Arginine Optical Probe

Abstract

The present invention relates to an arginine fluorescent optical probe. Specifically, the present invention provides an arginine-sensitive polypeptide, which is a mutant of an arginine-binding protein, and (1) has the sequence set forth in SEQ ID NO:1 and has mutations at one, two, three, or more sites selected from the following: (1) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (2) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (3) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (4) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (5) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (6) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (7) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (8) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (9) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (10) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (11) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (12) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (13) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (14) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (15) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (16) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (17) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (18) a fluorescent probe formed from a wild-type arginine-sensitive polypeptide, (19 ...

Description

[Technical Field]

[0001] The present invention relates to the technical field of optical probes, and in particular to an arginine optical probe and its preparation method and application. [Background technology]

[0002] Arginine, one of the 20 naturally occurring amino acids, was first isolated and extracted from lupine seedlings by Schluss in 1886. Its molecular structure was elucidated in the early 20th century, and it became possible to synthesize it artificially. Arginine fulfills its biological functions in vivo as the physiologically active L-arginine. Arginine is not only a component of proteins in the body, but also a precursor for the synthesis of various biologically active substances such as polyamines and NO. It stimulates the secretion of certain hormones, thereby participating in biological processes such as endocrine regulation and body-specific immune control. It also functions as an intermediate in the urea cycle, alleviating ammonia poisoning and preventing metabolic disorders caused by excess ammonia. Furthermore, it plays an important role in the homogeneous metabolism of the body, being utilized in various metabolic pathways such as arginase, nitric oxide synthase, arginine / glycine guanidinotransferase, and arginyl-tRNA synthetase. Therefore, arginine's multiple biological functions have attracted widespread attention from researchers, making it a hot topic in amino acid research.

[0003] There are two main pathways for arginine metabolism in mammals: first, arginase decomposes arginine into ornithine and urea, which are precursors for the synthesis of polyamines, which are important for the regulation of cell growth and development; second, nitric oxide synthase decomposes arginine into equal molecular weights of citrulline and NO, which is widely involved in intercellular and intracellular signal transduction as a messenger molecule for intracellular, intercellular, and neurotransmitter functions. Furthermore, arginine, glycine, and methionine act together as amidine donors in the amidinotransfer reaction to synthesize guanidinoacetic acid and phosphocreatine. Gamma-guanidinobutyric acid is also synthesized from arginine in nervous tissue (Delforge J et al., Eur J Biochem. 1975, 57(1):231-239 (Non-Patent Document 1); Fernandez ML et al., J. Bacteriol. 2004, 186(18):6142-6149 (Non-Patent Document 2); Fernandez ML et al., J. Bacteriol. 2008, 190(18):3018-3025 (Non-Patent Document 3)).

[0004] Arginine is one of the three substrates that form creatine, an important nutrient involved in ascites formation (its deficiency can lead to mental retardation) and a signaling molecule in the body. Arginine is an intermediate in the urea cycle (which includes L-ornithine, L-citrulline, and argininosuccinate) and the nitric oxide cycle (which includes ornithine and argininosuccinate), generating polyamine structures that can regulate cellular function via ornithine. Arginine deficiency (which can be induced by increasing the activity of arginase, the enzyme that converts arginine to ornithine) can impair B-cell function (immunity), inhibit hair and muscle growth, and impair neuromuscular function. Arginase is known to be overexpressed in patients with type 2 diabetes, a risk factor for cardiovascular disease in this cohort, and its reduction may be present in patients with renal failure.

[0005] Because arginine has the important functions mentioned above, measuring arginine content is particularly important. Commonly used methods for detecting arginine include capillary electrophoresis (Li Xt et al., Chem Res Chin Univ 2013, 29(3):434-438 (Non-Patent Document 4); Meng J et al., The Analyst 2010, 135(7):1592-1599 (Non-Patent Document 5)), high-performance liquid chromatography (Tateda N et al., Analytical sciences: the international journal of the Japan Society for Analytical Chemistry 2001, 17(6):775-778 (Non-Patent Document 6); Wadud S et al., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374 (Non-Patent Document 7)), enzyme-linked immunosorbent assay, and ultraviolet-visible spectrophotometry (Hortpro MA et al., J Am Chem So 2003, 125(1):20-21 (Non-Patent Document 8); Pu F et al., Anal Chem 2010, 82(19):8211-8216 (Non-Patent Document 9); Du J et al., Chemical communications (Cambridge, England) 2013, 49(47):5399-5401 (Non-Patent Document 10); Engeser M et al., Chemical Communications 1999, (13):1191-1192 (Non-Patent Document 11)) and fluorescence spectroscopy (Engeser M et al., Chemical Communications 1999, (13):1191-1192 (Non-Patent Document 11)).

[0006] However, these detection methods for live cell research require time-consuming sample processing steps, such as cell disruption, separation, extraction, and purification, and have major drawbacks: they cannot perform in situ, real-time, dynamic, high-throughput, and high-spatiotemporal resolution detection in live cells and subcellular organelles. There remains a need in this field for real-time, in situ, quantitative, and high-throughput methods for intracellular and extracellular detection of arginine. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Delforge J et al., Eur J Biochem. 1975, 57(1):231-239 [Non-patent document 2] Fernandez ML et al., J. Bacteriol. 2004, 186(18):6142-6149 [Non-patent document 3] Fernandez ML et al., J. Bacteriol. 2008, 190(18):3018-3025 [Non-patent document 4] Li Xt et al., Chem Res Chin Univ 2013, 29(3):434-438 [Non-Patent Document 5] Meng J et al., The Analyst 2010, 135(7):1592-1599 [Non-patent document 6] Tateda N et al., Analytical sciences: the international journal of the Japan Society for Analytical Chemistry 2001, 17(6):775-778 [Non-Patent Document 7] Wadud S et al., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374 [Non-patent document 8] Hortpro MA et al., J Am Chem So 2003, 125(1):20-21 [Non-Patent Document 9] Pu F et al., Anal Chem 2010, 82(19):8211-8216 [Non-Patent Document 10] Du J et al., Chemical communications (Cambridge, England) 2013, 49(47):5399-5401 [Non-Patent Document 11] Engeser M et al., Chemical Communications 1999, (13):1191-1192 Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above, an object of the present invention is to provide an arginine fluorescent probe for real-time localization, high-throughput, and quantitative detection of arginine inside and outside cells. [Means for solving the problem]

[0009] In order to achieve the above object of the invention, the present invention provides the following technical solutions: In a first aspect of the present invention, there is provided an arginine-sensitive polypeptide, the arginine-sensitive polypeptide being a mutant of an arginine-binding protein, (1) having the sequence set forth in SEQ ID NO: 1 and having amino acid mutations, including amino acid modifications, substitutions, or deletions, at one, two, or three or more sites selected from the following: T32, E37, F71, G89, M90, D91, R96, E135, T138, T139, H140, D177; or (2) A sequence having at least 70% sequence identity with the sequence described in (1), having the mutation described in (1), and retaining arginine sensitivity.

[0010] In one or more embodiments, the mutations are one, two, or three or more selected from T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, D177A.

[0011] The present invention further provides an arginine optical probe comprising an arginine-sensitive polypeptide and an optically active polypeptide or a functional mutant thereof, wherein the optically active polypeptide or a functional mutant thereof is located within the sequence of the arginine-sensitive polypeptide, and the arginine-sensitive polypeptide is divided into a first portion and a second portion by the optically active polypeptide or a functional mutant thereof.

[0012] The present invention provides an arginine optical probe comprising an arginine-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the arginine-sensitive polypeptide B, and the arginine-sensitive polypeptide B is divided into a first portion B1 and a second portion B2, forming a probe structure of the formula B1-A-B2.

[0013] In one embodiment, the arginine-sensitive polypeptide is (1) having the sequence set forth in SEQ ID NO: 1 and having amino acid mutations, including amino acid modifications, substitutions, or deletions, at one, two, or three or more sites selected from the following: T32, E37, F71, G89, M90, D91, R96, E135, T138, T139, H140, D177; or (2) A sequence having at least 70% sequence identity with the sequence described in (1), having the mutation described in (1), and retaining arginine sensitivity.

