WO2024109819A1 - 一种磷酸烯醇式丙酮酸光学探针及其制备方法和应用 - Google Patents

一种磷酸烯醇式丙酮酸光学探针及其制备方法和应用 Download PDF

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WO2024109819A1
WO2024109819A1 PCT/CN2023/133332 CN2023133332W WO2024109819A1 WO 2024109819 A1 WO2024109819 A1 WO 2024109819A1 CN 2023133332 W CN2023133332 W CN 2023133332W WO 2024109819 A1 WO2024109819 A1 WO 2024109819A1
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phosphoenolpyruvate
polypeptide
optically active
optical probe
sequence
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French (fr)
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杨弋
赵玉政
孙玉莹
李写
邹叶君
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East China University of Science and Technology
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East China University of Science and Technology
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Publication of WO2024109819A1 publication Critical patent/WO2024109819A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to the technical field of optical probes, and in particular to a phosphoenolpyruvic acid optical probe and a preparation method and application thereof.
  • Phosphoenolpyruvate is a high-energy intermediate in the glycolysis pathway and a key metabolite connecting glycolysis and gluconeogenesis.
  • the high-energy phosphate bonds contained in PEP enable it to play the role of a phosphate donor in bacteria, plants, and mammalian cells, and play an important role in maintaining intracellular ATP content, glucose homeostasis, glucose metabolism regulation, tumor immunity, and cell protection.
  • the object of the present invention is to provide a probe and method for real-time localization, high-throughput and quantitative detection of phosphoenolpyruvate inside and outside cells.
  • the first aspect of the present invention provides a phosphoenolpyruvate binding protein variant, which:
  • (b) is a sequence having at least 70% sequence identity with the sequence of (a) and having the mutation described in (1) and retaining the ability to bind to phosphoenolpyruvate.
  • the phosphoenolpyruvate binding protein variant has the sequence shown in SEQ ID NO: 1, and: (I) has a mutation at 1, 2, 3, 4 or more sites selected from the following: I253, Q254, R255, G256, G257, and/or, (II) has a mutation at 1, 2, 3, 4, 5 or more sites selected from the following: R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320.
  • the mutated site in (I) includes Q254.
  • the mutated site in (I) includes a site selected from any one or more of the following groups: (I.1) I253, Q254, (I.2) I253, Q254, R255, G256, (I.3) Q254, R255, G256, (I.4) I253, Q254, G256, G257
  • the mutated sites in (II) include sites selected from any one or more of the following groups: (II.1) R21, (II.2) V54, (II.3) R55, (II.4) V57, (II.5) A58, (II.6) I60, (II.7) N59, (II.8) R212, (II.9) G213, (II.10) K215, (II.11) S216, (II.12) L320, (II.13) R72, (II.14) V54, R72.
  • groups selected from any one or more of the following groups: (II.1) R21, (II.2) V54, (II.3) R55, (II.4) V57, (II.5) A58, (II.6) I60, (II.7) N59, (II.8) R212, (II.9) G213, (II.10) K215, (II.11) S216, (II.12) L
  • the phosphoenolpyruvate binding protein variant has the sequence shown in SEQ ID NO: 1, and: (I) has mutations at the following sites: I253, Q254, G256 and G257, and (II) has mutations at sites selected from any one or more of the following groups: (II.1) R21, (II.2) V54, (II.3) R55, (II.4) V57, (II.5) A58, (II.6) I60, (II.7) N59, (II.8) R212, (II.9) G213, (II.10) K215, (II.11) S216, (II.12) L320, (II.13) R72, (II.14) V54, R72.
  • the phosphoenolpyruvate binding protein variant has the sequence shown in SEQ ID NO: 1, and has mutations at the following positions: (II.15) V54, I253, Q254, R255, G256, (II.16) N59, I253, Q254, R255, G256, (II.17) V54, I253, Q254, G256, G257, (II.18) N59, I253, Q254, G256, G257, (II.19) R212, I253, Q254, G256, G257, (II.20) G213, I253, Q254, G256, G257, (II.21) K215, I253, Q254, G256, G257, (II.22) L320, I253, Q254, G256, G257, (II.23) S216, I253, Q254, G256, G257.
