketoglutarate concentration; its spatial conformation also changes during dynamic changes in α-ketoglutarate concentration. When describing the optical probes of this invention (e.g., when describing insertion sites or mutation sites), the amino acid residue numbers are all referenced to SEQ ID NO:1.

[0064] A protein-based "optically active peptide" is a peptide capable of emitting fluorescence. Fluorescence is an optical property of an optically active peptide, which can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitude at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient to determine the utility of the fluorescent protein substrate of the present invention in an activity assay. The measurable difference can be determined by determining the amount of any quantitative fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states. The optically active peptides described herein may also include their functional variants. Functional variants of optically active peptides include, but are not limited to, variants that can undergo the same or similar fluorescence property changes as the parent optically active peptide.

[0065] In this article, "response fold" refers to the normalized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the normalized ratio is measured by detecting the change in the ratio of fluorescence intensity at 528nm emission after excitation at 420nm to fluorescence intensity at 528nm emission after excitation at 485nm. 420 / 485 The response factor is calculated as follows:

[0066] Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells not expressing the probe protein. pH-sensitive interference was eliminated by dividing the change in the ratio of probe fluorescence intensity in parallel experimental groups by the change in the ratio of control fluorescence intensity. F = F0 sample -F BLK

[0067] F represents fluorescence intensity. sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK This represents the background fluorescence intensity of samples without expressed fluorescent probes. F 485 F represents the fluorescence intensity of a fluorescent protein sample excited at 485 nm and emitted at 528 nm. 420 This represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 420 nm. The Normalized Ratio represents the ratio of fluorescence intensity of the probe to the α-KG response. controlThe pH-corrected Normalized Ratio represents the ratio of fluorescence intensity of the fluorescent probe to the α-KG response of the pH control probe. It is the fold change in probe or response after pH correction. 420 / 485 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0068] "Connector" or "linking region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the α-ketoglutarate-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2. When the recombinant optical probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The connector sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0069] As used herein, the terms "chromophore," "fluorophore," and "fluorescent protein" are synonymous, referring to proteins that emit fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); as well as the commonly used red fluorescent protein RFP in this field, and its cyclically rearranged derivatives such as cpmApple, cpmOrange, and cpmKate. The sequences of exemplary fluorescent proteins are shown in any of SEQ ID NO:2-9.

[0070] Green fluorescent protein (GFP) was originally extracted from the bioluminescent jellyfish *Aequorea Victoria*. It consists of 238 amino acids and has a molecular weight of approximately 26 kDa. GFP has a unique barrel-shaped structure formed by 12 β-sheet chains, encapsulating a chromophore tripeptide (Ser65-Tyr66-Gly67). In the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxyphenylmethylene imidazolinone, producing fluorescence. GFP fluorescence does not require cofactors and is very stable, making it an excellent imaging tool. GFP has two excitation peaks: a main peak at 395 nm produces emission light at 508 nm, while an excitation peak at 475 nm produces emission light at 503 nm. An exemplary cpGFP is shown in SEQ ID NO:6.

[0071] Yellow fluorescent protein (YFP) is derived from green fluorescent protein (GFP), and its amino acid sequence shares over 90% homology with GFP. The key difference between YFP and GFP lies in the mutation of amino acid position 203 from threonine to tyrosine (T203Y). Compared to the original AvGFP, the main excitation wavelength of YFP is red-shifted to 514 nm, while the emission wavelength changes to 527 nm. Based on this, a site-directed mutation (S65T) at amino acid position 65 of YFP yields the fluorescence-enhanced yellow fluorescent protein (EYFP). cpYFP is obtained by linking the original N-terminus and C-terminus of GFP with a flexible short peptide chain. A new N-terminus and C-terminus are created near the chromophore of the original GFP. Amino acids 145–238 of the original protein are used as the N-terminus of the new protein, and amino acids 1–144 of the original protein are used as the C-terminus. The two fragments are linked by 5–9 flexible short peptide chains. In this invention, the proximal chromophore is preferably located at amino acids Y144 and N145; the flexible short peptide chain is preferably VDGGSGGTG or GGSGG. An exemplary cpYFP sequence is shown in SEQ ID NO:2.

[0072] Red fluorescent protein (RFP) was initially extracted from marine corals. Wild RFP is an oligomeric protein, which is not conducive to fusion expression in organisms. Subsequently, red fluorescent proteins of different color bands were derived from RFP, the most commonly used being mCherry and mKate. An example of mCherry is shown in SEQ ID NO:4 or 8. An example of mCherry is shown in SEQ ID NO:5.

[0073] In other embodiments, the fluorescent protein may also be one or more of the following: blue fluorescent protein cpBFP with an amino acid sequence as shown in SEQ ID NO:7, orange fluorescent protein cpmOrange with an amino acid sequence as shown in SEQ ID NO:3, and apple red fluorescent protein cpmApple with an amino acid sequence as shown in SEQ ID NO:9.

[0074] The α-ketoglutarate optical probe of this invention comprises an α-ketoglutarate-sensitive polypeptide B, such as α-ketoglutarate-binding protein or a variant thereof, and an optically active polypeptide A, such as fluorescent protein or a variant thereof. The optically active polypeptide A is inserted into the α-ketoglutarate-sensitive polypeptide B, dividing B into two parts, B1 and B2, forming a probe structure of type B1-A-B2. The interaction between the α-ketoglutarate-sensitive polypeptide B and α-ketoglutarate leads to an increase in the optical signal of the optically active polypeptide A.

[0075] In the optical probe of this invention, the optically active polypeptide can be located at any position of the α-ketoglutarate-sensitive polypeptide. In one embodiment, the optically active polypeptide is located at any position of the α-ketoglutarate-sensitive polypeptide in the NC direction. Specifically, the optically active polypeptide is located in a flexible region of the α-ketoglutarate-sensitive polypeptide. This flexible region refers to specific structures, such as loop domains, present in the higher-order structure of the protein. These domains have higher mobility and flexibility compared to other higher-order structures of the protein, and their spatial conformation can dynamically change after the protein binds to the ligand. The flexible region described in this invention mainly refers to the region where the insertion site is located in the α-ketoglutarate-binding protein, such as the region of amino acid residues 43-53. For example, the optically active peptide is located at amino acid sequences 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 ...54, 45 / 55, 45 / 51, 45 / 52, 5 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and / or 52 / 53. In a preferred embodiment, the optically active polypeptide is located at amino acid positions 43 / 50, 44 / 45, 44 / 52, 44 / 53, 45 / 46, 45 / 50, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51 and 48 / 49 of the α-ketoglutarate binding protein, as shown in SEQ ID NO:10-14.

