Lysine optical probe and application thereof

Abstract

The invention relates to a lysine optical probe as well as a preparation method and application thereof. In one aspect, the present invention relates to an optical probe comprising a lysine-sensitive polypeptide or functional variant thereof and an optically active polypeptide or functional variant thereof wherein the optically active polypeptide or functional variant thereof is located within the sequence of the lysine-sensitive polypeptide or functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in detection of lysine.

Description

Technical Field

[0001] The present invention relates to the technical field of optical probes, and particularly to a lysine optical probe, a preparation method thereof, and an application thereof. Background Art

[0002] Common methods for detecting lysine include high-performance liquid chromatography, gas chromatography-mass spectrometry, mass spectrometry, etc. These methods have high sensitivity and accuracy, but require complex sample processing and professional instrument equipment, and cannot perform high-throughput detection. Utilizing the property that the free primary amine of lysine can react with ninhydrin reagent to produce a color reaction, colorimetry can be used for determination, but this method is interfered by other free amino acids (Ameen S et al., J Nanobiotechnology, 2016, 14(1):49.). In addition, these methods cannot monitor the distribution and metabolic dynamics of lysine in real time. Therefore, it is of great significance to develop a new method with good specificity, fast response speed, and capable of real-time quantitative detection of lysine in vivo. Summary of the Invention

[0003] The purpose of the present invention is to provide a probe and method for real-time localization, high-throughput, and quantitative detection of lysine inside and outside cells.

[0004] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] In the first aspect of the present invention, a lysine-binding protein variant is provided, which:

[0006] (1) has the sequence shown in SEQ ID NO:1 and has mutations at 1, 2, 3, or 4 of the following sites: D11, T12, Y14, A15, K23, E25, I27, Q42, A49, F52, S58, S69, S70, L71, S72, D75, R77, D91, A97, L117, S120, T121, Q122, D128, N129, T132, A139, A141, S148, D161, A174, E177, S184, K188, F191, G192, D193, G194, T195, G196, T215, Q219, N234, and the mutations include amino acid modification, substitution, or deletion,

[0007] (2) is a sequence having at least 70% sequence identity with the sequence of (1), having the mutations described in (1), and retaining the ability to bind lysine.

[0008] In one or more embodiments, the mutation includes a mutation at a site selected from any one of the following groups: (1) F52, (2) F52 and T121, (3) F52 and Q122, (4) F52 and S72, (5) F52, D193 and G194, (6) F52, F191 and G192, (7) F52, T195 and G196, (8) T12, F52, D193 and G194, (9) F52 and D193.

[0009] In one or more embodiments, T12 is mutated to P. In one or more embodiments, F52 is mutated to T, S or H, preferably to T. In one or more embodiments, S72 is mutated to E. In one or more embodiments, T121 is mutated to S or I, preferably to I. In one or more embodiments, Q122 is mutated to A or H, preferably to H. In one or more embodiments, F191 is mutated to G or Y, preferably to G. In one or more embodiments, G192 is mutated to W or D, preferably to D. In one or more embodiments, D193 is mutated to E, K, L or T, preferably to T. In one or more embodiments, G194 is mutated to D, R or S, preferably to R. In one or more embodiments, T195 is mutated to D. In one or more embodiments, G196 is mutated to R or Y, preferably to Y.

[0010] In one or more embodiments, the mutation includes a mutation selected from any one of the following groups: (1) F52T, (2) F52H, (3) F52T and Q122H, (4) F52T and T121S, (5) F52T and T121I, (6) F52T and Q122A, (7) F52T and S72E, (8) F52T, D193L and G194D, (9) F52T, F191G and G192D, (10) F52T, D193K, (11) F52T, D193E and G194S, (12) F52T, F191Y and G192W, (13) F52T, D193T and G194R, (14) F52T, D193E and G194R, (15) F52T, D195D and G196R, (16) F52T, D195D and G196Y, (17) T12P, F52T, D193T and G194R, (18) F52S.

[0011] On the other hand, the present invention provides a lysine optical probe, comprising a lysine-sensitive polypeptide or its functional variant and an optically active polypeptide or its functional variant, wherein the optically active polypeptide or its functional variant is located within the sequence of the lysine-sensitive polypeptide or its functional variant. The lysine-sensitive polypeptide or its functional variant is divided into a first part and a second part by the optically active polypeptide or its functional variant.

[0012] In one or more embodiments, the lysine optical probe includes a lysine-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the lysine-sensitive polypeptide B, dividing the lysine-sensitive polypeptide B into a first part B1 and a second part B2, forming a probe structure of the B1-A-B2 type.

[0013] In one or more embodiments, the lysine-sensitive polypeptide includes a lysine-binding protein or a functional variant thereof. In one or more embodiments, the lysine-sensitive polypeptide is derived from Salmonella enterica subsp. enterica serovar Typhimurium.

[0014] In one or more embodiments, the lysine-sensitive polypeptide has:

[0015] (1) The sequence shown in SEQ ID NO: 1, or a sequence having at least 70% sequence identity with them and retaining lysine-binding activity,

[0016] (2) The sequence of the lysine-binding protein variant according to any one of the embodiments of the first aspect herein, or

[0017] (3) A sequence having at least 70% sequence identity with the sequence described in (2), having the mutation described in (2), and retaining lysine sensitivity.

[0018] 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 protein (cpYFP as shown in SEQ ID NO: 2), green fluorescent protein (cpGFP as shown in SEQ ID NO: 3), blue fluorescent protein (cpBFP as shown in SEQ ID NO: 4), and mApple fluorescent protein (cpmApple as shown in SEQ ID NO: 5). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has the sequence shown in any one of SEQ ID NOs: 2-5.

[0019] In one embodiment, the optically active polypeptide is located in the 85-91 and / or 192-196 regions of the lysine-sensitive polypeptide, numbered corresponding to the full length of the lysine-sensitive polypeptide. Preferably, the optically active polypeptide is located in one or more of the following regions of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196. More preferably, the optically active polypeptide is located in one or more of the following sites of the lysine-sensitive polypeptide: 86 / 90, 193 / 194, 194 / 195, 192 / 193, 192 / 195, 193 / 195.

[0020] In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one or more embodiments, the linker flanking the optically active polypeptide comprises no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids. In one or more embodiments, the linker flanking the optically active polypeptide comprises the amino acid Y. In one or more embodiments, the linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is as follows: the first part B1 of the lysine-sensitive polypeptide - Y - the optically active polypeptide A - the second part B2 of the lysine-sensitive polypeptide. In one or more embodiments, the optical probe of the present invention does not comprise a linker.

[0021] In one or more embodiments, the optical probe of the present invention further comprises a targeting sequence for targeting the probe to a specific organelle such as a cell.

[0022] In one or more embodiments, the optically active polypeptide is cpYFP, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 88, 86 / 87, 86 / 90, 87 / 90, 88 / 89, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, wherein the lysine-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a lysine-binding protein variant according to any one of the embodiments of the first aspect herein; preferably, cpYFP is located at any one or more of the following sites of the lysine-sensitive polypeptide: 86 / 87, 86 / 90, 87 / 90, 89 / 91, 192 / 193, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195.

[0023] In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 89, 85 / 90, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196, 195 / 196, wherein the lysine-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a lysine-binding protein variant according to any one of the embodiments of the first aspect herein; preferably, cpGFP is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 87, 85 / 89, 86 / 87, 86 / 89, 86 / 90, 87 / 91, 88 / 89, 88 / 90, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196, 195 / 196.

[0024] In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 91, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196, 195 / 196, wherein the lysine-sensitive polypeptide (1) is as shown in SEQ ID NO: 1, or (2) is a lysine-binding protein variant according to any one of the embodiments of the first aspect herein; preferably, cpBFP is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 86, 85 / 88, 85 / 89, 85 / 91, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 89, 87 / 90, 88 / 89, 88 / 91, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196.

[0025] In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 91, 88 / 89, 88 / 91, 89 / 90, 89 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196, wherein the lysine-sensitive polypeptide (1) is as shown in SEQ ID NO: 1, or (2) is a lysine-binding protein variant according to any one of the embodiments of the first aspect herein; preferably, cpmApple is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 89, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 91, 88 / 91, 89 / 90, 89 / 91, 192 / 193, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195.