[0014] In one or more embodiments, the mutations are one, two, or three or more selected from T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, D177A.

[0015] In one embodiment, the optically active polypeptide is a fluorescent protein, or a functional fragment or mutant thereof. In one embodiment, the fluorescent protein is a yellow fluorescent protein derived from YFP. Preferably, the fluorescent protein is cpYFP as shown in SEQ ID NO:3.

[0016] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. In one embodiment, the flanking of the optically active polypeptide comprises a linker of 5 or fewer amino acids, for example, a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as shown below: a first portion B1 of the arginine-sensitive polypeptide -Y-optically active polypeptide A-a second portion B2 of the arginine-sensitive polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker.

[0017] In one embodiment, the optically active polypeptide is located between residues 200-203 of the arginine-sensitive polypeptide, where the numbering corresponds to the full-length of the arginine-binding protein. In one embodiment, the optically active polypeptide substitutes one or more amino acids within residues 200-203 of the arginine-sensitive polypeptide, where the numbering corresponds to the full-length of the arginine-binding protein.

[0018] In one embodiment, the optically active polypeptide is located at the 200 / 203 position of the arginine-sensitive polypeptide.

[0019] In one or more embodiments, the optically active polypeptide is located at position 200 / 203 of the arginine-sensitive polypeptide, and the arginine-sensitive polypeptide contains one or more mutations at positions selected from the following: F71, G89, M90, R96, E135, T139, and D177. These probes have a response to arginine that exceeds that of the control.

[0020] In one or more embodiments, the optically active polypeptide is located at position 200 / 203 of the arginine-sensitive polypeptide, and the arginine-sensitive polypeptide contains one or more mutations at positions selected from the following: T32, E37, D91, T138, H140. These probes respond to arginine more than three-fold compared to the control.

[0021] In one or more embodiments, the optical probe comprises a cpYFP insertion at the 200 / 203 site of the arginine-sensitive polypeptide and one or more mutations selected from the group consisting of T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A.

[0022] In one or more embodiments, the optical probe has a sequence set forth in any of SEQ ID NOs: 4-15, or a sequence having at least 70% sequence identity thereto.

[0023] The present invention further provides fusion polypeptides comprising an optical probe described herein and another polypeptide. In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to various organelles or subcellular compartments, a tag for purification, or a tag for immunoblotting.

[0024] The present invention further provides a nucleic acid molecule, the nucleic acid molecule comprising: (a) a coding sequence for a polypeptide or probe described in any one embodiment herein; or (b) a complementary sequence of (a); or (c) a fragment of (a) or (b).

[0025] In one embodiment, the nucleic acid sequence of the present invention is selected from the group consisting of: (1) a coding sequence for an amino acid sequence set forth in any of SEQ ID NOs:4-15 or a complementary sequence thereof; (2) a sequence having at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, or at least 50% identity to (1); and (3) a fragment of (1) or (2).

[0026] In one or more embodiments, the fragment is a primer.

[0027] The present invention further relates to variants of the above nucleic acid molecules, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and mutants of the optical probes or fusion proteins of the present invention, or their complementary sequences.

[0028] The present invention further provides a nucleic acid construct comprising a nucleic acid molecule described herein, wherein the nucleic acid sequence encodes an optical probe or fusion polypeptide described in the present invention.

[0029] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0030] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0031] In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0032] The present invention further provides a host cell, the host cell (1) expressing an optical probe or fusion polypeptide described in any one of the embodiments of the present invention, (2) comprising a nucleic acid molecule described in any one of the embodiments of the present invention, or (3) comprising a nucleic acid construct described in any one of the embodiments of the present invention. The host cell is preferably Escherichia coli.

[0033] The present invention further provides a kit for detecting arginine, the kit comprising an optical probe or a fusion polypeptide or polynucleotide described herein or an optical probe prepared by the methods described herein.

[0034] In one or more embodiments, the kit further comprises one or more reagents selected from the following: a buffer, a medium, and an arginine standard.

[0035] The present invention provides a method for preparing an optical probe described herein, the method comprising: providing a host cell that expresses an optical probe or fusion polypeptide described herein, culturing the host cell under conditions such that the cell expresses the optical probe or fusion polypeptide, and isolating the optical probe or fusion polypeptide.

[0036] In one or more embodiments, the method comprises the steps of: 1) incorporating a nucleic acid molecule encoding the arginine 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 expression of the expression vector; and 3) isolating the arginine optical probe.

[0037] The present invention further provides a method for detecting arginine in a sample, the method comprising: contacting the sample with an optical probe or fusion polypeptide or host cell described herein; and detecting an optical change in the optically active polypeptide. The detection may be performed in vivo, ex vivo, intracellularly, or in situ. The sample may be, for example, blood or urine.

[0038] In one or more embodiments, the contacting is carried out at a pH of 6 to 8 or 7 to 8. Preferably, the contacting is carried out at a pH of about 7.

[0039] In one or more embodiments, the contacting is carried out at a temperature of 20-40°C.

[0040] The present specification further provides a method for quantifying arginine in a sample, the method comprising: contacting an optical probe or fusion polypeptide or host cell described herein with a sample; detecting an optical change in the optically active polypeptide; and quantifying arginine in the sample based on the optical change in the optically active polypeptide.

[0041] In one or more embodiments, the contacting is carried out at a pH of 6 to 8 or 7 to 8. Preferably, the contacting is carried out at a pH of about 7.

[0042] In one or more embodiments, the contacting is carried out at a temperature of 20-40°C.

[0043] The present invention further provides a method for screening compounds (e.g., pharmaceuticals), the method comprising: contacting an optical probe or fusion polypeptide or host cell described herein with a candidate compound in a system containing arginine, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide. The method allows for high-throughput screening of compounds.

[0044] In one or more embodiments, a host cell described herein is contacted with a candidate compound in a system comprising arginine, and an optical change in the optically active polypeptide indicates whether the candidate compound can modulate the cellular uptake of arginine.

[0045] In one or more embodiments, the system is a solution system, a cellular system, or an intracellular system.

[0046] In one or more embodiments, the contacting is carried out at a pH of 6 to 8 or 7 to 8. Preferably, the contacting is carried out at a pH of about 7.

[0047] In one or more embodiments, the contacting is carried out at a temperature of 20-40°C.

[0048] The present invention further provides uses of the arginine optical probes or fusion polypeptides or host cells described herein for detecting arginine in a sample, screening compounds, and / or localizing arginine intracellularly and / or extracellularly. In one or more embodiments, the localization is real-time localization.

[0049] In one or more embodiments, the intracellular and / or extracellular localization of arginine comprises contacting a system containing arginine with an optical probe or fusion polypeptide or host cell described herein and detecting an optical change in the optically active polypeptide. [Effects of the Invention]

[0050] Benefits of the Invention: The arginine fluorescent probe provided by the present invention comprises an arginine-sensitive polypeptide mutant and fluorescent protein A inserted into its sequence. The arginine fluorescent probe provided by the present invention is easily maturated. Compared to probes formed with wild-type arginine-sensitive polypeptides, the fluorescent probe of the present invention has a larger dynamic change in fluorescence, excellent specificity, and can be expressed intracellularly using genetic engineering methods, enabling real-time localization, high-throughput, and quantitative detection of arginine inside and outside of cells. Time-consuming sample processing steps are eliminated. Experimental results show that the arginine optical probe provided by the present application has a maximum response to arginine that is more than 15-fold higher than the control. It can be used to detect arginine locally in intracellular structures such as the cytoplasm, mitochondria, nucleus, Golgi apparatus, peroxisomes, and lysosomes, and can also be used for high-throughput compound screening and quantitative detection of arginine in blood and urine. [Brief explanation of the drawings]