[0076] In this paper, at sites represented in the "X / Y" format, the optically active polypeptide has portions of α-ketoglutarate-sensitive polypeptide at both ends. The N-terminus of the optically active polypeptide consists of the N-terminal starting amino acid (e.g., any amino acid from position 1 to 12) to the Xth amino acid of the α-ketoglutarate-sensitive polypeptide sequence, and the C-terminus consists of the Yth amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 112) of the α-ketoglutarate-sensitive polypeptide sequence. If the two numbers in the "X / Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, the insertion site 44 / 45 indicates that the optically active polypeptide is located between amino acids 44 and 45 of the α-ketoglutarate-sensitive polypeptide. If the two numbers in the site represented by “X / Y” are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acid between the amino acids indicated by the number. For example, insertion site 47 / 49 indicates that the optically active polypeptide replaces amino acid 48 of the α-ketoglutarate-sensitive polypeptide, and insertion site 43 / 50 indicates that the optically active polypeptide replaces amino acids 44-49 of the α-ketoglutarate-sensitive polypeptide.

[0077] In one or more embodiments, the optical probe comprises, from N-terminus to C-terminus, residues 1-X of SEQ ID NO:1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO:2-9, and residues Y-112 of SEQ ID NO:1, wherein X and Y are selected from any group of the following: (1) X is 43, Y is 44, (2) X is 43, Y is 45, (3) X is 43, Y is 46, (4) X is 43, Y is 47, (5) X is 43, Y is 48, (6) X is 43, Y is 49, (7) X is 43, Y is 50, (8) X is 43, Y is 51, (9) X is 43, Y is 52, (10) X is 43, Y is 53, (11) X is 44, Y is 45, (12) X is 44, Y is 46, (13) X is 44. Y is 47, (14) X is 44, Y is 48, (15) X is 44, Y is 49, (16) X is 44, Y is 50, (17) X is 44, Y is 51, (18) X is 44, Y is 52, (19) X is 44, Y is 53, (20) X is 45, Y is 46, (21) X is 45, Y is 47, (22) X is 45, Y is 48, (23) X is 45, Y is 49, (24) X is 45, Y is 50, (25) X is 45, Y is 51, (26) X is 45, Y is 52, (27) X is 45, Y is 53, (28) X is 46, Y is 47, (29) X is 46, Y is 48, (30) X is 46, Y is 49, (31) X is 46, Y is 50, (32) X is 46, Y is 51, (33) X is 46, Y is 52, (34) X is 46, Y is 53, (35) X is 47, Y is 48, (36) X is 47, Y is 49, (37) X is 47, Y is 50, (38) X is 47, Y is 51, (39) X is 47, Y is 52, (40) X is 47, Y is 53, (41) X is 48, Y is 4 9, (42) X is 48, Y is 50, (43) X is 48, Y is 51, (44) X is 48, Y is 52, (45) X is 48, Y is 53, (46) X is 49, Y is 50, (47) X is 49, Y is 51, (48) X is 49, Y is 52, (49) X is 49, Y is 53, (50) X is 50, Y is 51, (51) X is 50, Y is 52, (52) X is 50, Y is 53, (53) X is 51, Y is 52, (54) X is 51, Y is 53, (55) X is 52, Y is 53.

[0078] Preferably, the optically active polypeptide is located at one or more sites selected from the following groups of the α-ketoglutarate-sensitive polypeptide: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 4 7, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53, and 52 / 53, exemplary optically active peptides located at amino acid positions 44 / 45 of the α-ketoglutarate-binding protein, the sequence of which is shown in SEQ ID NO:10.

[0079] When referring to a polypeptide or protein, the term "variant" or "mutant" as used in this invention includes variants that have the same function as the polypeptide or protein but with a different sequence. These variants include, but are not limited to, deletions, insertions, and / or substitutions of one or more amino acids (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) in the sequence of the polypeptide or protein, and sequences obtained by adding one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to its carboxyl terminus and / or amino terminus. It is not intended to be theoretically limited to changes in amino acid residues that do not alter the overall conformation and function of the polypeptide or protein, i.e., functionally conserved mutations. For example, in the art, substitution with amino acids of similar or comparable properties generally does not change the function of the polypeptide or protein. In the art, amino acids of similar properties often refer to amino acid families with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of the polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids. As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic sites of factor Xa, thrombin, or enterokinase. Variants of polypeptides or proteins can include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants.These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein. An exemplary GlnK1 protein is the full-length amino acid sequence shown in SEQ ID NO:1, which retains its binding function to α-ketoglutarate and does not affect the changes in optical properties of the inserted optically active polypeptide in response to binding with α-ketoglutarate.

[0080] The optical probe of the present invention may comprise a mutated α-ketoglutarate-sensitive polypeptide, wherein a mutated α-ketoglutarate-binding protein variant of SEQ ID NO:1 or a truncated variant thereof exhibits binding activity different from α-ketoglutarate at sites selected from the following: E44, R45, Y46, Y51, I52, V53, D54. The amino acid mutation includes modification, substitution, or deletion of amino acids. In a preferred embodiment, the mutation of the α-ketoglutarate-binding protein variant includes mutations at sites selected from any of the following groups: (1) E44, (2) R45 and Y46, (3) Y51 and I52, (4) V53 and D54.

[0081] In one or more embodiments, as an example, in SEQ ID NO:1 or a truncated variant thereof, E44 mutates to I. In one or more embodiments, R45 mutates to V. In one or more embodiments, Y46 mutates to S. In one or more embodiments, Y51 mutates to L. In one or more embodiments, I52 mutates to W. In one or more embodiments, V53 mutates to G, A, N, H, L, Q, S, or M. In one or more embodiments, D54 mutates to H, F, N, W, K, or V.