[0026] In one or more embodiments, the optically active polypeptide is cpYFP, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 192 / 195, 193 / 195, 194 / 195, 192 / 193, and the lysine-sensitive polypeptide (i) is as shown in SEQ ID NO:1, or (ii) is a variant of (i) having a mutation selected from any one of the following groups: (1) F52T, (2) F52H, (3) F52T and Q122H, (4) F52T and T121S, (5) F52T and T121I, (6) F52T and Q122A, (7) F52T and S72E, (8) F52T, D193L and G194D, (9) F52T, F191G and G192D, (10) F52T, D193K, (11) F52T, D193E and G194S, (12) F52T, F191Y and G192W, (13) F52T, D193T and G194R, (14) F52T, D193E and G194R, (15) F52T, D195D and G196R, (16) F52T, D195D and G196Y, (17) T12P, F52T, D193T and G194R, (18) F52S.

[0027] On the other hand, the present invention also provides a fusion polypeptide, comprising the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides that localize the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. There may be a linker between the optical probe and the other polypeptides in the fusion polypeptide described herein.

[0028] On the other hand, the present invention also provides a nucleic acid molecule, which comprises: (1) the coding sequence of the polypeptide or probe described in any embodiment herein, or (b) the complementary sequence of (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0029] In one or more embodiments, the nucleic acid sequence comprises the coding sequence of any of the amino acid sequences shown in SEQ ID NOs: 6-9. Preferably, the nucleic acid sequence comprises the nucleotide sequence SEQ ID NO:10 or a variant thereof. More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to the nucleotide sequence SEQ ID NO:10; or comprises a nucleotide sequence that is substantially similar or identical to any of the nucleotide sequences SEQ ID NO:10.

[0030] The present invention also relates to the complementary sequence of the above nucleic acid sequence or its variant, which may comprise a nucleic acid sequence encoding a fragment, analogue, derivative, soluble fragment and variant of the optical probe or fusion protein of the present invention or its complementary sequence.

[0031] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein. This nucleic acid sequence encodes the optical probe or fusion polypeptide described in the present invention.

[0032] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

[0033] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0034] In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors.

[0035] The present invention also provides a host cell comprising: (1) expressing the optical probe or fusion polypeptide according to any embodiment of the present invention; (2) comprising the nucleic acid molecule according to any embodiment of the present invention; or (3) comprising the nucleic acid construct according to any embodiment of the present invention. The host cell is preferably Escherichia coli.

[0036] On the other hand, the present invention also provides a lysine detection kit, comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe prepared by the method described herein.

[0037] In one or more embodiments, the kit further comprises one or more reagents selected from the following: buffer, culture medium, lysine standard.

[0038] The present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions for expression in the cell, and isolating the optical probe or fusion polypeptide.

[0039] In one or more embodiments, the method for preparing the lysine optical probe or fusion polypeptide described herein comprises the following steps: 1) transferring an expression vector encoding the lysine optical probe described herein into a host cell; 2) culturing the host cell under conditions suitable for the expression of the expression vector, and 3) isolating the lysine optical probe.

[0040] The present invention also provides a method for detecting lysine in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, and detecting the change of the optically active polypeptide. The detection can be carried out in vivo, in vitro, subcellularly or in situ. The sample is, for example, blood.

[0041] The present invention also provides a method for quantifying lysine in a sample, including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, detecting the change of the optically active polypeptide, and quantifying lysine in the sample according to the change of the optically active polypeptide.

[0042] The present invention also provides a method for screening a compound (such as a drug), including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with a candidate compound in a lysine-containing system, detecting the change of the optically active polypeptide, and screening the compound according to the change of the optically active polypeptide. The method can screen compounds in a high-throughput manner.

[0043] In one or more embodiments, contacting the host cell described herein with a candidate compound in a lysine-containing system, and the optical change of the optically active polypeptide indicates whether the candidate compound can regulate the cell's uptake of lysine.

[0044] On the other hand, the present invention also provides a method for intracellular and / or extracellular localization of the lysine, including: contacting a lysine-containing system with the optical probe or the host cell, and detecting the optical change of the optically active polypeptide.

[0045] In one or more embodiments, the system is a solution system, a cell system, or a subcellular system.

[0046] On the other hand, the present invention also provides the use of the lysine optical probe or fusion polypeptide or host cell described herein in detecting lysine in a sample, screening a compound, or intracellular / extracellular localization of lysine. In one or more embodiments, the localization is real-time localization.

[0047] Advantages of the present invention: The lysine optical probe provided by the present invention is easy to mature, has a large fluorescence dynamic change, good specificity, and can be expressed in cells by genetic manipulation methods. It can perform real-time localization, high-throughput, and quantitative detection of lysine inside and outside cells, eliminating the time-consuming sample processing steps. The experimental results show that the lysine optical probe provided by this application has a maximum response to lysine more than 2.3 times that of the control, and can perform localization, qualitative, and quantitative detection of cells in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, and Golgi apparatus, and can also perform high-throughput compound screening and quantitative detection of lysine in blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 SDS-PAGE diagram of the exemplary lysine optical probe described in Example 1;

[0049] Figure 2Exemplary lysine optical probe comprising cpYFP and lysine binding protein for the lysine response change graph described in Example 2;

[0050] Figure 3 Exemplary lysine optical probe comprising cpGFP and lysine binding protein for the lysine response change graph described in Example 3;

[0051] Figure 4 Exemplary lysine optical probe comprising cpBFP and lysine binding protein for the lysine response change graph described in Example 4;

[0052] Figure 5 Exemplary lysine optical probe comprising cpmApple and lysine binding protein for the lysine response change graph described in Example 5;

[0053] Figure 6 Fluorescence spectral property graph of the exemplary lysine optical probe described in Example 7;

[0054] Figure 7 Titration curve of the exemplary lysine optical probe described in Example 7 for different concentrations of lysine;

[0055] Figure 8 Bar graph of the specific detection of the exemplary lysine optical probe described in Example 7 for 20 amino acids;

[0056] Figure 9 Photographs of the subcellular localization of the exemplary lysine optical probe in mammalian cells described in Example 8;

[0057] Figure 10 Schematic diagram for dynamically monitoring the lysine concentration in the cytoplasm of the exemplary lysine optical probe in mammalian cells described in Example 9;

[0058] Figure 11 Dot plot of the high-throughput compound screening of the exemplary lysine optical probe at the live cell level described in Example 10;

[0059] Figure 12 Bar graph of the quantification of lysine in human blood by the exemplary lysine optical probe described in Example 11. Detailed implementation manners

[0060] When giving a numerical value or range, the term "about" as used herein means within 20%, within 10%, and within 5% of the given numerical value or range.

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

[0062] As used herein, the term "lysine-sensitive polypeptide" or "lysine-responsive polypeptide" refers to a polypeptide that responds to lysine, and the response includes any response of the chemical, biological, electrical or physiological parameters of the polypeptide related to the interaction with the sensitive polypeptide. The response includes small changes, for example, changes in the orientation of the amino acid or peptide fragment of the polypeptide and, for example, changes in the primary, secondary or tertiary structure of the polypeptide, 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; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include the transition from an α-helix to a β-sheet or from a β-sheet to an α-helix. It is understood that as long as the fluorescence of the fluorescent protein moiety is changed, the detectable change does not need to be a conformational change. The lysine-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of lysine-sensitive polypeptides include, but are not limited to, variants that can interact with lysine and thus undergo the same or similar changes as the parental lysine-sensitive polypeptide.

[0063] The lysine-sensitive polypeptides of the present invention include, but are not limited to, the lysine-binding protein LAO or variants having more than 90% homology thereto. The exemplary lysine-binding protein LAO of the present invention is derived from Salmonella enterica subsp. enterica serovar Typhimurium. The lysine-binding protein can sense changes in lysine concentration, and the spatial conformation of the lysine-binding protein also changes during the dynamic change of lysine concentration. The exemplary LAO protein is shown in SEQ ID NO:1. When describing the optical probe or lysine-binding protein of the present invention (for example, when describing the insertion site or mutation site), the amino acid residue numbers are all referenced to SEQ ID NO:1.