[0051] The present invention will be further described below with reference to the drawings and examples. [Figure 1] FIG. 1 is an SDS-PAGE analysis diagram of the arginine fluorescent probe according to Example 2. [Figure 2] Figure 2 shows the change in response to arginine in the yellow fluorescent protein cpYFP, ​​which was mutated at the T32, E37, F71, G89, M90, D91, R96, E135, E138, T139, H140, and D177 sites based on an arginine fluorescent probe inserted into the 200 / 203 insertion site of the arginine-binding protein. [Figure 3]FIG. 3 shows the titration curve of a point-mutated probe based on an arginine fluorescent probe in which the yellow fluorescent protein cpYFP was inserted into the 200 / 203 insertion site of the arginine-binding protein, against different concentrations of arginine. [Figure 4] FIG. 4 is a fluorescence spectrum diagram of an exemplary arginine fluorescent probe in which the yellow fluorescent protein cpYFP is mutated at the E37 site based on the arginine fluorescent probe inserted into the 200 / 203 insertion site of the arginine binding protein. [Figure 5A] FIG. 5 shows the specificity of mutant probes based on an exemplary arginine fluorescent probe in which the yellow fluorescent protein cpYFP is inserted into the 200 / 203 insertion site of an arginine binding protein. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above. [Figure 5J] Same as above. [Figure 6] FIG. 6 shows the affinity to arginine at different pH values ​​of an arginine fluorescent probe mutated at the E37 site based on an exemplary arginine fluorescent probe in which the yellow fluorescent protein cpYFP was inserted into the 200 / 203 insertion site of an arginine binding protein. [Figure 7] Figure 7 shows the change in response to arginine at different temperatures of an arginine fluorescent probe mutated at the E37 site based on an exemplary arginine fluorescent probe in which the yellow fluorescent protein cpYFP was inserted into the 200 / 203 insertion site of an arginine binding protein. [Figure 8]FIG. 8 shows the affinity to arginine at different temperatures of an arginine fluorescent probe mutated at the E37 site based on an exemplary arginine fluorescent probe in which the yellow fluorescent protein cpYFP was inserted into the 200 / 203 insertion site of an arginine binding protein. [Figure 9] FIG. 9 shows the localization and performance within intracellular organelles of an arginine fluorescent probe mutated at the E37 site based on an arginine fluorescent probe in which an exemplary yellow fluorescent protein, cpYFP, ​​was inserted into the 200 / 203 insertion site of an arginine-binding protein. [Figure 10] FIG. 10 shows dynamic monitoring of transmembrane transport of arginine using an exemplary yellow fluorescent protein, cpYFP, ​​mutated at the E37 site based on an arginine fluorescent probe inserted into the 200 / 203 insertion site of an arginine-binding protein. [Figure 11] FIG. 11 shows a high-throughput compound screening at the cell level using an exemplary yellow fluorescent protein, cpYFP, ​​based on an arginine fluorescent probe inserted into the 200 / 203 insertion site of an arginine-binding protein and mutated at the E37 site. [Figure 12] FIG. 12 shows the quantitative detection of arginine in human blood and urine by an arginine fluorescent probe mutated at the E37 site based on an exemplary yellow fluorescent protein cpYFP inserted into the 200 / 203 insertion site of an arginine binding protein. DETAILED DESCRIPTION OF THE INVENTION

[0052] When a numerical value or range is disclosed, the term "about" as used herein means that the numerical value or range is within 20%, within 10%, and within 5% of the stated numerical value or range.

[0053] As used herein, the terms "comprise", "include", and their equivalents (including "contain" and "consist of") mean that, for example, a composition "comprising" X may consist solely of X, but may also contain other substances, e.g., X+Y.

[0054] As used herein, the term "arginine-sensitive polypeptide" or "arginine-responsive polypeptide" refers to a polypeptide that responds to arginine, and the response refers to any response in a chemical, biological, electrical, or physiological parameter of the polypeptide related to the interaction of the sensitive polypeptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the polypeptide, and changes in the primary, secondary, or tertiary structure of the polypeptide (including changes in protonation, electrochemical potential, and / or conformation). "Conformation" refers to the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing pendant groups. A change in the three-dimensional structure of a molecule results in a change in conformation. Examples of conformational changes include a transition from α-helix to β-sheet or a transition from β-sheet to α-helix. It should be understood that the detected change does not necessarily have to be a conformational change, as long as the fluorescence of the fluorescent protein moiety changes. The arginine-sensitive polypeptides described herein may also include functional mutants thereof. Functional mutants of arginine-sensitive polypeptides include, but are not limited to, mutants that are able to interact with arginine and cause the same or similar changes as the parent arginine-sensitive polypeptide.

[0055] The arginine-sensitive polypeptides of the present invention include mutants of the arginine-binding protein STM4351 derived from Salmonella Typhimurium. An exemplary STM4351 protein is set forth in SEQ ID NO:1. The inventors have discovered that STM4351 mutants have a higher binding affinity for arginine. Such mutations include those at positions 32 (T), 37 (E), 71 (F), 89 (G), 90 (M), 91 (D), 96 (R), 135 (E), 138 (T), 139 (T), 140 (H), and 177 (D) of STM4351. Illustratively, the mutations at these positions are to alanine (A) or similar amino acids, such as valine, leucine, isoleucine, arginine, phenylalanine, methionine, or tryptophan.

[0056] STM4351 consists of an arginine-binding / regulatory domain and a DNA-binding domain. Therefore, the arginine-sensitive polypeptide of the present invention may be a fragment of the above-mentioned STM4351 protein mutant containing the arginine-binding domain (e.g., one that does not contain the DNA-binding domain).

[0057] In an exemplary embodiment, the optical probe of the present invention may be a probe having cpYFP inserted at the 200 / 203 site of an arginine binding protein and one or more mutations selected from the group consisting of T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A.

[0058] As used herein, the term "optical probe" refers to an arginine-sensitive polypeptide fused to an optically active polypeptide. The inventors have discovered that the conformational change induced by the specific binding of an arginine-sensitive polypeptide, such as an arginine-binding protein, to physiological concentrations of arginine induces a conformational change in the optically active polypeptide (e.g., a fluorescent protein), thereby causing a change in the optical properties of the optically active polypeptide. The presence and / or level of arginine can be detected and / or analyzed by plotting a standard curve using the fluorescence of the fluorescent protein measured at different arginine concentrations. When describing the optical probes of the present invention (e.g., when describing insertion or mutation sites), all amino acid residue numbers refer to SEQ ID NO:1.

[0059] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operably inserted into an arginine-sensitive polypeptide. A protein "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is one of the optical properties of an optically active polypeptide and can be used as a means of detecting the responsiveness of the optical probes of the present invention. Preferably, the protein substrate is selected to have readily distinguishable fluorescent properties between its unactivated and activated conformational states. The optically active polypeptides described herein may also be functional mutants thereof. Functional mutants of optically active polypeptides include, but are not limited to, mutants that can induce changes in fluorescent properties that are the same as or similar to those of the parent optically active polypeptide.

[0060] The term "fluorescent protein" as used herein refers to a protein that emits fluorescence when irradiated with excitation light. Fluorescent proteins are a fundamental detection method in the field of biological science, and include, for example, green fluorescent protein (GFP), which is commonly used in the field of biotechnology, and circularly rearranged blue fluorescent protein (cpBFP), circularly rearranged green fluorescent protein (cpGFP), circularly rearranged yellow fluorescent protein (cpYFP), etc., which are derived by mutation of this protein; also includes red fluorescent protein (RFP), which is commonly used in this field, and circularly rearranged proteins derived from this protein (cpmApple, cpmOrange, cpmKate, etc.). Fluorescent proteins and their sequences that can be used in the present invention are well known in the field. Illustratively, cpYFP is shown in SEQ ID NO:3.