[0082] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) E44I, (2) R45V, (3) Y46S, (4) Y51L, (5) I52W, (6) V53G, (7) V53A, (8) V53N, (9) V53H, (10) V53L, (11) V53Q, (12) V53S, (13) V53M, (14) D54H, (15) D54F, (16) D54N, (17) D54W, (18) D54K, (19) D54V, (20) R45V and Y46S, (21) Y51L and I52W, (22) V53G and D54H, (23) V53A and D54F, (24) V53G and D54F, (25) V53N and D54H, (26) V53H and D54N, (27) V53L and D54W, (28) V53Q and D54K, (29) V53L and D54H, (30) V53G and D54V, (31) V53S and D54N, (32) V53Q and D54N, (33) V53L and D54F, (34) V53M and D54N. This invention provides α-ketoglutarate-binding protein variants having these mutations, as well as optical probes containing such α-ketoglutarate-binding protein variants as α-ketoglutarate-sensitive peptides.

[0083] The optical probe of the present invention may comprise a mutated optically active polypeptide. In some embodiments, the mutated optically active polypeptide has a mutation at the Y1 site, the mutation including modification, substitution, or deletion of an amino acid, and in one or more embodiments, the mutation is Y1V or Y1E.

[0084] In one or more embodiments, the optical probe comprises any of the amino acid sequences SEQ ID NO:10-14 or variations thereof. In one or more embodiments, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-14. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar to or identical to any of the amino acid sequences SEQ ID NO:11-14.

[0085] In one or more embodiments, the fluorescent protein has the sequence shown in SEQ ID NO:2 or a variant having a mutation at any one or more of the following sites at its first amino acid position: 1V and 1E. The optically active polypeptide is located at one or more sites selected from the following sites of the α-ketoglutarate-sensitive polypeptide: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 4 7, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53.

[0086] In some embodiments, the optically active polypeptide is located at position 44 / 53 of the α-ketoglutarate-binding protein, and the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity. The optically active polypeptide has SEQ ID NO:1. The α-ketoglutarate has any of the sequences shown in NO:2-9, and has mutations selected from any of the following: (1) V53G and D54H, (2) V53A and D54F, (3) V53G and D54F, (4) V53N and D54H, (5) V53H and D54N, (6) V53L and D54W, (7) V53Q and D54K, (8) V53L and D54H, (9) V53G and D54V, (10) V53S and D54N, (11) V53Q and D54N.

[0087] In some embodiments, the optically active polypeptide is located at position 45 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the α-ketoglutarate has a mutation selected from any of the following: (1) E44I; (2) Y51L and I52W; and optionally the optically active polypeptide is mutated to (3) Y1V.

[0088] In some embodiments, the optically active polypeptide is located at position 46 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has the sequence shown in any of SEQ ID NO:2-9, and the α-ketoglutarate has a mutation selected from any of the following: (1) R45V and Y46S; and optionally the optically active polypeptide is mutated to (2) Y1E.

[0089] In some embodiments, the optically active polypeptide is located at position 46 / 53 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the α-ketoglutarate has a mutation selected from any of the following: (1) V53L and D54F; (2) V53M and D54N.

[0090] In one or more embodiments, the optically active polypeptide is located at position 44 / 53 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53G and D54H of the α-ketoglutarate-sensitive polypeptide, (2) V53A and D54F of the α-ketoglutarate-sensitive polypeptide, (3) V53G and D54F of the α-ketoglutarate-sensitive polypeptide, (4) V53N and D54H of the α-ketoglutarate-sensitive polypeptide, (5) V53H of the α-ketoglutarate-sensitive polypeptide. (6) V53L and D54W of α-ketoglutarate-sensitive peptides, (7) V53Q and D54K of α-ketoglutarate-sensitive peptides, (8) V53L and D54H of α-ketoglutarate-sensitive peptides, (9) V53G and D54V of α-ketoglutarate-sensitive peptides, (10) V53S and D54N of α-ketoglutarate-sensitive peptides, (11) V53Q and D54N of α-ketoglutarate-sensitive peptides.

[0091] In one or more embodiments, the optically active polypeptide is located at position 45 / 51 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) E44I of the α-ketoglutarate-sensitive polypeptide and Y1V of the optically active polypeptide; (2) Y51L and I52W of the α-ketoglutarate-sensitive polypeptide.

[0092] In one or more embodiments, the optically active polypeptide is located at position 46 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) R45V and Y46S of the α-ketoglutarate-sensitive polypeptide and Y1E of the optically active polypeptide.

[0093] In one or more embodiments, the optically active polypeptide is located at position 46 / 53 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53L and D54F of the α-ketoglutarate-sensitive polypeptide; (2) V53M and D54N of the α-ketoglutarate-sensitive polypeptide.

[0094] In two or more polypeptide or nucleic acid sequences, the term "identity" or "percentage of identity" refers to the similarity of two or more sequences or subsequences, or the similarity of a certain percentage of amino acid residues or nucleotides in a specified region, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, through manual alignment and visual inspection (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, within a comparison window or specified region). For example, preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0095] As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.

[0096] The optical probe provided by this invention comprises any of the amino acid sequences SEQ ID NO:10-13 or variations thereof. In one embodiment, the optical probe provided by this invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-13. In a preferred embodiment, the optical probe provided by this invention comprises a sequence substantially similar to or identical to any of the amino acid sequences SEQ ID NO:10-13.

[0097] As used herein, the terms “functional variant,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., GlnK1 protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins of this invention (e.g., GlnK1 protein or fluorescent protein) may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (e.g., a secreted sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional variants, derivatives, and analogs are within the scope well known to those skilled in the art.

[0098] The difference between the analogue and the original polypeptide or protein can be a difference in amino acid sequence, a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0099] The analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the α-ketoglutarate-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the protein to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolysis or optimize their solubility are also included.

[0100] The present invention also provides a method for preparing the above-mentioned α-ketoglutarate optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the α-ketoglutarate optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the α-ketoglutarate optical probe.

[0101] As used herein, the term "nucleic acid" or "nucleotide" can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" as used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acids of this invention may comprise a nucleotide sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the described nucleic acid sequence. This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used herein, a “nucleic acid fragment” contains at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0102] The full-length sequence or fragments of the optical probe or fusion protein of this invention can generally be obtained by PCR amplification, artificial synthesis, or recombinant methods. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in this invention, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequence. When the nucleotide sequence is greater than 2500 bp, it is preferable to perform 2 to 6 PCR amplifications, and then splice the fragments from each amplification in the correct order. This invention does not impose any special limitations on the PCR amplification procedure and system; conventional PCR amplification procedures and systems in the art can be used. Recombinant methods can also be used to obtain the relevant sequence in large quantities. This typically involves cloning it into a vector, transforming it into cells, and then isolating and purifying the relevant polypeptide or protein from the proliferated host cells using conventional methods. Furthermore, artificial synthesis methods can be used to synthesize the relevant sequence, especially when the fragment length is short. In this invention, when the nucleotide sequence of the optical probe is less than 2500 bp, artificial synthesis methods can be used. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, without other special requirements. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of this invention (or its functional variants, derivatives, or analogues) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of this invention using methods such as mutagenic PCR or chemical synthesis.