[0064] As used herein, the term "optical probe" refers to a lysine-sensitive polypeptide fused with an optically active polypeptide. The inventors have found that the conformational change caused by the specific binding of a lysine-sensitive polypeptide, such as a lysine-binding protein, to physiological concentrations of lysine will cause a conformational change in the optically active polypeptide (such as a fluorescent protein), and thus lead to a change in the optical properties of the optically active polypeptide. By plotting a standard curve using the fluorescence of the fluorescent protein measured at different lysine concentrations, the presence and / or level of lysine can be detected and analyzed.

[0065] In the optical probe of the present invention, an optically active polypeptide (such as a fluorescent protein) is operably inserted into a lysine-sensitive polypeptide. A "protein-based optically active polypeptide" is a polypeptide having the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide, which can be used as a means to detect the responsiveness of the optical probe of the present invention. As used herein, the term "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the amplitude of excitation at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime or fluorescence anisotropy. A measurable difference in any of these properties between the active and inactive states is sufficient for 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 fluorescent property, for example, the amount of fluorescence at a specific wavelength or the integration of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent characteristics that are easily distinguishable in the unactivated and activated conformational states. The optically active polypeptides described herein may also include functional variants thereof. Functional variants of optically active polypeptides include, but are not limited to, variants that can undergo the same or similar changes in fluorescent properties as the parental optically active polypeptide.

[0066] As used herein, the terms "fluorescent protein", "chromophore" and "fluorophore" are synonymous and refer to a protein that emits fluorescence upon irradiation with excitation light. Fluorescent proteins are a basic detection means in the field of biological sciences. For example, the commonly used green fluorescent protein GFP in the field of biotechnology and the circularly permuted blue fluorescent protein (cpBFP), circularly permuted green fluorescent protein (cpGFP), circularly permuted yellow fluorescent protein (cpYFP), etc. derived from the mutation of this protein; there are also the commonly used red fluorescent protein RFP in the field of the present technology and the circularly permuted proteins derived from this protein, such as cpmApple, cpmOrange, cpmKate, etc. Those skilled in the art know the fluorescent proteins and their sequences that can be used in the present invention. Exemplarily, cpYFP is shown as SEQ ID NO:2; cpGFP is shown as SEQ ID NO:3; cpBFP is shown as SEQ ID NO:4; cpmApple is shown as SEQ ID NO:5.

[0067] "Linker" 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 lysine-sensitive polypeptide and the optically active polypeptide is selected from 0 to 3, and the number of amino acids at the carboxyl terminus is selected from 0 to 2; when the recombinant optical probe is connected to a functional protein as a basic unit, it can be fused to the amino or carboxyl terminus of the recombinant optical probe. The linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0068] The lysine optical probe described in the present invention includes a lysine-sensitive polypeptide (B), such as a lysine-binding protein or a variant thereof, and an optically active polypeptide (A), such as a fluorescent protein. The optically active polypeptide (A) is inserted into the lysine-sensitive polypeptide (B), dividing B into two parts, B1 and B2, to form a probe structure of the B1-A-B2 type; the interaction between the lysine-sensitive polypeptide B and lysine causes the optical signal of the optically active polypeptide (A) to become stronger.

[0069] In the optical probe of the present invention, the optically active polypeptide can be located at any position of the lysine-sensitive polypeptide. In one embodiment, the optically active polypeptide is located in the N-C direction at any position of the lysine-sensitive polypeptide in the N-C direction. Specifically, the optically active polypeptide is located in the flexible region of the lysine-sensitive polypeptide, and the flexible region refers to some specific structures such as loop domains existing 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 the spatial structure conformation of this region can change dynamically after the protein binds to the ligand. The flexible region described in the present invention mainly refers to the region where the insertion site is located in the lysine-binding protein, such as the amino acid residue 85-91 and / or 192-196 region. Exemplarily, the optically active polypeptide is located at one or more sites selected from the following in the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196. In this article, if the two numbers in the site represented in the form of "X / Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids represented by these numbers. For example, the insertion site 85 / 86 means that the optically active polypeptide is located between amino acids 85 and 86 of the lysine-sensitive polypeptide. If the two numbers in the site represented in the form of "X / Y" are not consecutive integers, it means that the optically active polypeptide replaces the amino acids between the amino acids represented by these numbers. For example, the insertion site 192 / 194 means that the optically active polypeptide replaces amino acid 193 of the lysine-sensitive polypeptide.

[0070] In one or more embodiments, the optical probe sequentially comprises, from the N-terminus to the C-terminus, the residues at positions 1-X of SEQ ID NO:1, the optically active polypeptide shown in any one of SEQ ID NOs:2-5, and the residues at positions Y-238 of SEQ ID NO:1, wherein X and Y are selected from any one of the following groups: (1) X is 85 and Y is 86; (2) X is 85 and Y is 87; (3) X is 85 and Y is 88; (4) X is 85 and Y is 89; (5) X is 85 and Y is 90; (6) X is 85 and Y is 91; (7) X is 86 and Y is 87; (8) X is 86 and Y is 88; (9) X is 86 and Y is 89; (10) X is 86 and Y is 90; (11) X is 86 and Y is 91; (12) X is 87 and Y is 88; (13) X is 87 and Y is 89; (14) X is 87 and Y is 90; (15) X is 87 and Y is 91; (16) X is 88 and Y is 89; (17) X is 88 and Y is 90; (18) X is 88 and Y is 91; (19) X is 89 and Y is 90; (20) X is 89 and Y is 91; (21) X is 90 and Y is 91; (22) X is 192 and Y is 193; (23) X is 192 and Y is 194; (24) X is 192 and Y is 195; (25) X is 192 and Y is 196; (26) X is 193 and Y is 194; (27) X is 193 and Y is 195; (28) X is 193 and Y is 196; (29) X is 194 and Y is 195; (30) X is 194 and Y is 196; or (31) X is 195 and Y is 196.

[0071] Preferably, the optically active polypeptide is located at one or more sites selected from the following of the lysine-sensitive polypeptide: 86 / 90, 193 / 194, 194 / 195, 192 / 193, 192 / 195, 193 / 195.

[0072] When referring to a certain polypeptide or protein, the term "variant" or "mutant" used in the present invention includes variants having the same function of the polypeptide or protein but different sequences. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and / or amino terminal. These variants may also include polypeptides or proteins having a sequence identity of 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% with the polypeptide or protein. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of a polypeptide or protein, i.e., a function conservative mutation. For example, in the art, when amino acids with similar or similar properties are substituted, the function of a polypeptide or protein is usually not changed. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which are clearly 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 non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, lysine, methionine, lysine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, lysine, lysine, histidine). For another example, adding one or more amino acids to the amino terminus and / or carboxyl terminus will generally not change the function of a polypeptide or protein. Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids.

[0073] In two or more polypeptide or nucleic acid molecule sequences, the terms "identity" or "percent identity" refer to the maximum correspondence when comparing and aligning two or more sequences or subsequences by known methods in the art such as sequence comparison algorithms, through manual alignment and visual inspection, over a comparison window or specified region, where the two or more sequences are identical or a certain percentage of the amino acid residues or nucleotides are identical in the specified region (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical). For example, preferred algorithms for determining percent sequence identity and percent 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.

[0074] As is well known to those skilled in the art, in gene cloning operations, it is often necessary to design appropriate restriction enzyme sites, which will inevitably introduce one or more irrelevant residues at the ends of the expressed polypeptide or protein, and this does not affect the activity of the target polypeptide or protein. Another example is that in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein. For example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.