  • I253 is mutated to V, N, F, C, E, G, S, K, H, D, P, L, R, T, or Q.
  • Q254 is mutated to L, V, A, R, P, M, K, I.
  • R255 is mutated to L, I, or F.
  • G256 is mutated to H, F, Y, V.
  • R21 is mutated to K.
  • V54 is mutated to G, K, Q.
  • R55 is mutated to S.
  • V57 is mutated to M.
  • A58 is mutated to G.
  • N59 is mutated to D, H, or E.
  • I60 is mutated to W.
  • R72 is mutated to A.
  • R212 is mutated to I, E, M.
  • G213 is mutated to T.
  • K215 is mutated to H, D, E.
  • S216 is mutated to D, L, Y, I, M, F, or Q.
  • L320 is mutated to Y, N, or F.
  • the mutation comprises one or more selected from the group consisting of: I253K, Q254L, R255L and/or G256F.
  • the mutation comprises a mutation selected from any one of the following groups: (1) I253Y, Q254I, (2) I253V, Q254L, R255L, G256H, (3) Q254L, R255L, G256H, (4) Q254V, R255I, G256H, (5) I253N, Q254L, R255L, G256F, (6) I253F, Q254L, R255L, G256H 256F, (7) I253V, Q254A, R255F, G256Y, (8) I253V, Q254L, R255F, G256Y, (9) I253C, Q254L, R255L, G256F, (10) I253E, Q254L, R255L, G256F, (11) I253G, Q254L, R255L, G256F, (12) I253I, Q254L, R255L , G256F, (13) I253S, Q254
  • the first aspect of the present invention also provides a phosphoenolpyruvate optical probe, comprising a phosphoenolpyruvate sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the phosphoenolpyruvate sensitive polypeptide.
  • the phosphoenolpyruvate sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.
  • the phosphoenolpyruvate-sensitive polypeptide has:
  • SEQ ID NO: 1 (2) a functional variant of the sequence shown in SEQ ID NO: 1, which has a mutation within 5 (preferably 3, more preferably 2) amino acids at the site where it is connected to the optically active polypeptide, and/or has a mutation at 1, 2, 3, 4, 5 or more sites selected from the group consisting of R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320,
  • (2) is the sequence shown in SEQ ID NO: 1 and has a mutation at 1, 2, 3, 4, 5 or more sites selected from the group consisting of: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320, wherein the mutation comprises an amino acid modification, substitution or deletion.
  • (2) is the sequence of the phosphoenolpyruvate binding protein variant described in any embodiment of the first aspect of the present invention.
  • the optically active polypeptide is located at or replaces residues 68-71, 168-173 and/or 253-257 of the phosphoenolpyruvate-sensitive polypeptide, numbered corresponding to the full length of the phosphoenolpyruvate-sensitive polypeptide.
  • the optically active polypeptide is located at any one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide: 68/69, 68/70, 69/70, 68/71, 69/71, 70/71, 168/169, 168/170, 169/170, 168/171, 169/171, 170/171, 168/172, 169 More preferably, the optically active polypeptide is located at any one or more of the following sites of the phosphoenolpyruvate-sensitive polypeptide: 69/71, 170/171, 171/172, 254/255, 254/256 and 255/257.
  • the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 (preferably 2) amino acids at the junction with the optically active polypeptide.
  • the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein.
  • the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9, preferably SEQ ID NOs: 2, 6, 7 or 9.
  • the functional variant of the fluorescent protein has a sequence as shown in any one of SEQ ID NOs: 2-9 and has a mutation at the amino acid position 1 corresponding to SEQ ID NO: 2, and the mutation is preferably mutated to S, G, N, T, P, A, E, or H.
  • the functional variant of the fluorescent protein has a sequence as shown in any one of SEQ ID NOs: 2-9 and has a mutation at the amino acid position 246 corresponding to SEQ ID NO: 2, and the mutation is preferably mutated to F, C, W, or H.