[0103] After obtaining the nucleotide sequence encoding an optical probe, this invention incorporates the nucleotide sequence encoding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. These vectors can replicate and stably express in a host cell. An important characteristic of these recombinant vectors is that they typically contain an expression control sequence. The term "expression control sequence" used herein refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. Recombinant vectors applicable in this invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operative linking" refers to the connection of the target nucleotide sequence to a regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the retroviral LTR, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector can be a commercially available pCDF vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and XhoI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

[0104] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc., including but not limited to the aforementioned host cells. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, such cells are well known and commonly used in the art. An exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0105] The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MgCl2 method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0106] This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the α-ketoglutarate optical probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0107] In this invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the α-ketoglutarate fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the optical probe is separated using His-tagged affinity chromatography.

[0108] This invention also provides the application of the α-ketoglutarate optical probe in real-time localization, quantitative detection, and high-throughput compound screening of α-ketoglutarate. In one aspect, the α-ketoglutarate optical probe is preferably linked to signal peptides at different sites within the cell, transferred into the cell, and its real-time localization is achieved by detecting the intensity of fluorescence signals within the cell; the corresponding α-ketoglutarate is then quantitatively detected using a standard titration curve. The standard titration curve of α-ketoglutarate described in this invention is plotted based on the fluorescence signals obtained by the α-ketoglutarate optical probe at different concentrations of α-ketoglutarate. The α-ketoglutarate optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of α-ketoglutarate, thus improving accuracy. In high-throughput compound screening, the α-ketoglutarate optical probe of this invention adds different compounds to the cell culture medium, measures changes in α-ketoglutarate content, and thereby screens out compounds that affect changes in α-ketoglutarate content. The application of the α-ketoglutarate optical probe described in this invention in the real-time localization, quantitative detection, and high-throughput compound screening of α-ketoglutarate is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.

[0109] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0110] Partial Specific Implementation Plan

[0111] Project 1. A variant of an α-ketoglutarate-binding protein, which:

[0112] (a) Having the sequence shown in SEQ ID NO: 1 and having mutations at one, two, three, four, five or more sites selected from the following: E44, R45, Y46, Y51, I52, V53, D54, said mutations including amino acid modifications, substitutions or deletions.

[0113] (b) is a sequence that has at least 70% sequence identity with (a) and has the mutation described in (1) and retains the ability to bind to α-ketoglutarate.

[0114] Preferably, the α-ketoglutarate binding protein variant mutation includes mutations selected from any one, two, three, four, five or more of the following sites: E44, R45, Y46, Y51, I52, V53, D54;

[0115] More preferably, the mutation includes mutations at sites selected from any of the following groups: (1) E44, (2) R45 and Y46, (3) Y51 and I52, (4) V53 and D54;

[0116] More preferably, the E44 mutation is I; the R45 mutation is V; the Y46 mutation is S; the Y51 mutation is L; the I52 mutation is W; the V53 mutation is G, A, N, H, L, Q, S or M; and the D54 mutation is H, F, N, W, K or V.

[0117] More preferably, the mutation comprises mutations selected from any of the following groups: (1) E44I, (2) R45V, (3) Y46S, (4) Y51L, (5) I52W, (6) V53G, (7) V53A, (8) V53N, (9) V53H, (10) V53L, (11) V53Q, (12) V53S, (13) V53M, (14) D54H, (15) D54F, (16) D54N, (17) D54W, (18) D54K, (19) D54V, (20) R45V and Y46S ,(21)Y51L and I52W,(22)V53G and D54H,(23)V53A and D54F,(24)V53G and D54F,(25)V53N and D54H,(26)V53H and D54N,(27)V53L and D54W,(28)V53Q and D54K,(29)V53L and D54H,(30)V53G and D54V,(31)V53S and D54N,(32)V53Q and D54N,(33)V53L and D54F,(34)V53M and D54N。

[0118] Project 2. An optical probe comprising an α-ketoglutarate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located at one or more sites of the α-ketoglutarate-sensitive polypeptide selected from the following: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 5 2, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53, wherein the α-ketoglutarate-sensitive polypeptide is an α-ketoglutarate-binding protein or a functional variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof.

[0119] The α-ketoglutarate-sensitive polypeptide has the following characteristics:

[0120] (i) The sequence shown in SEQ ID NO:1, or a sequence that has at least 70% sequence identity with it and retains its activity of binding to α-ketoglutarate,

[0121] (ii) The sequence of the α-ketoglutarate-binding protein variant described in Project 1, or

[0122] (iii) Having at least 70% sequence identity with the sequence described in (ii) and having the mutation described in (ii) while retaining the sequence sensitive to α-ketoglutarate,

[0123] Preferably, the optically active polypeptide has:

[0124] (a) Any of the sequences shown in SEQ ID NO:2-9

[0125] (b) The sequence shown in SEQ ID NO:2 and having a mutation at the Y1 site, the mutation including modification, substitution, or deletion of an amino acid; preferably, the mutation is selected from any one or more of the following: Y1V and Y1E, or

[0126] (c) A variant sequence that has at least 70% sequence identity with (a) or (b) and retains the function of the fluorescent protein.

[0127] Item 3. The optical probe as described in Item 2, characterized in that the optically active polypeptide is located at position 44 / 53 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence having at least 70% sequence identity with it and retaining α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53G and D54H of the α-ketoglutarate-sensitive polypeptide, (2) V53A and D54F of the α-ketoglutarate-sensitive polypeptide, (3) V53G and D54F of the α-ketoglutarate-sensitive polypeptide, (4) V53N and D54H of the α-ketoglutarate-sensitive polypeptide, (5) V53H and D54F of the α-ketoglutarate-sensitive polypeptide. D54N, (6) V53L and D54W of α-ketoglutarate-sensitive peptides, (7) V53Q and D54K of α-ketoglutarate-sensitive peptides, (8) V53L and D54H of α-ketoglutarate-sensitive peptides, (9) V53G and D54V of α-ketoglutarate-sensitive peptides, (10) V53S and D54N of α-ketoglutarate-sensitive peptides, (11) V53Q and D54N of α-ketoglutarate-sensitive peptides; or

[0128] The optically active polypeptide is located at position 45 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) E44I of the α-ketoglutarate-sensitive polypeptide and Y1V of the optically active polypeptide; (2) Y51L and I52W of the α-ketoglutarate-sensitive polypeptide; or

[0129] The optically active polypeptide is located at position 46 / 51 of the α-ketoglutarate-binding protein. The α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity. The optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9. Furthermore, the optical probe contains the following mutations: R45V and Y46S of the α-ketoglutarate-sensitive polypeptide and Y1E of the optically active polypeptide; or

[0130] The optically active polypeptide is located at position 46 / 53 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53L and D54F of the α-ketoglutarate-sensitive polypeptide; (2) V53M and D54N of the α-ketoglutarate-sensitive polypeptide.