[0075] The optical probe of the present invention may comprise a lysine-sensitive polypeptide with mutations. The mutations are, for example, mutations at 1, 2, 3, 4 or more sites among D11, T12, Y14, A15, K23, E25, I27, Q42, A49, F52, S58, S69, S70, L71, S72, D75, R77, D91, A97, L117, S120, T121, Q122, D128, N129, T132, A139, A141, S148, D161, A174, E177, S184, K188, F191, G192, D193, G194, T195, G196, T215, Q219, N234. Exemplarily, the mutations are selected from 1, 2, 3, 4 or more of F52T, F52H, F52S, S72E, T121S, T121I, Q122H, Q122A, F191G, F191Y, G192D, G192W, D193E, D193K, D193L, D193T, G194D, G194R, G194S, T195D, G196R, G196Y. In one or more embodiments, the mutations comprise mutations selected from any one of the following groups: (1) F52T, (2) F52H, (3) F52T and Q122H, (4) F52T and T121S, (5) F52T and T121I, (6) F52T and Q122A, (7) F52T and S72E, (8) F52T, D193L and G194D, (9) F52T, F191G and G192D, (10) F52T, D193K, (11) F52T, D193E and G194S, (12) F52T, F191Y and G192W, (13) F52T, D193T and G194R, (14) F52T, D193E and G194R, (15) F52T, T195D and G196R, (16) F52T, T195D and G196Y, (17) T12P, F52T, D193T and G194R, (18) F52S.

[0076] Among them, as an example in the examples, in SEQ ID NO:1, F52 is mutated to T, S or H, Q122 is mutated to H or A, T121 is mutated to I or S, S72 is mutated to E, D193 is mutated to L, K, E or T, F191 is mutated to G or Y, G194 is mutated to D, S or R, G192 is mutated to D or W, G196 is mutated to R or Y, and T12 is mutated to P.

[0077] In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be a probe in which cpYFP is inserted at one or more sites selected from 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196 of LAO and having one or more mutations selected from the following: F52T, F52H, F52S, S72E, T121S, T121I, Q122H, Q122A, F191G, F191Y, G192D, G192W, D193E, D193K, D193L, D193T, G194D, G194R, G194S, T195D, G196R, G196Y. Preferably, the optical probe is a probe in which cpYFP is inserted at one or more sites selected from 193 / 194, 194 / 195, 192 / 193, 192 / 195, 193 / 195, 86 / 90 of LAO and having one or more mutations selected from the following: F52T, F52H, F52S, S72E, T121S, T121I, Q122H, Q122A, F191G, F191Y, G192D, G192W, D193E, D193K, D193L, D193T, G194D, G194R, G194S, T195D, G196R, G196Y.

[0078] The optical probe provided by the present invention comprises any one of the amino acid sequences SEQ ID NO: 6-9 or a variant thereof. In one embodiment, the optical probe provided by the present invention comprises a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with any one of the amino acid sequences SEQ ID NO: 6-9. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence that is substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 6-9.

[0079] In some specific embodiments, the lysine-sensitive polypeptide in the optical probe is as shown in SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at the 194 / 195 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide has a mutation selected from any one of the following: (1) F52T, (2) F52H, (3) F52T and Q122H, (4) F52T and Q122A, (5) F52T and T121I, (6) F52T and T121S, (7) F52T and S72E, (8) F52T, D193L and G194D, (9) F52T, D193K, (10) F52T, D193E and G194S, (11) F52T, T195D and G196R, (12) F52T, T195D and G196Y, (13) F52S.

[0080] In some specific embodiments, the lysine-sensitive polypeptide in the optical probe is as shown in SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at the 193 / 195 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide has a mutation selected from any one of the following: (14) F52H, (15) F52T, (16) F52S.

[0081] In some specific embodiments, the lysine-sensitive polypeptide in the optical probe is as shown in SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at the 192 / 193 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide has a mutation selected from any one of the following: (17) F52T, F191G, G192D, (18) F52T, F191Y, G192W, (19) F52T, D193T, G194R, (20) F52T, D193E, G194R, (21) T12P, F52T, D193T, G194R, (22) F52T, (23) F52S.

[0082] In some specific embodiments, the lysine-sensitive polypeptide in the optical probe is as shown in SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at the 193 / 194 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide has a mutation selected from any one of the following: (24) F52T.

[0083] As used herein, the terms "functional fragment", "functional variant", "derivative" and "analogue" refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (such as LAO protein or fluorescent protein). Functional variants, derivatives or analogues of the polypeptides or proteins of the present invention (such as LAO protein or fluorescent protein) can be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusing the mature protein with another compound (such as a compound that prolongs the protein half-life, such as polyethylene glycol), or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a secretion sequence or a sequence used to purify this protein or a proprotein sequence, or a fusion polypeptide formed with an antigen IgG fragment). According to the teachings herein, these functional variants, derivatives and analogues are within the scope well-known to those skilled in the art.

[0084] The differences between the analogue and the original polypeptide or protein can be differences in the amino acid sequence, or differences in the modified form that do 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 by radiation or exposure to mutagens, and can also be obtained by site-directed mutagenesis or other known molecular biology techniques.

[0085] The fusion polypeptides of the present invention comprise the optical probes described herein and other polypeptides. In some embodiments, the optical probes described herein further comprise other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probes. The other polypeptides can be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides that localize the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. There may be a linker between the optical probe and the other polypeptides in the fusion polypeptides described herein.

[0086] As used herein, the "response multiple" is the normalized fluorescence ratio. The greater the deviation of the response multiple of the probe from 1 (whether it becomes larger or smaller), the greater the change multiple or response ability of the probe relative to the control. As described in many places in the examples, "the change in the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission". This detection method is well-known in the art and is briefly described as follows:

[0087] Determination of the change multiple of the probe: The fluorescence signal value is corrected by subtracting the detection signal value of the cells that do not express the probe protein. The detection signal of the probe in the parallel experimental group is divided by the control detection signal to eliminate the pH-sensitive interference and obtain the corrected data.

[0088] F = F sample -F BLK

[0089]

[0090]

[0091]

[0092]

[0093] F represents the fluorescence intensity (Fluorescence intensity), F sample represents the total fluorescence intensity of the sample expressing the fluorescent probe, F BLK represents the background fluorescence intensity of the sample not expressing the fluorescent probe, F cpYFP represents the fluorescence intensity of the sample used as a pH control. F 485 represents the fluorescence intensity of the fluorescent protein sample excited at 485 nm and emitted at 528 nm, F 420 represents the fluorescence intensity of the fluorescent protein sample excited at 420 nm and emitted at 528 nm. Ratio sensor represents the fluorescence intensity ratio of the probe, Ratio cpYFP represents the fluorescence intensity ratio of the pH control fluorescent protein corresponding to the probe.

[0094] In this article, the fold change or response fold of the probe refers to the Normalized Ratio 485 / 420 (i.e., the normalized fluorescence ratio). When the Normalized Ratio of the probe 485 / 420 deviates more from 1 (whether it becomes larger or smaller), it indicates a greater fold change or response fold of the probe.

[0095] The subcellular organelles described in this article include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosome, peroxisome, etc. In some embodiments, the tags for purification or the tags for immunoblotting include 6 histidines (6*His), glutathione S-transferase (GST), Flag.

[0096] The expression vector of the present invention comprises the nucleic acid sequence of the present invention or its complementary sequence operably linked to an expression control sequence, and the nucleic acid sequence encodes the optical probe or fusion polypeptide of the present invention. In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors. In some embodiments, the prokaryotic expression vector is preferably obtained by operably linking the plasmid pCDF with the nucleic acid sequence described herein. In some embodiments, the expression control sequence includes an origin of replication, a promoter, an enhancer, an operator, a terminator, and a ribosome binding site.

[0097] The present invention also provides a method for preparing the above lysine optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the lysine optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for the expression of the expression vector, and 3) isolating the lysine optical probe.

[0098] As used in the present invention, the terms "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. When referring to a nucleic acid, the term "variant" as used herein may 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. The nucleic acid of the present invention may comprise a nucleotide sequence having a sequence identity of at least about 50%, at least about 60%, 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% with the nucleic acid sequence. The present invention also relates to nucleic acid fragments that hybridize to the above sequences. In an exemplary embodiment, the nucleic acid sequence is as shown in SEQ ID NO: 10, which represents the coding sequence of a probe with cpYFP inserted at positions 194 / 195 of the functional fragment of the lysine binding protein and having mutations of F52T, D193E, and G194S. As used herein, the "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR).