[0061] The term "linker" or "connecting region" refers to an amino acid or nucleotide sequence that connects two parts of a polypeptide, protein, or nucleic acid of the present invention. Illustratively, in the present invention, the number of amino acids at the amino terminus of the connecting region between the arginine-sensitive polypeptide and the optically active polypeptide is selected to be 0 to 3, and the number of amino acids at the carboxyl terminus is selected to be 0 to 2; when the recombinant optical probe is connected to a functional protein as a basic unit, the linker sequence can be fused to the amino terminus or carboxyl terminus of the recombinant optical probe. The linker sequence is a short peptide chain composed of one or more flexible amino acids, such as Y.

[0062] In the optical probe of the present invention, the optically active polypeptide is located at the 200 / 203 position in the N-C direction of the arginine-sensitive polypeptide. Herein, when the two numbers in the "X / Y" format are not consecutive integers, this means that the optically active polypeptide substitutes the amino acids between the amino acids indicated by those numbers. For example, insertion site 200 / 203 indicates that the optically active polypeptide substitutes amino acids 201-202 of the arginine-sensitive polypeptide.

[0063] The terms "mutant" or "variant" used herein when referring to a polypeptide or protein include variants that have similar functions to the polypeptide or protein but differ in sequence. Mutant polypeptides or proteins may include homologous sequences, conservative mutants, allelic variants, naturally occurring mutants, and induced mutants. These variants include, but are not limited to, sequences obtained by deleting, inserting, and / or substituting one or more amino acids (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5) in the sequence of the polypeptide or protein and adding one or more amino acids (usually up to 20, preferably up to 10, more preferably up to 5) to the carboxyl and / or amino termini. These variants may also include polypeptides or proteins that share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein. Without being bound by theory, the change of amino acid residue that does not change the overall configuration and function of polypeptide or protein is called function-conserving mutation.For example, in the field, when an amino acid is substituted with an amino acid that has close or similar properties, it usually does not change the function of polypeptide or protein.In the field, amino acids with similar properties often refer to a family of amino acids that have similar side chains, and are clearly defined in the field.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, addition of one or more amino acids, for example, to the amino and / or carboxyl termini, generally does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for commonly 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.

[0064] With respect to two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences being identical, or having a certain percentage of amino acid residues or nucleotides identical over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical), when the two or more sequences or subsequences are compared and aligned for maximum correspondence using methods known in the art, such as sequence comparison algorithms, over a comparison window or designated region. For example, preferred algorithms suitable 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.

[0065] It is well known to those skilled in the art that gene cloning procedures often require the design of appropriate enzyme cleavage sites, inevitably introducing one or more extraneous residues at the termini of the expressed polypeptide or protein, but which do not affect the activity of the target polypeptide or protein. Furthermore, for the purposes of constructing fusion proteins, promoting recombinant protein expression, obtaining recombinant proteins that are automatically secreted outside host cells, or conveniently purifying recombinant proteins, it is often necessary to add several amino acids to the N-terminus, C-terminus, or other appropriate regions of the recombinant protein, including, but not limited to, appropriate linker peptides, signal peptides, leader peptides, terminal extensions, tags such as glutathione S-transferase (GST), maltose E-binding protein, protein A, 6His, or Flag, or proteolytic enzyme sites for factor Xa, thrombin, or enterokinase.

[0066] As used herein, the terms "functional fragment," "derivative," and "analog" refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., arginine-binding protein or fluorescent protein). Functional mutants, derivatives, or analogs of the polypeptides or proteins (e.g., arginine-binding protein or fluorescent protein) of the present invention may be (i) proteins with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted (such substituted amino acid residues may or may not be those encoded by genetic codons), or (ii) proteins with substitutions at one or more amino acid residues, or (iii) proteins formed by fusing the mature protein with another compound (e.g., a compound such as polyethylene glycol to extend the half-life of the protein), or (iv) proteins formed by fusing additional amino acid sequences to the protein sequence (e.g., secretory sequences, sequences used for purifying the protein, proprotein sequences, or fusion proteins formed with antigenic IgG fragments). Based on the teachings herein, these functional mutants, derivatives, and analogs fall within the scope known to those skilled in the art. These analogs also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids) or non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the arginine-sensitive polypeptides of the present invention are not limited to the exemplary proteins, mutants, derivatives, and analogs listed above. Modified forms (which do not usually alter the primary structure) include chemically derivatized forms of proteins in vivo or in vitro, such as acetylation and carboxylation. Modifications further include glycosylation, including proteins that have been glycosylated during protein synthesis and processing or in further processing steps. Such modifications can be achieved by exposing the protein to glycosylating enzymes (e.g., mammalian glycosylases or deglycosylases).Modified forms further include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins modified to increase resistance to proteolysis or to optimize solubility.

[0067] The fusion polypeptides of the present invention comprise an optical probe described herein and another polypeptide. In some embodiments, the optical probe described herein further comprises another polypeptide fused thereto. The other polypeptide described herein does not affect the properties of the optical probe. The other polypeptide may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to various organelles or subcellular compartments, a tag for purification, or a tag for immunoblotting. A linker may be present between the optical probe and the other polypeptide in the fusion polypeptide described herein.

[0068] Subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, plasma membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the purification tag or immunoblotting tag includes hexahistidine (6*His), glutathione S-transferase (GST), and Flag.

[0069] The present invention includes nucleic acid molecules encoding the arginine-sensitive polypeptides or optical probes described herein. The terms "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be the coding strand or the non-coding strand. When referring to nucleic acids, the term "mutant" as used herein may refer to naturally occurring allelic mutants or non-naturally occurring mutants. These nucleotide mutants include complementary synonymous mutants (degenerate mutants), substitution mutants, deletion mutants, and insertion mutants. As known in the art, allelic mutants are alternative forms of nucleic acids that may contain one or more nucleotide substitutions, deletions, or insertions, but do not substantially alter the function of the encoded protein. Nucleic acids of the present invention may comprise nucleotide sequences having 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% sequence identity to the sequences of the nucleic acids. The present invention further relates to nucleic acid fragments that hybridize to the sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. Nucleic acid fragments are amenable to nucleic acid amplification techniques (e.g., PCR).

[0070] The full-length sequence or fragments of the fluorescent probe or fusion protein of the present invention can be obtained typically by PCR amplification, artificial synthesis, or recombinant techniques. Procedures and reagents used in general PCR, synthesis, and recombinant techniques are known in the art. Mutations can also be introduced into the protein sequences of the present invention by methods such as mutagenic PCR and chemical synthesis.

[0071] The present invention also relates to nucleic acid constructs, which comprise 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 a variety of ways to ensure expression of a polypeptide or protein. Depending on the expression vector's requirements, the nucleic acid construct may be manipulated before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0072] In some embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, including plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence for the protein or polypeptide of the present invention. Expression vectors can be provided to cells as bacterial or viral vectors. Typically, the polynucleotide of the present invention is expressed by operably linking the polynucleotide of the present invention to a promoter and incorporating the construct into the expression vector. The vector is suitable for replication and integration in eukaryotic cells. In one or more embodiments, the cloning vector and expression vector are combined into a single vector, i.e., a cloning-expression vector. The homologous recombination vector is used to integrate the expression cassette described herein into the host genome.

[0073] A typical expression vector contains an expression control sequence that can be used to control the expression of a desired nucleic acid sequence and is operably linked to the nucleic acid sequence of the present invention or its complementary sequence. As used herein, the term "expression control sequence" refers to an element operably linked to a target gene that regulates the transcription, translation, and expression of the target gene. It may be a replication origin, promoter, marker gene, or translation control element, including an enhancer, operon, terminator, ribosome binding site, etc., but the expression control sequence is selected depending on the host cell used. In a recombinant expression vector, "operably linked" refers to the linkage between the target nucleotide sequence and the regulatory sequence in a manner that allows expression of the nucleotide sequence. Methods that can be used to construct expression vectors containing a fusion protein coding sequence of the present invention and appropriate transcriptional / translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operatively linked to an appropriate promoter of the expression vector to direct mRNA synthesis. Representative examples of these promoters include the E. coli lac or trp promoter; the lambda phage PL promoter; the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, eukaryotic promoters including retroviral LTRs, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be the commercially available pET28a vector, without any other special requirements. For example, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and then the digestion products are ligated to obtain a recombinant expression vector. The present invention is not particularly limited by the specific procedures and parameters for enzymatic cleavage and ligation, and conventional procedures and parameters in the art can be used.