[0131] Item 4. A fusion polypeptide comprising the optical probe described in Item 2 or 3 and other polypeptides, said other polypeptides including a localization sequence, a tag for easy purification, or a tag for an immune response.

[0132] Item 5. A nucleic acid molecule comprising: (a) the coding sequence of the α-ketoglutarate-binding protein variant of Item 1, the coding sequence of the optical probe of Item 2 or 3, or the coding sequence of the fusion polypeptide of Item 4, or (b) the complementary sequence of (a).

[0133] Project 6. Nucleic acid constructs, including the nucleic acid molecules described in Project 5.

[0134] Preferably, the nucleic acid construct is a cloning vector, expression vector, or recombinant vector.

[0135] Item 7. A host cell, wherein the host cell:

[0136] (1) Contains, expresses or secretes the optical probe described in item 2 or 3 or the fusion polypeptide described in item 4;

[0137] (2) Contains the nucleic acid molecules described in item 5; and / or

[0138] (3) Includes the nucleic acid constructs described in Project 6.

[0139] Item 8. A test kit comprising:

[0140] (1) The optical probe described in item 2 or 3 or the fusion peptide described in item 4.

[0141] (2) The nucleic acid molecules described in Project 5

[0142] (3) The nucleic acid constructs described in Project 6

[0143] (4) The host cell described in Project 7.

[0144] The detection kit may optionally also include other reagents required for the detection of α-ketoglutarate using an optical probe.

[0145] Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and α-ketoglutarate standard.

[0146] Project 9. A method for preparing the optical probe described in Project 2 or 3 or the fusion polypeptide described in Project 4, comprising: culturing the host cell described in Project 7, and isolating the optical probe or fusion polypeptide from the culture.

[0147] Item 10. The use of the optical probes described in Item 2 or 3, the fusion peptides described in Item 4, the nucleic acid molecules described in Item 5, the nucleic acid constructs described in Item 6, and / or the host cells described in Item 7 in detecting α-ketoglutarate in samples, screening compounds, or intracellular and / or extracellular localization of α-ketoglutarate.

[0148] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0149] Example

[0150] The α-ketoglutaric acid optical probe provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0151] I. Experimental Materials and Reagents

[0152] The embodiments primarily employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well-known to those skilled in the art. Examples include: Jane Rothcomems et al.'s *Molecular Cloning: A Laboratory Manual* (3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s *Animal Cell Culture: A Basic Technique Guide* (5th edition, translated by Zhang Jingbo, Xu Cunshuan et al.); and J.S. Bonnie Fesnon, M. Dassault et al.'s *A Concise Laboratory Manual of Cell Biology* (translated by Zhang Jingbo et al.). Those skilled in the art can readily implement this invention by making minor modifications and variations based on the following embodiments, and all such modifications and variations fall within the scope of the claims of this application.

[0153] The pCDF-cpYFP and pCDF-α-ketoglutarate binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all sequenced by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase from Tiangen Biotech (Beijing) Co., Ltd., and the PrimeSTAR DNA polymerase from TaKaRa. Corresponding polymerase buffers and dNTPs were included with each purchase. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligase and T4 phosphorylase (T4 PNK), were purchased from Fermentas, and corresponding buffers were included with each purchase. The Lip2000 transfection kit was purchased from Invitrogen. Amino acids, including α-ketoglutarate, were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salts, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from WHB.

[0154] The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0155] The main instruments used in the examples include: Biotek Synergy 2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0156] II. Molecular Biology Methods and Cellular Experimental Methods

[0157] II.1 Polymerase Chain Reaction (PCR):

[0158] 1. PCR amplification of the target fragment:

[0159] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system for PCR amplification is shown in Table 1, and the amplification program is shown in Table 2.

[0160] Table 1: PCR amplification reaction system

[0161] Table 2. PCR amplification program

[0162] 2. Long fragment (>2500bp) amplification PCR:

[0163] The long-fragment amplification used in this invention is primarily a reverse PCR amplification vector, a technique used in the following embodiments to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.

[0164] Table 3. PCR reaction system for long fragment (>2500bp) amplification

[0165] Table 4. PCR amplification program for long fragments (>2500bp)

[0166] Table 5. PCR amplification program for long fragments (>2500bp)

[0167] II.2 Endonuclease digestion reaction:

[0168] The systems for double enzyme digestion of plasmid vectors are shown in Table 6, where n represents the amount of sterile ultrapure water (μL) required to bring the system to its total volume.

[0169] Table 6. Plasmid vector double enzyme digestion system

[0170] II.3 Phosphorylation of DNA fragments at the 5' end

[0171] Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is shown in Table 7, where T4 PNK is an abbreviation for T4 polynucleotide kinase, used for the addition reaction of the 5' phosphate group of the DNA molecule.

[0172] Table 7. Phosphorylation reaction system

[0173] II.4 Ligation reaction between target fragment and vector

[0174] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0175] 1. Blunt-end ligation of blunt-ended short fragments and linearized vectors

[0176] The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

[0177] Table 8. Reaction systems for blunt-ended fragment ligation

[0178] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0179] DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is shown in Table 9, where the mass ratio of PCR product fragment to vector double digestion product is approximately between 2:1 and 6:1.

[0180] Table 9. Viscous end-linking reaction system

[0181] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0182] The 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system is shown in Table 10.

[0183] Table 10. Self-cyclization linkage reaction systems

[0184] II.5 Preparation and Transformation of Competent Cells

[0185] Preparation of competent cells:

[0186] 1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with a shaker.