[0099] The full-length sequence or fragment of the optical probe or fusion polypeptide of the present invention can generally be obtained by PCR amplification, artificial synthesis or recombination. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the nucleotide sequence is greater than 2500 bp, it is preferably amplified by PCR 2 to 6 times, and then the fragments amplified each time are spliced together in the correct order. The present invention has no special limitation on the PCR amplification program and system, and the conventional PCR amplification program and system in the art can be used. The relevant sequences can also be obtained in large quantities by recombination. This is usually to clone it into a vector, then transfer it into cells, and then isolate and purify the relevant polypeptide or protein from the proliferated host cells by conventional methods. In addition, the relevant sequences can also be synthesized by artificial synthesis, especially when the fragment length is short. In the present invention, when the nucleotide sequence of the optical probe is less than 2500 bp, the artificial synthesis method can be used for synthesis. The artificial synthesis method is the conventional DNA artificial synthesis method in the art without other special requirements. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. At present, it is already possible to completely obtain the DNA sequence encoding the protein (or its functional variants, derivatives or analogs) of the present invention by chemical synthesis. Then this DNA sequence can 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 the present invention by methods such as mutant PCR or chemical synthesis.

[0100] The present invention also provides a detection kit comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe or fusion polypeptide prepared by the method described herein. The kit may also optionally contain other reagents required for detecting lysine using the optical probe. Those skilled in the art know the conventional other reagents.

[0101] The present invention also relates to nucleic acid constructs that contain the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid construct can be manipulated according to the differences or requirements of the expression vector before being inserted into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0102] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector, such as a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The cloning vector can be used to provide the coding sequence of the protein or polypeptide of the present invention. The expression vector can be provided to cells in the form of a bacterial vector or a viral vector. Generally, the expression of the polynucleotides of the present invention is achieved by operably linking the polynucleotides of the present invention to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence. The gene knock-in vector is used to knock in or integrate the expression cassette described herein into the host genome.

[0103] As used herein, the term "expression control sequence" refers to elements that can be operably linked to a gene of interest and regulate the transcription, translation, and expression of the gene of interest, which can be an origin of replication, a promoter, a marker gene, or a translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. In a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed. Those skilled in the art are familiar with the methods for constructing expression vectors containing the coding sequence of the fusion polypeptide of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses, and some other known promoters that can control the expression of genes 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 the commercially available pCDF vector without other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are digested with HindIII and XhoI respectively, and then the digested products of the two are ligated to obtain a recombinant expression vector. The present invention has no special limitations on the specific steps and parameters of digestion and ligation, and 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 comprising a fusion protein. Such a transfer process can be carried out by conventional techniques well known to those skilled in the art such as transformation or transfection. The host cell described in the present invention refers to a cell capable of receiving and accommodating a recombinant DNA molecule and is a site for recombinant gene amplification. An ideal recipient cell should meet two conditions: being easy to obtain and proliferate. The "host cell" of the present invention can include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells. The host cell is preferably various cells that are beneficial to the expression of gene products or fermentation production, and such cells are well known and commonly used in the art. Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers and host cells.

[0105] The method of transferring to a host cell described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfer or electroporation. When the host is a prokaryote such as Escherichia coli, the preferred method is treatment with the CaCl2 method or the 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 selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0106] After the expression vector is transferred into the host cell in the present invention, the host cell into which the expression vector has been transferred is subjected to amplified expression culture, and a lysine optical probe is isolated. The amplified expression culture of the host cell can be carried out by a conventional method. According to the type of host cell used, the culture medium used in the culture can be various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell.

[0107] In the present invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties. The present invention places no special limitation on the method for separating the lysine fluorescent protein, and a conventional method for separating a fusion protein 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 renaturation treatment, salting-out method, centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. In one embodiment, the His-tag affinity chromatography method is used for the separation of the optical probe.

[0108] The present invention also provides the application of the lysine optical probe in the real-time localization, quantitative detection of lysine, and high-throughput compound screening. In one aspect, the lysine optical probe is preferably linked to signal peptides at different parts of cells and transferred into cells. By detecting the intensity of the fluorescence signal in the cells, the real-time localization of lysine is carried out; the quantitative detection of the corresponding lysine is carried out through a lysine standard addition curve. The lysine standard addition curve of the present invention is drawn based on the fluorescence signals of the lysine optical probe under different concentrations of lysine. The lysine optical probe of the present invention is directly transferred into cells. During the real-time localization and quantitative detection of lysine, there is no need for a time-consuming sample treatment process, which is more accurate. When the lysine optical probe of the present invention is used for high-throughput compound screening, different compounds are added to the cell culture medium, and the change in the lysine content is measured, so as to screen out the compounds that have an impact on the change in the lysine content. In the application of the lysine optical probe of the present invention in the real-time localization, quantitative detection of lysine, and high-throughput compound screening, it is for non-diagnostic and non-therapeutic purposes and does not involve the diagnosis and treatment of diseases.

[0109] In this text, concentrations, contents, percentages, and other numerical values can be expressed in the form of ranges. It should also be understood that using this range form is only for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly mentioned in the upper and lower limits of the range, and should also include all individual numerical values or sub-ranges included in this range.

[0110] Examples

[0111] The following examples are used to illustrate in detail the lysine optical probe provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0112] I. Experimental materials and reagents

[0113] In the examples, conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, etc. are mainly used. These methods are well-known to those of ordinary skill in the art. For example: "Molecular Biology Experiment Reference Manual" by Jan Roskams et al., "Molecular Cloning: A Laboratory Manual" (Third Edition, August 2002, published by Science Press, Beijing) by J. Sambrook, D.W. Russell, translated by Huang Peitang et al.; "Animal Cell Culture: A Practical Approach" (Fifth Edition) by Freshney et al., translated by Zhang Jingbo, Xu Canshuan, etc.; "A Compendium of Cell Biology Experiments" by J.S. Bonifacino, M. Dasso, etc., translated by Zhang Jingbo, etc.

[0114] In the examples, the plasmids pCDF-cpYFP and pCDF-lysine-binding protein were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and correctly identified by mass spectrometry by Shanghai Generay Biotech Co., Ltd. The expression plasmids constructed in the examples were all subjected to sequence determination, which was completed by BGI and Generay Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotech, the pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa. When purchasing the three polymerases, the corresponding polymerase buffer and dNTP were given as gifts. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 polynucleotide kinase (T4 PNK) were purchased from Fermentas, and the corresponding buffer etc. were attached when purchasing. The transfection reagent Lip2000 Kit was purchased from Invitrogen. Amino acids such as lysine were all purchased from Sigma. Unless otherwise specified, chemical reagents such as inorganic salts were all purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Ameresco. The 96-well detection blackboard and 384-well fluorescence detection blackboard were purchased from Grenier.

[0115] In the examples, the DNA purification kit was purchased from BBI (Canada), and the ordinary plasmid miniprep kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloning strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column packing materials were all from GE healthcare.

[0116] 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), Microfuge22R bench-top high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic cell disruptor (Ningbo Xinzhi), nucleic acid electrophoresis apparatus (Shenergy), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), inverted fluorescence microscope (Nikon, Japan).

[0117] II. Molecular Biology Methods and Cell Experiment Methods

[0118] II.1 Polymerase Chain Reaction (PCR):

[0119] 1. Amplification PCR of target fragment:

[0120] This method is mainly used for gene fragment amplification and identification of positive clones by colony PCR. The reaction system for the PCR amplification is as follows: 0.5 - 1 μL of template sequence, 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 5 μL of 10×pfu buffer, 0.5 μL of pfu DNA polymerase, 1 μL of dNTP (10 mM), 41.5 - 42 μL of sterilized ultrapure water (ddH2O), with a total volume of 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 2 - 10 minutes, 30 cycles (94 - 96°C for 30 - 45 seconds, 50 - 65°C for 30 - 45 seconds, 72°C for a certain time (600 bp / min)), and extension at 72°C for 10 minutes.

[0121] 2. Amplification PCR of long fragment (>2500 bp):

[0122] For the long fragment amplification used in the present invention, it is mainly reverse PCR amplification of the vector, which is a technique used to obtain site-directed mutagenesis in the following examples. Reverse PCR primers are designed at the mutation site, and the 5'-end of one of the primers contains the mutated nucleotide sequence. The amplified product will contain the corresponding mutation site. The reaction system for long fragment amplification PCR is as follows: 1 μL of template sequence (10 pg - 1 ng), 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 10 μL of 5×PrimerSTAR buffer, 0.5 μL of PrimerSTAR DNA polymerase, 4 μL of dNTP (2.5 mM), 33.5 μL of sterilized ultrapure water (ddH2O), with a total volume of 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50 - 68°C for 5 - 15 seconds, 72°C for a certain time (1000 bp / min)), and extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000 bp / min)), and extension at 72°C for 10 minutes.