[0074] After obtaining a recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide, including a fusion protein. This transfer process can be carried out using conventional techniques, such as transformation and transfection, well known to those skilled in the art. The term "host cell" as used herein refers to a cell capable of receiving and accommodating a recombinant DNA molecule and serving as a site for recombinant gene amplification. Ideal recipient cells must be easily accessible and easily propagated. The "host cell" of the present invention includes prokaryotic and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells, and mammalian cells. Specific examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells such as Drosophila S2 or Sf9, and animal cells such as CHO, COS, HEK293, HeLa, and Bowes' melanoma cells, but are not limited to the host cells listed above. The host cells are preferably cells advantageous for the expression or fermentation of gene products, and such cells are well known and commonly used in the art. An exemplary host cell used in the examples of the present invention is the E. coli JM109-DE3 strain. It is well known to those skilled in the art how to select appropriate vectors, promoters, enhancers and host cells.

[0075] The methods for introducing DNA into host cells described in the present invention are conventional methods in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfection, or electroporation. When the host is a prokaryotic organism such as E. coli, the method is preferably the CaCl2 method or the MgCl2 method, and the procedures used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be applied: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

[0076] In the present invention, an expression vector is introduced into a host cell, and then the host cell is amplified, expressed, and cultured to isolate and obtain an arginine optical probe. Conventional methods can be used for amplifying, expressing, and culturing the host cell. Depending on the type of host cell used, various conventional media may be used for culturing. The host cell is cultured under conditions suitable for its growth.

[0077] In the present invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated or purified by various separation methods based on its physical, chemical, and other properties. In the present invention, the method for isolating the arginine fluorescent protein is not particularly limited, and any conventional fusion protein isolation method known in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, cell disruption by osmosis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), other liquid chromatography techniques, and combinations of these methods. In one embodiment, affinity chromatography using a His tag is used to isolate the optical probe.

[0078] The present invention further provides uses of the arginine optical probe in real-time localization, quantitative detection, and high-throughput compound screening of arginine. In one aspect, the arginine optical probe is preferably linked to a signal peptide at a different site in a cell, introduced into the cell, and the intensity of the fluorescent signal in the cell is detected to perform real-time localization of arginine; the corresponding arginine is quantitatively detected by combining the change in the fluorescent signal with a standard arginine titration curve. The change in the fluorescent signal is represented, for example, by a normalized fluorescent signal ratio. In an embodiment involving cpYFP, ​​the ratio is the ratio between the 485 nm fluorescent signal and the 420 nm fluorescent signal of the sample and the corresponding ratio of the control. The arginine standard titration curve described in the present invention is plotted based on the fluorescent signal of the arginine optical probe under different arginine concentrations. The arginine fluorescent probe of the present invention is directly introduced into the cell, eliminating the need for time-consuming sample processing during the real-time localization and quantitative detection of arginine, making it more accurate. When using the arginine fluorescent probe of the present invention for high-throughput compound screening, different compounds are added to cell culture media, changes in arginine content are measured, and compounds that affect the changes in arginine content are screened. The applications of the arginine optical probe described in the present invention for real-time localization, quantitative detection of arginine, and high-throughput compound screening are both for non-diagnostic and therapeutic purposes, and do not include the diagnosis and treatment of diseases.

[0079] The present invention further provides a detection kit comprising the optical probe, nucleic acid molecule, nucleic acid construct, and / or cell described herein. The kit may further comprise other reagents necessary for the detection of arginine. Such other reagents are well known in the art and include, for example, buffers, cell culture media, and arginine standards. An exemplary buffer is, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0080] Some specific embodiments: Item 1. An arginine-sensitive polypeptide, wherein the arginine-sensitive polypeptide is a mutant of an arginine-binding protein; (1) having the sequence set forth in SEQ ID NO: 1 and having amino acid mutations, including amino acid modifications, substitutions, or deletions, at one, two, or three or more sites selected from the following: T32, E37, F71, G89, M90, D91, R96, E135, T138, T139, H140, D177; or (2) An arginine-sensitive polypeptide having at least 70% sequence identity with the sequence described in (1), having the mutation described in (1), and retaining arginine sensitivity.

[0081] Preferably, the mutations are one, two or three or more selected from T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, D177A.

[0082] Item 2. An optical probe comprising the arginine-sensitive polypeptide described in Item 1 and an optically active polypeptide or a functional mutant thereof, wherein the optically active polypeptide or a functional mutant thereof is located within the sequence of the arginine-sensitive polypeptide.

[0083] Preferably, the optically active polypeptide is located between residues 200 and 203 of the arginine-sensitive polypeptide.

[0084] More preferably, the optically active polypeptide is located at the 200 / 203 position of the arginine-sensitive polypeptide.

[0085] Item 3: The optical probe according to Item 2, wherein the optically active polypeptide is a fluorescent protein or a functional fragment thereof.

[0086] Preferably, the fluorescent protein is cpYFP as shown in SEQ ID NO:3.

[0087] Item 4: The optical probe according to Item 2 or 3, wherein cpYFP is inserted into the 200 / 203 site of the arginine-sensitive polypeptide, and the optical probe has one or more mutations selected from the group consisting of T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A.

[0088] Preferably, the optical probe has a sequence set forth in any of SEQ ID NOs: 4-15, or a sequence having at least 70% sequence identity thereto.

[0089] Item 5, a nucleic acid molecule comprising: (a) a coding sequence for an optical probe according to any one of items 2-4; or (b) the complement of (a); or (c) A fragment of (a) or (b).

[0090] Preferably, said fragment is a primer.

[0091] Item 6. A nucleic acid construct comprising the nucleic acid molecule according to Item 5.

[0092] Preferably, said nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

[0093] Item 7, (1) expressing the optical probe described in any one of items 2-4; (2) comprising the nucleic acid molecule described in item 5; or (3) The nucleic acid construct according to item 6 is included. A host cell characterized by:

[0094] Item 8. A method for preparing an optical probe according to any one of Items 2 to 4, comprising providing a host cell according to Item 7, culturing the host cell under conditions in which the optical probe is expressed, and isolating the optical probe or fusion polypeptide.

[0095] Item 9: Use of the optical probe according to any one of items 2-4, the nucleic acid sequence according to item 5, the nucleic acid construct according to item 6, or the host cell according to item 7 in detecting arginine in a sample, screening compounds, and intracellular and / or extracellular localization of arginine.

[0096] Preferably, detecting arginine in the sample includes contacting the optical probe or the host cell with a sample, detecting an optical change in the optically active polypeptide, and quantifying arginine in the sample based on the optical change in the optically active polypeptide; Screening the compound includes contacting the optical probe or the host cell with a candidate compound in a system containing arginine, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide; The intracellular and / or extracellular localization of arginine comprises contacting a system containing arginine with the optical probe or the host cell and detecting an optical change in the optically active polypeptide.

[0097] Item 10, (1) The optical probe according to any one of Items 2-4, (2) The nucleic acid sequence described in item 5; (3) A nucleic acid construct according to item 6, or (4) a host cell according to item 7, and Other reagents required for the detection of arginine by optical probe, A detection kit comprising:

[0098] Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer solution, a medium, and an arginine standard.

[0099] In this document, concentrations, contents, percentages, and other numerical values ​​may all be expressed in range format. It should be understood that this range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the values ​​expressly recited at the upper and lower limits of the range, but also all individual values ​​or subranges encompassed within the range. [Example]

[0100] The arginine fluorescent probe provided in the present invention will be described in detail below in conjunction with examples, which should not be construed as limiting the scope of protection of the present invention.