[0187] 2. Take 0.5-1 mL of the overnight culture and transfer it to 50 mL of LB medium. Incubate at 37°C and 220 rpm for 3 to 5 hours until the OD600 reaches 0.5.

[0188] 3. Pre-cool the cells in an ice bath for 2 hours.

[0189] 4. Centrifuge at 4000 rpm for 10 minutes at 4℃.

[0190] 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, and add resuspending buffer to a final volume of 50 mL after homogenization.

[0191] 6. Ice bath for 45 minutes.

[0192] 7. Centrifuge at 4000 rpm for 10 minutes at 4℃, and resuspend the bacteria in 5 mL of ice-cold storage buffer.

[0193] 8. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

[0194] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0195] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0196] Transformation of competent cells:

[0197] 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0198] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0199] 3. Place the bacterial solution in a 42°C water bath for 90 seconds to heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

[0200] 4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

[0201] 5. Centrifuge the bacterial culture at 4000 rpm for 3 minutes, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0202] II.6 Protein Expression, Purification, and Fluorescence Detection

[0203] 1. Transform the expression vector (e.g., the pCDF-based α-ketoglutarate optical probe expression vector) into BL21(DE3) cells, incubate upside down overnight, pick clones from the plate into 250ml Erlenmeyer flasks, place them on a shaker at 37℃ and incubate at 220rpm until OD=0.4-0.8, add 1 / 1000 (v / v) of IPTG (1M), and induce expression at 18℃ for 24-36 hours.

[0204] 2. After induction of expression, centrifuge at 4000 rpm for 30 minutes to collect the bacteria. Resuspend the bacterial pellet in 50 mM phosphate buffer and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0205] 3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 100mM HEPES buffer (pH 7.4).

[0206] 4. After SDS-PAGE identification of the purified protein, the probe was diluted with assay buffer (100mM HEPES, 100mM NaCl, pH 7.4) to a final concentration of 0.2-5μM. α-Ketoglutarate was prepared into a stock solution with a final concentration of 50mM using assay buffer (100mM HEPES, 100mM NaCl, pH 7.4).

[0207] 5. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add α-ketoglutarate titration, and measure the fluorescence intensity of the protein at 528 nm emission after excitation by 420 nm light and at 528 nm emission after excitation by 485 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0208] 6. Take 100 μl of 1 μM protein solution, incubate at 37 °C for 10 minutes, add α-ketoglutarate, and measure the absorption and fluorescence spectra of the protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0209] II.7 Transfection and Fluorescence Detection of Mammalian Cells

[0210] 1. The pCDNA3.1+-based α-ketoglutarate optical probe plasmid was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) and cultured in a 37°C, 5% CO2 cell culture incubator. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

[0211] 2. After the expression was induced, the adherent HeLa cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

[0212] Example 1: α-Ketoglutarate binding protein particles

[0213] The GlnK1(1-112) gene (SEQ ID NO:1) from *Methanococcus japonicus* was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with BamHI and XhoI enzymes. The pCDF vector was also double-digested with the corresponding enzymes. After ligation with T4 DNA ligase, the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

[0214] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0215] In this embodiment, the following sites were selected for insertion into cpYFP based on pCDF-GlnK1 to obtain the corresponding pCDF-GlnK1-cpYFP plasmids: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 4 9, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53, 52 / 53.

[0216] The cpYFP DNA fragment was generated using PCR, and a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-GlnK1 vector, whose 5' and 3' ends contained sequences (15 bp–25 bp) completely identical to those at the cpYFP terminals. The linearized pCDF-GlnK1 and cpYFP fragments underwent homologous recombination using Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 50 μg / mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee or BGI Genomics.

[0217] After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 41 kDa. This size is consistent with the size of the pCDF-cpYFP-GlnK1 fusion protein containing the His-tag purified label. The results are shown in Figure 1.

[0218] The supernatant of fragmented *E. coli* expressing the GlnK1-cpYFP fusion protein was used for α-ketoglutarate response screening. The detection signal of the fusion fluorescent protein containing 10 mM α-ketoglutarate was divided by the detection signal of the fusion fluorescent protein without α-ketoglutarate. The results are shown in Table 11. The detection results showed that optical probes with an α-ketoglutarate response greater than 1.3 times or less than 0.7 times compared to the control were inserted at sites 43 / 47, 43 / 50, 43 / 51, 44 / 45, 44 / 52, 44 / 53, 45 / 46, 45 / 50, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 48 / 49, and 48 / 53.

[0219] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0220] Following the method described in Example 2, cpYFP was replaced with cpGFP to construct an α-ketoglutarate green fluorescent protein probe. As shown in Table 11, the detection results indicate that optical probes with a response to α-ketoglutarate greater than 1.3 times or less than 0.7 times compared to the control were inserted at sites 43 / 50, 43 / 51, 44 / 53, 45 / 46, 45 / 50, 45 / 51, 46 / 47, 46 / 52, 46 / 53, 47 / 49, and 47 / 50.

[0221] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0222] Following the method described in Example 2, cpYFP was replaced with cpBFP to construct an α-ketoglutarate blue fluorescent protein probe. As shown in Table 11, the detection results indicate that optical probes with a response to α-ketoglutarate greater than 1.3 times or less than 0.7 times compared to the control were inserted at sites 43 / 47, 43 / 50, 43 / 51, 44 / 45, 44 / 52, 44 / 53, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 49, 47 / 50, and 47 / 51.

[0223] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0224] Following the method described in Example 2, cpYFP was replaced with cpmApple to construct an α-ketoglutarate red fluorescent protein probe. As shown in Table 11, the detection results indicate that optical probes with a response to α-ketoglutarate greater than 1.2 times or less than 0.8 times compared to the control were inserted at sites 43 / 50, 43 / 51, 43 / 53, 44 / 45, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 51, 46 / 47, 46 / 51, 46 / 52, 46 / 53, 47 / 49, and 50 / 51.

[0225] Table 11

[0226] Standardized fluorescence signal ratio

[0227] Example 6: Expression and detection of mutated cpYFP optical probe

[0228] For the optical probes obtained in Example 2 that responded to α-ketoglutarate more than 1.5 times or less than 0.7 times, namely the eight optical probes inserted at sites 44 / 45, 44 / 53, 45 / 46, 45 / 51, 46 / 51, 46 / 53, 47 / 48, and 47 / 49, the probes were linearized by reverse PCR, and the sequences of the mutation sites were introduced into the primers. The resulting PCR products were homologously recombinated using Hieff Clone Enzyme to establish a mutant library. The recombinant plasmid of the mutant library was transformed into BL21(DE3) to induce expression. The response to α-ketoglutarate was screened using the supernatant of E. coli expressing the probe protein. The detection signal of the fusion fluorescent protein containing 10 mM α-ketoglutarate was divided by the detection signal of the fusion fluorescent protein without α-ketoglutarate (the system contained 2 mM Mg-ATP). The results are shown in Table 12. The detection results show that the optical probes that responded to α-ketoglutarate more than 4 times are shown below.