[0123] II.2 Restriction enzyme digestion reaction:

[0124] The system for double digestion of plasmid vector is as follows: 20 μL of plasmid vector (about 1.5 μg), 5 μL of 10× buffer, 1 - 2 μL of restriction enzyme 1, 1 - 2 μL of restriction enzyme 2, and made up to a total volume of 50 μL with sterilized ultrapure water. The reaction condition is 37°C for 1 - 7 hours.

[0125] II.3 5'-end phosphorylation reaction of DNA fragment

[0126] Plasmids or genomic ends extracted from microorganisms contain phosphate groups, while PCR products do not. Therefore, a phosphate group addition reaction needs to be carried out on the 5'-end bases of the PCR products. Only DNA molecules with phosphate groups at the ends can undergo ligation reactions. The phosphorylation reaction system is as follows: 5 - 8 μL of the DNA sequence of the PCR product fragment, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), 0 - 3 μL of sterilized ultrapure water, with a total volume of 10 μL. The reaction conditions are 37 °C for 30 minutes - 2 hours, followed by inactivation at 72 °C for 20 minutes.

[0127] II.4 Ligation reaction of the target fragment and the vector

[0128] The ligation methods vary between different fragments and vectors. Three ligation methods are used in the present invention

[0129] 1. Blunt-end ligation of blunt-end short fragments and linearized vectors

[0130] The principle of this method is that after the blunt-end product obtained by PCR undergoes phosphorylation reaction on the 5'-end of the DNA fragment under the action of T4 PNK, it is ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain the recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of the DNA fragment treated with T4 PNK, 4 μL of the linearized vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, with a total of 10 μL. The reaction conditions are 22 °C for 30 minutes.

[0131] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends

[0132] DNA fragments cut by restriction endonucleases usually produce protruding sticky ends, so they can be ligated with vector fragments with complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1 - 7 μL of the PCR product fragment DNA after digestion, 0.5 - 7 μL of the plasmid after digestion, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterilized ultrapure water is added to make up the total volume to 10 μL. The reaction conditions are 16 °C for 4 - 8 hours.

[0133] 3. Ligation reaction of self-cyclization of the DNA fragment product phosphorylated at the 5'-end after introducing site-directed mutation by inverse PCR

[0134] The 3'-end and 5'-end of the linearized vector are ligated through self-cyclization ligation reaction of the 5'-end phosphorylated DNA fragment to obtain the recombinant plasmid. The self-cyclization ligation reaction system is as follows: 10 μL of the phosphorylation reaction system, 0.5 μL of T4 ligase (5 U / μL), with a total volume of 10.5 μL. The reaction conditions are 16 °C for 4 - 16 hours.

[0135] II.5 Preparation and Transformation of Competent Cells

[0136] Preparation of Competent Cells:

[0137] 1. Pick a single colony (such as Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight on a shaker at 37 °C.

[0138] 2. Transfer 0.5 - 1 mL of the overnight culture into 50 mL of LB medium. Incubate at 37 °C with shaking at 220 rpm for 3 to 5 hours until the OD600 reaches 0.5.

[0139] 3. Pre - cool the cells in an ice bath for 2 hours.

[0140] 4. Centrifuge at 4000 rpm for 10 minutes at 4 °C.

[0141] 5. Discard the supernatant, resuspend the cells with 5 mL of pre - cooled buffer. After uniform suspension, add resuspension buffer to a final volume of 50 mL.

[0142] 6. Incubate in an ice bath for 45 minutes.

[0143] 7. Centrifuge at 4000 rpm for 10 minutes at 4 °C, and resuspend the bacteria with 5 mL of ice - pre - cooled storage buffer.

[0144] 8. Aliquot 100 μL of the bacterial suspension into each EP tube and store at - 80 °C or in liquid nitrogen.

[0145] Resuspension buffer: CaCl2 (100 mM), MgCl2 (70 mM), NaAc (40 mM)

[0146] 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

[0147] Transformation of Competent Cells:

[0148] 1. Take 100 μL of competent cells and thaw them on ice.

[0149] 2. Add an appropriate volume of the ligation product, gently pipette to mix well, and incubate on ice for 30 minutes. Usually, the volume of the ligation product added is less than 1 / 10 of the volume of the competent cells.

[0150] 3. Transfer the bacterial suspension to a 42 °C water bath for heat shock for 90 seconds, and then quickly transfer it to an ice bath and place it for 5 minutes.

[0151] 4. Add 500 μL of LB and incubate at 200 rpm on a 37 °C constant - temperature shaker for 1 hour.

[0152] 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes. Leave 200 μL of the supernatant, resuspend the bacterial cells evenly, and spread them evenly on the surface of an agar plate containing the appropriate antibiotic. Incubate the plate upside down overnight in a 37 °C constant temperature incubator.

[0153] II.6 Expression, purification, and fluorescence detection of proteins

[0154] 1. Transform the expression vector (e.g., a lysine optical probe expression vector based on pCDF) into BL21(DE3) cells. Incubate them upside down overnight. Pick colonies from the plate into a 250 ml conical flask and culture them on a shaker at 37 °C and 220 rpm until OD = 0.4 - 0.8. Add 1 / 1000 (v / v) IPTG (1 M) and induce expression at 18 °C for 24 - 36 hours.

[0155] 2. After the induction of expression is completed, centrifuge to collect the bacteria at 4000 rpm for 30 minutes. Add 50 mM phosphate buffer to resuspend the bacterial cell pellet and sonicate until the bacteria are clarified. Centrifuge at 9600 rpm at 4 °C for 20 minutes.

[0156] 3. Purify the protein from the centrifuged supernatant through a self - assembled nickel column affinity chromatography column. The protein after nickel column affinity chromatography is then obtained through a self - assembled desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4).

[0157] 4. After the purified protein is identified by SDS - PAGE, dilute the probe with the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a protein solution with a final concentration of 0.2 - 5 μM. Prepare a stock solution of lysine with a final concentration of 50 mM using the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).

[0158] 5. Take 100 μl of the 1 μM protein solution, incubate it at 37 °C for 10 minutes, add lysine for titration, and measure the fluorescence intensities of the protein emitted at 528 nm after excitation at 420 nm and at 528 nm after excitation at 485 nm. The fluorescence excitation and emission measurements of the samples are completed using a multi - functional microplate reader.

[0159] 6. Take 100 μl of the 1 μM protein solution, incubate it at 37 °C for 10 minutes, add lysine, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum measurements of the samples are completed using a spectrophotometer and a fluorescence spectrophotometer.

[0160] II.7 Transfection and fluorescence detection of mammalian cells

[0161] 1. The pCDNA3.1+-based lysine optical probe plasmid was transfected into HeLa using the transfection reagent Lipofectamine 2000 (Invitrogen), and cultured in a cell incubator at 37°C with 5% CO2. After 24 - 36 h of sufficient expression of the foreign gene, fluorescence detection was performed.

[0162] 2. After the induction of expression was completed, the adherent HeLa cells were rinsed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection respectively.

[0163] Example 1: Lysine-binding protein plasmid

[0164] The LAO gene in the Streptococcus pneumoniae gene was amplified by PCR. After gel electrophoresis of the PCR product, it was recovered and digested with HindIII and XhoI. At the same time, the pCDF vector was digested with the corresponding double enzymes. After ligation with T4 DNA ligase, the product was used to transform DH5α, and the transformed DH5α was spread on an LB plate (streptomycin 100 μg / mL) and cultured overnight at 37°C. After plasmid extraction of the growing DH5α transformants, PCR identification was performed. After the positive plasmid was sequenced correctly, subsequent plasmid construction was carried out.

[0165] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0166] In this example, the following sites were selected for inserting cpYFP based on pCDF-LAO to obtain the corresponding pCDF-LAO-cpYFP plasmids: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196.