[0101] I. Experimental materials and reagents In the examples, conventional genetic engineering, molecular biology, cloning methods, cell culture methods, imaging methods, etc. are mainly used, and these methods are well known to those skilled in the art, such as "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al.; "Molecular Cloning Laboratory Guide" by J. Sambrook, DW Russell, and translated by Huang Peitang et al. (3rd edition, August 2002, Science Press, Beijing); "Animal Cell Culture: A Guide to Basic Techniques" by Frasier et al., translated by Zhang Jingbo, Xu Cunshuan et al. (5th edition); and "Concise Cell Biology Laboratory Guide" by J.S. Bonifacion, M. Dassault et al., translated by Zhang Jingbo et al.

[0102] The pET28a-cpYFP and pET28a-arginine-binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pET28a plasmid vector was purchased from Invitrogen. All primers used in PCR were synthesized and purified by Shanghai Generay Biotech Co., Ltd. and correctly identified by mass spectrometry. All expression plasmids constructed in the examples were sequenced, and sequencing was completed by Hua Da Gene Co., Ltd. and Shanghai Genebioseq Co., Ltd. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotech Co., Ltd., pfu DNA polymerase was purchased from Tiangen Biotech (Beijing) Co., Ltd., and primeSTAR DNA polymerase was purchased from TaKaRa. The corresponding polymerase buffers and dNTPs were included as bonuses when purchasing the three polymerases. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, along with the appropriate buffers and other extras. The transfection reagent Lip2000 Kit was purchased from Invitrogen. Arginine and other reagents were purchased from Sigma. Unless otherwise noted, all chemical reagents, including inorganic salts, were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Ameresco; the 96-well detection black plate and the 384-well fluorescence detection black plate were purchased from Grenier.

[0103] The DNA purification kit used in the examples was purchased from BBI (Canada), and the general-purpose small-scale plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloning strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column fillers were both purchased from GE Healthcare.

[0104] The main equipment used in the examples is a Biotek Synergy 2 multifunction microplate reader (Bio-Tek, USA), an X-15R high-speed refrigerated centrifuge (Beckman, USA), a Microfuge22R tabletop high-speed refrigerated centrifuge (Beckman, USA), a PCR amplification device (Biometra, Germany), an ultrasonic homogenizer (Ningbo Xinzhi), a nucleic acid electrophoresis device (Shin-Neng Bocai), a fluorescence spectrophotometer (Varian, USA), a CO2 constant temperature cell incubator (SANYO), and an inverted fluorescence microscope (Nikon Japan).

[0105] II. Molecular Biology and Cell Experimental Methods II.1 Polymerase Chain Reaction (PCR): 1. Target fragment amplification PCR: This method is primarily used for amplifying gene fragments and identifying positive clones by colony PCR. The PCR amplification reaction system was as follows: 0.5-1 μL template sequence, 0.5 μL forward primer (25 μM), 0.5 μL reverse primer (25 μM), 5 μL 10× pfu buffer, 0.5 μL pfu DNA polymerase, 1 μL dNTP (10 mM), and 41.5-42 μL sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification program consisted of denaturation at 95°C for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, and 72°C for a fixed time (600 bp / min)), and extension at 72°C for 10 minutes.

[0106] 2. PCR for amplifying long fragments (>2500bp): The long fragment amplification used in this invention is primarily a technique for amplifying vectors by reverse PCR to obtain site-specific mutations in the following examples. Reverse PCR primers are designed for the mutation site, with one primer containing a mutated nucleotide sequence at its 5' end. The amplified product will then contain the corresponding mutation site. The PCR reaction system for long fragment amplification is as follows: 1 μL template sequence (10 pg-1 ng), 0.5 μL forward primer (25 μM), 0.5 μL reverse primer (25 μM), 10 μL 5x PrimerSTAR buffer, 0.5 μL PrimerSTAR DNA polymerase, 4 μL dNTPs (2.5 mM), 33.5 μL sterile ultrapure water (ddH2O), total volume 50 μL. The PCR amplification program was 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 fixed 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 fixed time (1000 bp / min)), and extension at 72°C for 10 minutes.

[0107] II.2 Endonuclease digestion reaction: The double enzyme digestion system for plasmid vectors was as follows: 20 μL (approximately 1.5 μg) of plasmid vector, 5 μL of 10x buffer, 11-2 μL of restriction enzyme 1, 1-2 μL of restriction enzyme 2, and sterile ultrapure water to a total volume of 50 μL. Reaction conditions were 37°C, 1-7 hours.

[0108] II.3 Phosphorylation of the 5' ends of DNA fragments While plasmids and genomes extracted from microorganisms all contain phosphate groups at their termini, PCR products do not. Therefore, a phosphate group must be added to the 5' end of the PCR product, and ligation only occurs with DNA molecules containing phosphate groups at their termini. The phosphorylation reaction system is as follows: 5-8 μL of PCR product fragment DNA sequence, 1 μL of 10x T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0109] II.4 Ligation reaction between target fragment and vector The ligation methods between different fragments and vectors are different, and in the present invention, the following three ligation methods were used.

[0110] 1. Blunt-end ligation of blunt-ended short fragments and linearized vector The principle of this method is to phosphorylate the blunt-ended PCR product at the 5' end of the DNA fragment using T4 PNK, then ligate it to a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10x T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. Reaction conditions: 22°C, 30 minutes.

[0111] 2. Ligation of sticky-ended DNA fragments with sticky-ended vector fragments DNA fragments digested with restriction enzymes typically generate protruding sticky ends, which can then be ligated to vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of restriction-digested PCR product fragment DNA, 0.5-7 μL of restriction-digested plasmid, 1 μL of 10x T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. Reaction conditions: 16°C, 4-8 hours.

[0112] 3. Self-circularization and ligation of the 5' phosphorylated DNA fragments containing site-specific mutations introduced by inverse PCR The 5'-phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via self-circularization ligation to obtain the recombinant plasmid. The self-circularization ligation reaction system was as follows: 10 μL of phosphorylation reaction system, 0.5 μL of T4 ligase (5 U / μL), and a total volume of 10.5 μL. Reaction conditions were 16°C, 4 to 16 hours.

[0113] II.5 Preparation of competent cells and transformation Preparation of competent cells: 1. A single colony (such as Mach1) was picked and inoculated into 5 mL of LB medium, and cultured overnight at 37°C in a shaker. 2. 0.5 to 1 mL of the overnight cultured bacterial solution was transferred to 50 mL of LB medium and cultured at 37°C, 220 rpm for 3 to 5 hours until the OD600 reached 0.5. 3. Cells were pre-cooled in an ice bath for 2 hours. The mixture was centrifuged at 4000 rpm for 10 minutes at 4.4°C. 5. The supernatant was discarded, and the cells were resuspended in 5 mL of pre-chilled buffer. After the cells were homogenized, resuspension buffer was added to bring the final volume to 50 mL. Ice bath for 6.45 minutes. 7. Centrifuge at 4000 rpm for 10 min at 4°C and resuspend the bacteria in 5 mL of ice-cooled storage buffer. 8. 100 μL of bacterial solution was placed in each EP tube and frozen and stored at -80°C or in liquid nitrogen.

[0114] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM) Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10x CaCl2 (1 M), 1 mL 10x MgCl2 (700 mM), 1 mL 10x NaAc (400 mM), 4.6 mL ddH2O

[0115] Transformation of competent cells: 1. Thaw 100 μL of competent cells in an ice bath. 2. Add an appropriate volume of ligation product, gently mix until homogenous, and place in an ice bath for 30 minutes. The volume of the ligation product added is usually less than 1 / 10 of the volume of the competent cells. 3. The bacterial solution was heat shocked in a 42°C water bath for 90 seconds, then immediately transferred to an ice bath and left for 5 minutes. 4. 500 μL of LB was added, and the mixture was cultured in a thermostatic shaker at 37°C and 200 rpm for 1 hour. 5. The bacterial solution was centrifuged at 4000 rpm for 3 minutes, 200 μL of the supernatant was retained, the bacteria were mixed uniformly by blowing, and the mixture was spread evenly on the surface of an agar plate containing an appropriate antibiotic. The plate was then inverted and incubated overnight in a 37°C incubator.