[0229] Table 12

[0230] Example 7: Performance of optical probe mutants

[0231] For example, purified α-ketoglutarate optical probes were treated with 0 mM and 5 mM α-ketoglutarate for 10 minutes, respectively, and then fluorescence spectra were detected using a fluorescence spectrophotometer. For excitation spectrum determination: with a fixed emission wavelength of 540 nm, the excitation spectrum from 350 to 500 nm was recorded, with readings every 1 nm. The results showed that the probe had two excitation peaks at 420 nm and 490 nm, as shown in Figure 2, A. For emission spectrum determination: with fixed excitation wavelengths of 420 nm and 490 nm, the emission spectrum from 500 to 600 nm was recorded, with readings every 1 nm. The results showed that the probe's emission peak was at 515 nm. After adding 5 mM α-ketoglutarate, the fluorescence intensity under 420 nm excitation decreased / increased by 1.6 times compared to the state without α-ketoglutarate; and under 490 nm excitation, the fluorescence intensity decreased / increased by 3.3 times compared to the state without α-ketoglutarate. (See Figures 2, B and 2, C).

[0232] The α-ketoglutarate optical probes listed in Table 12 of Example 6 were used for α-ketoglutarate detection at concentration gradients (0-10 mM). After treating the probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was measured. The probe titration results are shown in Figure 3, indicating that different mutants have different affinities for α-ketoglutarate.

[0233] The probes of some examples in Table 12, such as GlnK1-Y51L&I52W, GlnK1-V53L&D54F and GlnK1-V53M&D54N, were specifically tested for reactivity with substrates such as tricarboxylic acid cycle substrates and α-ketoglutaric acid analogs. The results showed that they had good specificity, as shown in Figure 4.

[0234] Example 8: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0235] In this embodiment, different localization signal peptides were fused with the optical probes GlnK1-Y51L&I52W to localize the optical probes to different organelles. HEK293 cells were transfected with plasmids fused with different localization signal peptides for 36 hours, washed with PBS, and placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. The results are shown in Figure 5. The α-ketoglutarate optical probes, by fusing with different specific localization signal peptides, can localize to subcellular organelles including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion. Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0236] HEK293 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours. After washing with PBS, the cells were placed in HBSS solution, and the changes in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission (485 nm excitation and 528 nm emission) were detected over a 30-minute period. The results are shown in Figure 6. After adding 200 μM and 2 mM DMKG, respectively, and continuing the detection for 30 minutes, the 485 / 420 ratio of the DMKG-added samples gradually increased, reaching a maximum of 1.68 times the initial value. After adding 1 μM of the mitochondrial respiration inhibitor oligomycin, and detecting for 30 minutes, the 485 / 420 ratio of the samples gradually decreased, reaching a minimum of 1.3 times the initial value.

[0237] Example 9: High-throughput compound screening in living cells based on optical probes

[0238] In this embodiment, we used HeLa cells expressing GlnK1-Y51L&I52W in the cytoplasm for high-throughput compound screening.

[0239] Transfected HeLa cells were washed with PBS, treated with HBSS solution (without α-ketoglutarate) for 1 hour, and then treated with 10 μM of the compound for 1 hour. α-ketoglutarate was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples without any compound treatment were used as controls for standardization. The results are shown in Figure 7. Of the 2000 compounds used, most had minimal effect on α-ketoglutarate uptake by cells. Six compounds increased cellular uptake of α-ketoglutarate, while three compounds significantly reduced it.

[0240] Example 10: Quantitative detection of α-ketoglutarate in blood using an optical probe

[0241] In this embodiment, purified GlnK1-V53L&D54W was used to analyze α-ketoglutarate in the blood supernatant of mice and humans.

[0242] After mixing GlnK1-V53L & D54W with diluted blood supernatant and treating for 10 minutes, the ratio of fluorescence intensity at 420nm excitation and 528nm emission to fluorescence intensity at 485nm excitation and 528nm emission was detected using an ELISA reader. The results are shown in Figure 8. The α-ketoglutarate content in mouse blood was approximately 5 μM, while the α-ketoglutarate content in human blood was approximately 10 μM.

[0243] As can be seen from the above embodiments, the α-ketoglutarate optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can be used to locate and quantify α-ketoglutarate in and out of cells in real time and can also be used for high-throughput compound screening.

[0244] Other implementation plans

[0245] This specification describes many embodiments. However, it should be understood that various modifications that may be learned by those skilled in the art upon reading this specification without departing from the spirit and scope of the invention should also be included within the scope of the appended claims.