[0167] The DNA fragment of cpYFP was generated by PCR. Meanwhile, the homologous sequences at the ends of cpYFP were introduced through the 5'-ends of the primers. The linearized vector of pCDF-lysine-binding protein was amplified by PCR, and the sequences (15 bp - 20 bp) exactly corresponding to the two ends of cpYFP were respectively carried at the 5'- and 3'-terminal most ends. The linearized pCDF-LAO and the cpYFP fragment underwent homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was spread on an LB plate (streptomycin 100 μg / mL) and cultured overnight at 37°C. After the positive clones were identified by PCR, the plasmids were extracted and sequenced. The sequencing was completed by Jie Li Sequencing Company.

[0168] After correct sequencing, the recombinant plasmid was transformed into BL21(DE3) for induced expression, and the protein was purified. The size was around 63 Kda by SDS-PAGE electrophoresis. This size was consistent with the size of the LAO-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-LAO-cpYFP. The results were as Figure 1 shown.

[0169] The lysine response screening was carried out with the broken supernatant of Escherichia coli expressing the LAO-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 1 mM lysine was divided by the detection signal of the fusion fluorescent protein without lysine. The results were as Figure 2 shown, among which the optical probes with a lysine response exceeding 1.3 times were the optical probes with insertions at the 194 / 195, 192 / 193, 192 / 195, 193 / 195, 86 / 90 sites or the corresponding amino acid sites of their family proteins.

[0170] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0171] According to the method in Example 2, cpYFP was replaced with cpGFP to construct a lysine green fluorescent protein fluorescent probe. The results were as Figure 3 shown, among which the detection results of the broken supernatant showed that the optical probes with a lysine response exceeding 1.3 times were the optical probes with insertions at the 192 / 193, 192 / 195, 193 / 195, 194 / 195 sites or the corresponding amino acid sites of their family proteins.

[0172] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0173] According to the method in Example 2, cpYFP was replaced with cpBFP to construct a lysine blue fluorescent protein fluorescent probe. The results were as Figure 4As shown, the optical probes with a lysine response exceeding 1.3 - fold in the broken supernatant detection results are optical probes with insertions at the 192 / 193, 192 / 194, 193 / 195, 194 / 195 sites or the corresponding amino acid sites of their family proteins.

[0174] Example 5: Expression and Detection of cpmApple Optical Probes at Different Insertion Sites

[0175] According to the method in Example 2, cpYFP was replaced with cpmApple to construct a lysine red fluorescent protein fluorescent probe. The results are as Figure 5 shown, where the broken supernatant detection results show that the optical probes with a lysine response exceeding 1.3 - fold are optical probes with insertions at the 192 / 193, 192 / 195, 193 / 195, 193 / 196, 194 / 195 sites or the corresponding amino acid sites of their family proteins.

[0176] Example 6: Expression and Detection of Mutant Optical Probes

[0177] Based on LAO - 192 / 195 - cpYFP, LAO - 193 / 195 - cpYFP, LAO - 194 / 195 - cpYFP, LAO - 192 / 193 - cpYFP, optical probe mutants were constructed. The plasmids LAO - 192 / 195 - cpYFP, LAO - 193 / 195 - cpYFP, LAO - 194 / 195 - cpYFP, LAO - 192 / 193 - cpYFP were linearized by PCR. NNK random mutations were set at the positions to be mutated in the primers. The obtained PCR products were phosphorylated and ligated under the action of PNK, T4 DNA ligase, and PEG4000 to obtain mutant plasmids at the D11, T12, Y14, A15, K23, E25, I27, Q42, A49, F52, S58, S69, S70, L71, S72, D75, R77, D91, A97, L117, S120, T121, Q122, D128, N129, T132, A139, A141, S148, D161, A174, E177, S184, K188, F191, G192, D193, G194, T195, G196, T215, Q219, N234 sites. The library was constructed by transformation and detected. Mutants with a lysine response greater than 1.6 - fold were screened, and sequencing was completed by Jie Li Sequencing Company. The sequences of some mutant optical probes are shown in the following table. An exemplary nucleic acid sequence is shown as SEQ ID NO:10 (LAO - 192 / 193 - cpYFP - T12P / F52T / D193T / G194R).

[0178] Table 1, Sequences of Mutated Optical Probes

[0179]

[0180]

[0181] Example 7. Spectral Properties, Titration Curves, and Specificity of Optical Probe Mutants

[0182] Exemplarily, three lysine optical probes numbered 9, 12, and 27 in Example 6 were purified and treated with 0 mM and 10 mM lysine for 10 minutes respectively, and then the fluorescence spectra were detected using a fluorescence spectrophotometer.

[0183] Measurement of the excitation spectrum: The excitation spectrum was recorded with an excitation range of 370 nm to 510 nm and an emission wavelength of 530 nm, and readings were taken every 5 nm. The results showed that the probe had two excitation peaks at approximately 425 and 490 nm.

[0184] Measurement of the emission spectrum: With the excitation wavelengths fixed at 420 nm and 460 nm respectively, the emission spectra in the ranges of 470 - 600 nm and 490 - 600 nm were recorded, and readings were taken every 5 nm. The excitation and emission spectra are as Figure 6 shown.

[0185] Twenty-two lysine optical probes numbered 6 - 27 in Example 6 were purified and detected for lysine with a concentration gradient (0 - 10 mM). After treating the purified probes for 10 minutes, the changes in the ratio of the fluorescence intensities at 420 nm excitation and 528 nm emission and at 485 nm excitation and 528 nm emission were detected. The results are as Figure 7 shown, and the Kd (binding constant) values of the 22 lysine optical probes were 23 μM - 1261 μM respectively.

[0186] The reactivity of the purified lysine optical probes numbered 9, 12, and 27 in Example 6 was detected with 20 similar substrates, and the results showed that they had good specificity, as Figure 8 shown.

[0187] Example 8: Subcellular Localization of Optical Probes and Their Performance within Subcellular Organelles

[0188] In this example, different localization signal peptides were fused with optical probe 27 (LAO - 192 / 193 - cpYFP - T12P / F52T / D193T / G194R) to localize the optical probe to different organelles.

[0189] After transfecting HeLa cells with an optical probe plasmid fused with different targeting signal peptides for 36 hours, rinse with PBS, place in HBSS solution, and perform fluorescence detection under the FITC channel using an inverted fluorescence microscope. The results are as Figure 9 shown. The lysine optical probe can be localized to subcellular organelles including the cytoplasm, cell outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion by fusing with different specific targeting signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of the fluorescence vary.

[0190] Example 9: Dynamic Monitoring of Lysine Transmembrane Transport

[0191] After transfecting HeLa cells with the cytoplasm-expressed optical probe 27 (LAO-192 / 193-cpYFP-T12P / F52T / D193T / G194R) plasmid for 36 hours, rinse with PBS, place in HBSS solution, and detect 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 within a 30-minute period. The results are as Figure 10 shown. Add 1 mM lysine and continue to detect for 30 minutes. The 485 / 420 of the sample with added lysine gradually increases, reaching a maximum of 1.49 times the initial value, while the 485 / 420 of the control group without added lysine remains unchanged at 0.68.

[0192] Example 10: High-Throughput Compound Screening Based on Optical Probes in Living Cells

[0193] In this example, we used HeLa cells expressing the cytoplasm optical probe 27 (LAO-192 / 193-cpYFP-T12P / F52T / D193T / G194R) for high-throughput compound screening.

[0194] The transfected HeLa cells were rinsed with PBS, placed in HBSS solution (without lysine) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Lysine was added dropwise to each sample. 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 recorded using a microplate reader. Standardization was performed using the sample without any compound treatment as a control. The results are as Figure 11 shown. Among the 2000 compounds used, the vast majority of the compounds had little effect on the entry of lysine into cells. Eleven compounds could enhance the uptake ability of cells for lysine, and another ten compounds could significantly reduce the uptake of lysine by cells.

[0195] Example 11: Quantitative Detection of Lysine in Blood Using Optical Probes

[0196] In this example, purified LAO-193 / 195-cpYFP-F52T was used to analyze lysine in the blood supernatants of mice and humans.