[0116] II.6 Protein expression, purification and fluorescence detection 1. The pET28a-based arginine probe plasmid was transformed into BL21(DE3) and grown upside down overnight. Clones were scraped from the plate into 250 mL Erlenmeyer flasks and cultured at 37°C in a shaker at 220 rpm until an OD of 0.4-0.8 was reached. 1 / 1000 (v / v) IPTG (1 M) was added and expression was induced at 18°C ​​for 24-36 h. 2. After the induction of expression was completed, the bacteria were collected by centrifugation at 4000 rpm for 30 minutes, the bacterial pellet was resuspended in 50 mM phosphate buffer, and the bacteria were sonicated until transparent. The mixture was centrifuged at 9600 rpm for 20 minutes at 4°C. 3. The supernatant was centrifuged and passed through a self-made nickel column affinity chromatography column to purify the protein. The nickel column affinity chromatography columned protein was further passed through a self-made desalting column to obtain the protein dissolved in 20 mM MOPS buffer (pH 7.4) or phosphate buffer PBS. 4. After identifying the purified arginine-binding protein mutants by SDS-PAGE, the probe was diluted in assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.3) or phosphate buffer PBS to a final concentration of 5–10 μM. Arginine was prepared in assay buffer (20 mM MOPS, pH 7.4) or phosphate buffer PBS to a final concentration of 1 M. 100 μl of the 5.5 μM protein solution was taken and incubated at 37°C for 5 minutes. Arginine was added to a final concentration of 100 mM, and the mixture was mixed uniformly. The light absorbance of the protein at 340 nm was measured using a multifunctional fluorescence microplate reader. 100 μl of the 6.1 μM fluorescent probe solution was incubated at 37°C for 5 minutes, and then titrated with arginine. The fluorescence intensity of the protein excited at 485 nm and emitted at 528 nm was measured. Fluorescence excitation and emission measurements of the samples were completed using a multifunctional fluorescence microplate reader. 100 μl of 7.1 μM fluorescent probe solution was taken, incubated at 37 °C for 5 minutes, arginine was added, and the absorption and fluorescence spectra of the probe protein were measured. The absorption and fluorescence spectra of the sample were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0117] II.7 Transfection into mammalian cells and fluorescence detection 1. The PAAV-based arginine optical probe plasmid was transfected into HEK293 cells using the transfection reagent Lipofectamine 2000 (Invitrogen) and cultured in a cell culture incubator at 37°C with 5% CO2. Fluorescence detection was performed 24–36 hours after full expression of the foreign gene. 2. After the induction of expression was completed, the adhered HEK293 cells were rinsed with PBS three times, placed in HBSS solution, and detected by fluorescence microscopy and microplate reader, respectively. [Example]

[0118] Example 1: Arginine-binding protein plasmid The STM4351 gene in Agrobacterium was amplified by PCR. The PCR product was isolated by gel electrophoresis and then digested with BamHI and EcoRI. At the same time, the pET28a vector was double-digested. After ligation with T4 DNA ligase, the ligation product was transformed into Trans5a. The transformed Trans5a was plated on an LB plate (100 μg / mL kanamycin) and cultured overnight at 37°C. Plasmids from the grown Trans5a transformants were extracted and identified by PCR. Positive plasmids were verified to be correct by sequencing before the next plasmid construction. [Example]

[0119] Example 2: Expression and detection of mutations at the cpYFP optical probe binding position in the 200 / 203 insertion site In this example, the following sites were selected for inserting cpYFP into pET28a-STM4351, resulting in the corresponding plasmid pET28a-STM4351-200 / 203-cpYFP. Mutations were performed on pET28a-STM4351-200 / 203-cpYFP to obtain plasmids with the following mutations: T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A. The sequences of exemplary optical probes are shown in Table 1.

[0120] [Table 1]

[0121] The cpYFP DNA fragment was generated by PCR, inactivated by 5'-end phosphorylation, and then amplified by reverse PCR to generate a pET28a-arginine binding protein linearized vector containing a different cleavage site. The linearized pET28a-STM4351 was ligated with the 5'-end phosphorylated cpYFP fragment using PEG4000 and T4 DNA ligase to generate the recombinant plasmid. These plates were placed in a Kodak multifunctional bioimaging system, and clones that emitted yellow fluorescence when excited in the FITC channel were scraped and sequenced by the Shanghai branch of Beijing Liuhehua University Gene Technology Co., Ltd.

[0122] The linearized plasmid pET28a-STM4351-200 / 203-cpYFP was amplified by reverse PCR using primers containing the desired mutation sites. The resulting PCR product was phosphorylated and ligated using PNK, T4 DNA ligase, and PEG4000 to obtain a site-directed mutated plasmid containing 12 mutation sites: T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A. Sequencing was completed by the Shanghai branch of Beijing Liuhehua University Gene Technology Co., Ltd.

[0123] After verifying the accuracy by sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis revealed that the protein size was approximately 55 kDa. This size was consistent with the size of the STM4351-cpYFP fusion protein containing a His-tag purification tag expressed by pET28a-STM4351-cpYFP. The results are shown in Figure 1.

[0124] An arginine-responsive screening was performed using purified STM4351-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 100 mM arginine was divided by the detection signal of the fusion fluorescent protein without arginine. The results are shown in Figure 2. The detection results showed that T32A, E37A, D91A, T138A, and H140A responded more than three-fold to arginine, except for E37A, which responded more than 15-fold to arginine. [Example]

[0125] Example 3. Titration curves of probes mutated at the cpYFP optical probe binding position in the 200 / 203 insertion site The E37A, G89A, and T138A probes were expressed and detected according to the method described in Example 2. The results are shown in Figure 3. Fluorescence detection revealed that the E37A probe responded to arginine by approximately 15-fold, was saturated with 10 mM arginine substrate, and had a substrate affinity of 170-210 μM; the G89A and T138A probes responded less to arginine, with response folds of approximately 1.29 and 3.14, respectively, and had substrate affinities of 23.57 μM and 189.8 μM, respectively. [Example]

[0126] Example 4. Spectral profile of probe E37A We purified the mutant STM4351-200 / 203-E37A-cpYFP, ​​which exhibits high response rates and excellent specificity. The purified arginine fluorescent probe was treated with 0 mM and 100 mM arginine for 10 minutes, and then its fluorescence spectrum was measured using a spectrofluorometer. The excitation wavelengths were fixed at 420 nm and 485 nm, respectively, and the emission spectrum was recorded from 360 to 540 nm, with readings taken every 5 nm. The spectral profile of the arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP is shown in Figure 4. When 500 mM arginine was added, the fluorescence intensity excited at 420 nm was almost unchanged compared to when 0 mM arginine was added; the fluorescence intensity excited at 485 nm increased approximately 15.0-fold compared to when 0 mM arginine was added. [Example]

[0127] Example 5. Determining Specificity of Arginine Probes Specificity was measured for the mutants T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, and H140A in Figure 2, and as a result, as shown in Figure 5, the mutant probes showed good specificity for arginine. [Example]

[0128] Example 6. Affinity of E37A probe with substrate under different acidity / basicity conditions STM4351-200 / 203-E37A-cpYFP was purified in the same manner as in Example 2, and its binding ability to the substrate arginine was measured under different pH conditions. As a result, as shown in Figure 6, the affinity to the substrate hardly changed under different pH conditions. [Example]

[0129] Example 7. Temperature sensitivity of the E37A probe STM4351-200 / 203-E37A-cpYFP was purified as in Example 2, and its binding ability to the substrate arginine was titrated under different temperature conditions. As shown in Figure 7, the binding response between the probe and substrate was best at 35°C, and the binding response fold between the probe and substrate was lowest at 20°C. [Example]

[0130] Example 8. Affinity of E37A probe with substrate at different temperatures STM4351-200 / 203-E37A-cpYFP was purified in the same manner as in Example 2, and its binding ability to the substrate arginine was measured under different temperature conditions. As a result, as shown in Figure 8, the affinity to the substrate hardly changed under different temperature conditions. [Example]