[0246] Part of the sequence in this article

Claims

1. A variant of an α-ketoglutarate-binding protein, wherein: (a) Having the sequence shown in SEQ ID NO: 1 and having mutations at one, two, three, four, five or more sites selected from the following: E44, R45, Y46, Y51, I52, V53, D54, said mutations including amino acid modifications, substitutions or deletions. (b) is a sequence that has at least 70% sequence identity with (a) and has the mutation described in (1) and retains the ability to bind to α-ketoglutarate. Preferably, the α-ketoglutarate binding protein variant mutation includes mutations selected from any one, two, three, four, five or more of the following sites: E44, R45, Y46, Y51, I52, V53, D54; More preferably, the mutation includes mutations at sites selected from any of the following groups: (1) E44, (2) R45 and Y46, (3) Y51 and I52, (4) V53 and D54; More preferably, the E44 mutation is I; the R45 mutation is V; the Y46 mutation is S; the Y51 mutation is L; the I52 mutation is W; the V53 mutation is G, A, N, H, L, Q, S or M; and the D54 mutation is H, F, N, W, K or V. More preferably, the mutation comprises mutations selected from any of the following groups: (1) E44I, (2) R45V, (3) Y46S, (4) Y51L, (5) I52W, (6) V53G, (7) V53A, (8) V53N, (9) V53H, (10) V53L, (11) V53Q, (12) V53S, (13) V53M, (14) D54H, (15) D54F, (16) D54N, (17) D54W, (18) D54K, (19) D54V, (20) R45V and Y46S ,(21)Y51L and I52W,(22)V53G and D54H,(23)V53A and D54F,(24)V53G and D54F,(25)V53N and D54H,(26)V53H and D54N,(27)V53L and D54W,(28)V53Q and D54K,(29)V53L and D54H,(30)V53G and D54V,(31)V53S and D54N,(32)V53Q and D54N,(33)V53L and D54F,(34)V53M and D54N。 2. An optical probe comprising an α-ketoglutarate-sensitive polypeptide and an optically active polypeptide, wherein, The optically active polypeptide is located at one or more sites selected from the following groups of the α-ketoglutarate-sensitive polypeptide: 43 / 44, 43 / 45, 43 / 46, 43 / 47, 43 / 48, 43 / 49, 43 / 50, 43 / 51, 43 / 52, 43 / 53, 44 / 45, 44 / 46, 44 / 47, 44 / 48, 44 / 49, 44 / 50, 44 / 51, 44 / 52, 44 / 53, 45 / 46, 45 / 47, 45 / 48, 45 / 49, 45 / 50, 45 / 51, 45 / 52, 45 / 53, 46 / 47, 46 / 48, 46 / 49, 46 / 50, 46 / 51, 46 / 52, 46 / 53, 47 / 48, 47 / 49, 47 / 50, 47 / 51, 47 / 52, 47 / 53, 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 49 / 50, 49 / 51, 49 / 52, 49 / 53, 50 / 51, 50 / 52, 50 / 53, 51 / 52, 51 / 53 and 52 / 53, wherein the α-ketoglutarate-sensitive polypeptide is an α-ketoglutarate-binding protein or a functional variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof. The α-ketoglutarate-sensitive polypeptide has the following characteristics: (i) The sequence shown in SEQ ID NO:1, or a sequence that has at least 70% sequence identity with it and retains its activity of binding to α-ketoglutarate, (ii) The sequence of the α-ketoglutarate-binding protein variant of claim 1, or (iii) Having at least 70% sequence identity with the sequence described in (ii) and having the mutation described in (ii) while retaining the sequence sensitive to α-ketoglutarate, Preferably, the optically active polypeptide has: (a) Any of the sequences shown in SEQ ID NO:2-9 (b) The sequence shown in SEQ ID NO:2 and having a mutation at the Y1 site, the mutation including modification, substitution, or deletion of an amino acid; preferably, the mutation is selected from any one or more of the following: Y1V and Y1E, or (c) A variant sequence that has at least 70% sequence identity with (a) or (b) and retains the function of the fluorescent protein.

3. The optical probe as described in claim 2, characterized in that, The optically active polypeptide is located at position 44 / 53 of the α-ketoglutarate-binding protein. The α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity. The optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9. Furthermore, the optical probe contains mutations selected from any of the following groups: (1) V53G and D54H of the α-ketoglutarate-sensitive polypeptide, (2) V53A and D54F of the α-ketoglutarate-sensitive polypeptide, (3) V53G and D54F of the α-ketoglutarate-sensitive polypeptide, (4) V53N and D54H of the α-ketoglutarate-sensitive polypeptide, (5) V53H and D54F of the α-ketoglutarate-sensitive polypeptide. D54N, (6) V53L and D54W of α-ketoglutarate-sensitive peptides, (7) V53Q and D54K of α-ketoglutarate-sensitive peptides, (8) V53L and D54H of α-ketoglutarate-sensitive peptides, (9) V53G and D54V of α-ketoglutarate-sensitive peptides, (10) V53S and D54N of α-ketoglutarate-sensitive peptides, (11) V53Q and D54N of α-ketoglutarate-sensitive peptides; or The optically active polypeptide is located at position 45 / 51 of the α-ketoglutarate-binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) E44I of the α-ketoglutarate-sensitive polypeptide and Y1V of the optically active polypeptide; (2) Y51L and I52W of the α-ketoglutarate-sensitive polypeptide; or The optically active polypeptide is located at position 46 / 51 of the α-ketoglutarate-binding protein. The α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity. The optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9. Furthermore, the optical probe contains the following mutations: R45V and Y46S of the α-ketoglutarate-sensitive polypeptide and Y1E of the optically active polypeptide; or The optically active polypeptide is located at position 46 / 53 of the α-ketoglutarate binding protein, the α-ketoglutarate-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a sequence that has at least 70% sequence identity with it and retains α-ketoglutarate binding activity, the optically active polypeptide has any of the sequences shown in SEQ ID NO:2-9, and the optical probe contains mutations selected from any of the following groups: (1) V53L and D54F of the α-ketoglutarate-sensitive polypeptide; (2) V53M and D54N of the α-ketoglutarate-sensitive polypeptide.

4. A fusion polypeptide comprising the optical probe of claim 2 or 3 and other polypeptides, said other polypeptides including a localization sequence, a tag for easy purification, or a tag for an immune response.

5. A nucleic acid molecule comprising: (a) the coding sequence of the α-ketoglutarate-binding protein variant of claim 1, the coding sequence of the optical probe of claim 2 or 3, or the coding sequence of the fusion polypeptide of claim 4, or (b) the complementary sequence of (a).

6. Nucleic acid constructs, including the nucleic acid molecule of claim 5. Preferably, the nucleic acid construct is a cloning vector, expression vector, or recombinant vector.

7. A host cell, wherein the host cell: (1) Containing, expressing or secreting the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4; (2) Contains the nucleic acid molecule as described in claim 5; and / or (3) It includes the nucleic acid construct of claim 6.

8. A test kit comprising: (1) The optical probe of claim 2 or 3 or the fusion polypeptide of claim 4. (2) The nucleic acid molecule according to claim 5, (3) The nucleic acid construct according to claim 6, (4) The host cell according to claim 7, The detection kit may optionally also include other reagents required for the detection of α-ketoglutarate using an optical probe. Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and α-ketoglutarate standard.

9. A method for preparing the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4, comprising: The host cells of claim 7 are cultured, and the optical probe or fusion polypeptide is isolated from the culture.

10. The use of the optical probe of claim 2 or 3, the fusion polypeptide of claim 4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6, and / or the host cell of claim 7 in detecting α-ketoglutarate in a sample, screening compounds, or intracellular and / or extracellular localization of α-ketoglutarate.

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