[0197] After mixing LAO-193 / 195-cpYFP-F52T with the diluted blood supernatant and treating for 10 minutes, the fluorescence intensity ratio at 420 nm excitation and 528 nm emission and the fluorescence intensity ratio at 485 nm excitation and 528 nm emission were detected using a microplate reader. The results are as Figure 12 shown. The lysine content in human blood is about 201 μM.

[0198] As can be seen from the above examples, the lysine optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has large fluorescence dynamic changes, good specificity, and can be expressed in cells by genetic manipulation methods. It can be used to locate and quantitatively detect lysine in real time inside and outside cells; and can also perform high-throughput compound screening.

[0199] Other embodiments

[0200] This specification describes many embodiments. However, it should be understood that various improvements learned by those skilled in the art from reading this specification without departing from the concept and scope of the present invention should also be included within the scope of the appended claims.

[0201] Partial sequences

[0202] SEQ ID NO:1 LAO

[0203] ALPQTVRIGTDTTYAPFSSKDAKGEFIGFDIDLGNEMCKRMQVKCTWVASDFDALIPSLKAKKIDAIISSLSITDKRQQEIAFSDKLYAADSRLIAAKGSPIQPTLESLKGKHVGVLQGSTQEAYANDNWRTKGVDVVAYANQDLIYSDLTAGRLDAALQDEVAASEGFLKQPAGKEYAFAGPSVKDKKYFGDGTGVGLRKDDTELKAAFDKALTELRQDGTYDKMAKKYFDFNVYGD

[0204] SEQ ID NO:2 cpYFP YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0205] SEQ ID NO:3 cpGFP

[0206] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0207] SEQ ID NO:4 cpBFP

[0208] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0209] SEQ ID NO:5 cpmApple

[0210] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA。

Claims

1. A lysine-binding protein variant, which: (1) has the sequence shown in SEQ ID NO:1 and has mutations at 1, 2, 3, or 4 of the following sites: D11, T12, Y14, A15, K23, E25, I27, Q42, A49, F52, S58, S69, S70, L71, S72, D75, R77, D91, A97, L117, S120, T121, Q122, D128, N129, T132, A139, A141, S148, D161, A174, E177, S184, K188, F191, G192, D193, G194, T195, G196, T215, Q219, N234, and the mutations include modifications, substitutions, or deletions of amino acids, (2) is a sequence that has at least 70% sequence identity with the sequence of (1), has the mutations described in (1), and retains the ability to bind lysine, Preferably, the mutations include mutations at sites selected from any one of the following groups: (1) F52, (2) F52 and T121, (3) F52 and Q122, (4) F52 and S72, (5) F52, D193, and G194, (6) F52, F191, and G192, (7) F52, T195, and G196, (8) T12, F52, D193, and G194, (9) F52 and D193, More preferably, T12 is mutated to P; F52 is mutated to T, S, or H; S72 is mutated to E; T121 is mutated to S or I; Q122 is mutated to A or H; F191 is mutated to G or Y; G192 is mutated to W or D; D193 is mutated to E, K, L, or T; G194 is mutated to D, R, or S; T195 is mutated to D; G196 is mutated to R or Y; More preferably, the mutations include mutations selected from any one of the following groups: (1) F52T, (2) F52H, (3) F52T and Q122H, (4) F52T and T121S, (5) F52T and T121I, (6) F52T and Q122A, (7) F52T and S72E, (8) F52T, D193L, and G194D, (9) F52T, F191G, and G192D, (10) F52T, D193K, (11) F52T, D193E, and G194S, (12) F52T, F191Y, and G192W, (13) F52T, D193T, and G194R, (14) F52T, D193E, and G194R, (15) F52T, D195D, and G196R, (16) F52T, D195D, and G196Y, (17) T12P, F52T, D193T, and G194R, (18) F52S.

2. A lysine optical probe, comprising a lysine-sensitive polypeptide and an optically active polypeptide, wherein the lysine-sensitive polypeptide has: (1) the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% sequence identity with them and retaining the lysine-binding activity, (2) the sequence of the lysine-binding protein variant described in claim 1, or (3) a sequence having at least 70% sequence identity with the sequence described in (2) and having the mutation described in (2) and retaining the sensitivity to lysine. The optically active polypeptide is a fluorescent protein, and the optically active polypeptide is located at one or more sites selected from the following of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196, Preferably, the optically active polypeptide is selected from any one of the following: cpYFP, cpGFP, cpBFP, cpmApple.

3. The optical probe according to claim 2, wherein, the optically active polypeptide is cpYFP, and it is located at one or more sites selected from the following of the lysine-sensitive polypeptide: 85 / 88, 86 / 87, 86 / 90, 87 / 90, 88 / 89, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, or the optically active polypeptide is cpGFP, and it is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 89, 85 / 90, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 91, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196, 195 / 196, or The optically active polypeptide is cpBFP, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 91, 86 / 87, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 90, 88 / 89, 88 / 90, 88 / 91, 89 / 90, 89 / 91, 90 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 194 / 195, 194 / 196, 195 / 196, or The optically active polypeptide is cpmApple, which is located at any one or more of the following sites of the lysine-sensitive polypeptide: 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 88, 87 / 89, 87 / 91, 88 / 89, 88 / 91, 89 / 90, 89 / 91, 192 / 193, 192 / 194, 192 / 195, 192 / 196, 193 / 194, 193 / 195, 193 / 196, 194 / 195, 194 / 196, 195 / 196.

4. The optical probe according to claim 2, wherein, The optically active polypeptide is cpYFP, which is located at the 193 / 195 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide contains one or more mutations selected from the following: F52T, F52H, F52S, The optically active polypeptide is cpYFP, which is located at the 194 / 195 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide contains one or more mutations selected from the following: F52T, F52H, Q122H, Q122A, T121I, T121S, S72E, D193L, D193K, D193E, G194D, G194S, T195D, G196R, G196Y, F52S, The optically active polypeptide is cpYFP, which is located at the 192 / 193 site of the lysine-sensitive polypeptide, and the lysine-sensitive polypeptide contains one or more mutations selected from the following: T12P, F52T, F52S, F191G, F191Y, G192D, G192W, D193T, D193E, G194R.

5. A nucleic acid molecule, which comprises: (a) The coding sequence of the optical probe according to any one of claims 2-4, (b) The complementary sequence of (a).

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5, Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

7. A host cell, the host cell: (1) Expresses the optical probe according to any one of claims 2-4; (2) Contains the nucleic acid molecule according to claim 5; or (3) Contains the nucleic acid construct according to claim 6.

8. A detection kit, which comprises: (1) The optical probe according to any one of claims 2-4, (2) The nucleic acid sequence according to claim 5, (3) The nucleic acid construct according to claim 6, or (4) The host cell according to claim 7, Optionally, the detection kit further comprises other reagents required for detecting lysine using an optical probe, Preferably, the detection kit further comprises one or more reagents selected from the following: buffer, culture medium, lysine standard.

9. A method for preparing the optical probe according to any one of claims 2-4, comprising: Providing the host cell according to claim 7, culturing the host cell under conditions for expression of the optical probe, and isolating the optical probe.

10. Use of the optical probe according to any one of claims 2-4, the nucleic acid sequence according to claim 5, the nucleic acid construct according to claim 6, or the host cell according to claim 7 in detecting lysine in a sample, screening compounds, and intracellular / extracellular localization of lysine. Preferably, detecting lysine in a sample comprises the steps of: contacting the optical probe or host cell with the sample, detecting the optical change of the optically active polypeptide, and detecting lysine in the sample based on the optical change of the optically active polypeptide, The screening of compounds comprises the steps of: contacting the optical probe or host cell with a candidate compound in a lysine-containing system, detecting the optical change of the optically active polypeptide, and screening the compound based on the optical change of the optically active polypeptide; preferably, the screening of compounds comprises the steps of: contacting the host cell with a candidate compound in a lysine-containing system, and the optical change of the optically active polypeptide indicates whether the candidate compound regulates the uptake of lysine by the cell, The intracellular / extracellular localization of lysine comprises the steps of: contacting a lysine-containing system with the optical probe or the host cell, and detecting the optical change of the optically active polypeptide, More preferably, the system is a solution system, a cell system or a subcellular system.

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