  • the functional variant of the fluorescent protein has a sequence as shown in any one of SEQ ID NOs: 2-9 and has a residue selected from the group consisting of: The following groups of mutations are shown: 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 2 or a variant thereof having any of the following mutations at the 1st and/or 246th amino acid: Y1S; Y1G; Y1N; Y1T; Y1P; Y1A; Y1E; Y1H; N246F; N246C; N246W; N246H; Y1S and N246F; Y1G and N246F; Y1H and N246F; Y1A and N246C; Y1G and N246C; Y1S and N246C; Y1T and N246C; Y1T and N246F, and the optically active polypeptide is located at one or more positions of the phosphoenolpyruvate-sensitive polypeptide selected from the following positions: 69/71, 170/171, 171/172, 254/255, 254/256 and 255/257.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 6 or a variant thereof having any of the following mutations at the amino acid corresponding to the 1st or 246th amino acid of SEQ ID NO: 2: 1S; 1G; 1N; 1T; 1P; 1A; 1E; 1H; 246F; 246C; 246W; 246H; 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F, and the optically active polypeptide is located at one or more positions of the phosphoenolpyruvate-sensitive polypeptide selected from the following positions: 171/172, 254/255 and 254/256.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 7 or a variant thereof having any of the following mutations at the amino acid corresponding to the 1st or 246th amino acid of SEQ ID NO: 2: 1S; 1G; 1N; 1T; 1P; 1A; 1E; 1H; 246F; 246C; 246W; 246H; 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F, and the optically active polypeptide is located at one or more positions of the phosphoenolpyruvate-sensitive polypeptide selected from the following positions: 170/171, 171/172, 254/255 and 254/256.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 10 or a variant thereof having any of the following mutations at the amino acid corresponding to the 1st or 246th amino acid of SEQ ID NO: 2: 1S; 1G; 1N; 1T; 1P; 1A; 1E; 1H; 246F; 246C; 246W; 246H; 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F, and the optically active polypeptide is located at one or more positions of the phosphoenolpyruvate-sensitive polypeptide selected from the following positions: 170/171, 171/172, 254/255 and 254/256.
  • the optical probe further comprises one or more linkers flanking the optically active polypeptide.
  • the linkers of the present invention can be any amino acid sequence of any length.
  • the optically active polypeptide flanks a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids.
  • the linker flanking the optically active polypeptide comprises amino acid Y.
  • linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide.
  • the optical probe is as follows: The first part B1 of the phosphoenolpyruvate-sensitive polypeptide, Y, the optically active polypeptide A, and the second part B2 of the phosphoenolpyruvate-sensitive polypeptide.
  • the optical probe of the present invention does not comprise a linker.
  • the optical probe of the present invention further comprises a localization sequence for localizing the probe to, for example, a specific organelle of a cell.
  • the phosphoenolpyruvate-sensitive polypeptide is as shown in SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9) or a variant thereof having any one or more mutations selected from the following positions at the amino acid corresponding to the 1st or 246th amino acid of SEQ ID NO: 2: 1S, 1G, 1N, 1T, 1P, 1A, 1E, 1H, 246F, 246C, 246W or 246H
  • the optically active polypeptide is located at any one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide: 68/69, 6 8/70, 69/70, 68/71, 69/71, 70/71, 168/169, 168/170, 169/170, 170/171, 168/172, 169/172, 1
  • the optically active polypeptide is located at any one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide selected from: 69/71, 170/171, 171/172, 254/255, 254/256 and 255/257.