[0131] Example 9. Localization of probes in intracellular organelles and their performance within intracellular organelles In this example, different localization signal peptides were fused to the C-terminus or N-terminus of the arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP, ​​and the arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP was localized to various organelles. HEK293 cells were transfected with the arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP gene plasmid fused with different localization signal peptides 36 hours later, washed with PBS, and then placed in HBSS solution. Fluorescence was detected in the FITC channel using an inverted fluorescence microscope. The arginine fluorescent probe FLIPpro, when fused with localization signal peptides of different specificities, was found to be able to localize to intracellular organelles, such as the cytoplasm, mitochondria, nucleus, and nucleoplasm (excluding nucleoli). As shown in Figure 9, fluorescence was observed in different intracellular structures, and the fluorescence distribution and intensity were different. [Example]

[0132] Example 10: Dynamic monitoring of arginine transmembrane transport 36 hours after transfection of HEK293 cells with the cytoplasmically expressed STM4351-200 / 203-E37A-cpYFP gene plasmid, the cells were washed with PBS and placed in HBSS. The ratio of the fluorescence intensity at 528 nm when excited at 420 nm to the fluorescence intensity at 528 nm when excited at 485 nm was measured over a 40-minute period. As shown in Figure 10, after 2 hours of starvation, 10 μM, 100 μM, and 1000 μM arginine were added and measurements were taken for 30 minutes. The 485 / 420 ratio gradually increased, reaching a maximum of 1.65-fold. [Example]

[0133] Example 11. High-throughput compound screening at the live cell level In this example, high-throughput compound screening was performed using HEK293 cells expressing the arginine probe STM4351-200 / 203-E37A-cpYFP in the cytoplasm.

[0134] HEK293 cells transfected with STM4351-200 / 203-E37A-cpYFP were washed with PBS, placed in HBSS solution (arginine-free), and then treated with 10 μM compounds for 1 hour. Arginine was then added dropwise to each cell. 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 was recorded using a microplate reader. A sample untreated with any compound served as the control. As shown in Figure 11, most of the 2,000 compounds treated with the cells had little effect on arginine entry into the cells. While 16 compounds were able to improve cellular arginine uptake, 11 compounds significantly reduced cellular arginine uptake. [Example]

[0135] Example 12. Quantitative detection of arginine in blood and urine using a probe In this example, purified arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP protein was used to analyze arginine in human blood supernatant and urine.

[0136] The arginine fluorescent probe STM4351-200 / 203-E37A-cpYFP fluorescent protein was mixed with the diluted blood supernatant for 10 minutes, and then the ratio of the fluorescence intensity excited at 420 nm and emitted at 528 nm to the fluorescence intensity excited at 485 nm and emitted at 528 nm was measured using a microplate reader. The urine detection experiment was performed in the same manner as for plasma detection. As a result, as shown in Figure 12, the arginine content in human blood was found to be approximately 150 μM, and the arginine content in human urine was found to be approximately 13.7 μM.

[0137] In some of the above examples, the probe with mutation E37A is selected as an example, but the probes with other mutations T32A, E37A, G89A, D91A, T138A, H140A also showed results corresponding to the arginine response results. As can be seen from the above examples, the arginine fluorescent probe provided by the present invention has a relatively small protein molecular weight, is easy to mature, has a large dynamic change in fluorescence, has excellent specificity, and can be expressed in cells using genetic engineering methods, enabling real-time localization and quantitative detection of arginine inside and outside the cells, and enabling high-throughput compound screening.

[0138] The above-described contents are merely preferred embodiments of the present invention, and those skilled in the art may make some improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also intended to fall within the scope of protection of the present invention.

[0139] Sequences herein 1 <stm4351ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDPDYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 2 <stm4351-200 203-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 3 <cpyfpYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN 4 <stm4351-200 203-t32a-cpyfp タンパク質配列ASVSARTLHFGTSAAYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 5 <stm4351-200 203-e37a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYAFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 6 <stm4351-200 203-f71a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSADSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 7 <stm4351-200 203-g89a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAAMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 8 <stm4351-200 203-m90a -cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGADMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 9 <stm4351-200 203-d91a -cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMAMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 10 <stm4351-200 203-r96a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKAEQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 11 <stm4351-200 203-e135a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLANGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 12 <stm4351-200 203-t138a -cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGATHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 13 <stm4351-200 203-t139a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTAHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 14 <stm4351-200 203-h140a-cpyfp タンパク質配列ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTAQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ 15 <stm4351-200 203-d177a-cpyfp タンパク質配列 ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLE NGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGAVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPD NHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLK CFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ< / stm4351-200>

Claims

1. An arginine-sensitive polypeptide, wherein the arginine-sensitive polypeptide is a mutant of an arginine-binding protein; (1) Having the sequence set forth in SEQ ID NO: 1 and having amino acid mutations, including amino acid modifications, substitutions, or deletions, at one, two, three, or more positions selected from the following: T32, E37, F71, G89, M90, D91, R96, E135, T138, T139, H140, D177; or (2) An arginine-sensitive polypeptide having at least 70% sequence identity with the sequence described in (1), having the mutation described in (1), and retaining arginine sensitivity. Preferably, the mutations are one, two or three or more selected from T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, D177A.

2. An optical probe comprising the arginine-sensitive polypeptide described in claim 1 and an optically active polypeptide or a functional mutant thereof, wherein the optically active polypeptide or a functional mutant thereof is located within the sequence of the arginine-sensitive polypeptide. Preferably, the optically active polypeptide is located between residues 200-203 of the arginine-sensitive polypeptide. More preferably, the optically active polypeptide is located at the 200 / 203 position of the arginine-sensitive polypeptide.

3. 3. The optical probe according to claim 2, wherein the optically active polypeptide is a fluorescent protein or a functional fragment thereof. Preferably, the fluorescent protein is cpYFP as shown in SEQ ID NO:

3.

4. The optical probe according to claim 2 or 3, characterized in that the optical probe has cpYFP inserted at the 200 / 203 site of the arginine-sensitive polypeptide and has one or more mutations selected from the group consisting of T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A. Preferably, the optical probe has a sequence set forth in any of SEQ ID NOs: 4-15, or a sequence having at least 70% sequence identity thereto.

5. A nucleic acid molecule comprising: (a) a coding sequence for an optical probe according to any one of claims 2-4, or (b) the complement of (a); or (c) A fragment of (a) or (b). Preferably, said fragment is a primer.

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

7. (1) Expressing an optical probe according to any one of claims 2 to 4; (2) comprising the nucleic acid molecule of claim 5; or (3) A nucleic acid construct according to claim 6. A host cell characterized by:

8. A method for preparing an optical probe according to any one of claims 2 to 4, comprising providing a host cell according to claim 7, culturing said host cell under conditions in which said optical probe is expressed, and isolating the optical probe or fusion polypeptide.

9. Use of an optical probe according to any one of claims 2 to 4, a nucleic acid sequence according to claim 5, a nucleic acid construct according to claim 6 or a host cell according to claim 7 in the detection of arginine in a sample, in the screening of compounds, in the intracellular and / or extracellular localization of arginine. Preferably, detecting arginine in the sample includes contacting the optical probe or the host cell with a sample, detecting an optical change in the optically active polypeptide, and quantifying arginine in the sample based on the optical change in the optically active polypeptide; Screening the compound includes contacting the optical probe or the host cell with a candidate compound in a system containing arginine, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide; The intracellular and / or extracellular localization of arginine comprises contacting a system containing arginine with the optical probe or the host cell and detecting an optical change in the optically active polypeptide.

10. (1) An optical probe according to any one of claims 2 to 4. (2) A nucleic acid sequence according to claim 5, (3) A nucleic acid construct according to claim 6, or (4) A host cell according to claim 7, and Other reagents required for the detection of arginine by optical probe, A detection kit comprising: Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer solution, a medium, and an arginine standard.

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