  • the optical probe comprises a phosphoenolpyruvate-sensitive polypeptide as shown in SEQ ID NO: 1 and having one or more of the following mutations: I253V, I253N, I253F, I253C, I253E, I253G, I253S, I253K, I253H, I253D, I253P, I253L, I253R, I253T, I253Q, Q254L, Q254V, Q254A, Q254R, Q254P, Q254M, Q254K, Q254I, R255L, R255I, R255F, G256H, G256F, G256I 256Y, G256V, G257L, R21K, V54G, V54K, V54Q, R55S, V57M, A58G, N59D, N59H, N59E, I60W, R72A, R212I, R212E, R
  • the mutation of the phosphoenolpyruvate-sensitive polypeptide comprises a mutation selected from any one of the following groups: (1) I253Y, Q254I, (2) I253V, Q254L, R255L, G256H, (3) Q254L, R255L, G256H, (4) Q254V, R255I, G256H, (5) I253N, Q254L, R255L, G256F, (6) I253F, Q254L, R255L, G256F, (7) I253V, Q254A, R255F, G256Y, (8) I253V, Q254L, R255F, G256Y, (9) I253C, Q254L, R255L, G256F, (10) I253E, Q254L, R255L, G256F, (11) I253G, Q254L, R255L, G256F, (12) I253I, Q254L, R255L, G256F, (11) I25
  • the phosphoenolpyruvate-sensitive polypeptide is as shown in SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9)
  • the optically active polypeptide is located at the 254/255 position of the phosphoenolpyruvate-sensitive polypeptide
  • the optical probe has the following mutations: (1) I253Y and Q254I of the phosphoenolpyruvate-sensitive polypeptide, (2) I253V, Q254L, R255L, G256H of the phosphoenolpyruvate-sensitive polypeptide, and 1S of a phosphoenolpyruvate-sensitive polypeptide, (3) Q254L, R255L, G256H of a phosphoenolpyruvate-sensitive polypeptide, and 1S of an optically active polypeptide, (4) Q254V, R255I, G256H of a phosphoenolpyruvate
  • an optically active polypeptide (10) I253E, Q254L, R255L, G256F of a phosphoenolpyruvate-sensitive polypeptide, and 1S of an optically active polypeptide, (11) I253G, Q254L, R255L, G256F of a phosphoenolpyruvate-sensitive polypeptide, and 1T of an optically active polypeptide, (12) I253I, Q254L, R255L, G256F of a phosphoenolpyruvate-sensitive polypeptide, and 1S of an optically active polypeptide, (13) I253S, Q254L, R255L, G256F of a phosphoenolpyruvate-sensitive polypeptide, and 1S of an optically active polypeptide; (14) I253G, Q254L, R255L, G256F of a phosphoenolpyruvate-sensitive polypeptide, and 1P of an optically active polypeptide; (14)
  • the phosphoenolpyruvate-sensitive polypeptide is as shown in SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9)
  • the optically active polypeptide is located at the 254/256 position of the phosphoenolpyruvate-sensitive polypeptide
  • the optical probe has the following mutations: (1) I253D, Q254L, G256V, G257L, and 1T, 246F of an optically active polypeptide, (2) I253S, Q254L, G256V, G257L, and 1T, 246F of a phosphoenolpyruvate-sensitive polypeptide, (3) I253Q, Q254L, G256V, G257L, and 1T, 246F of a phosphoenolpyruvate-sensitive polypeptide, (4) V54Q, I253Q, Q254L, G256V, G257L, and
  • the present invention also provides fusion polypeptides, comprising the optical probe described in any embodiment of the present invention and other polypeptides.
  • the other polypeptides are located at the N-terminus and/or C-terminus of the optical probe.
  • the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting.
  • the present invention also provides a nucleic acid molecule comprising: (a) a coding sequence of a protein variant, optical probe or fusion polypeptide as described in any embodiment of the present invention, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b).
  • the fragment is a primer.
  • the present invention also relates to variants of the above-mentioned nucleic acid molecules, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the protein variants, optical probes or fusion polypeptides of the present invention or their complementary sequences.
  • the present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein.
  • the nucleic acid sequence encodes the protein variant, optical probe or fusion polypeptide described in any embodiment of the present invention.
  • the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
  • the nucleic acid molecule is operably linked to an expression control sequence.
  • the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.
  • Another aspect of the present invention provides a host cell, wherein the host cell: (1) expresses the optical probe or fusion polypeptide according to any embodiment of the present invention; (2) comprises the nucleic acid molecule according to any embodiment of the present invention; or (3) comprises the nucleic acid construct according to any embodiment of the present invention.
  • the host cell is preferably Escherichia coli.
  • Another aspect of the present invention provides a phosphoenolpyruvate detection kit, comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe prepared by the method described herein.
  • the kit further comprises one or more reagents selected from the group consisting of a buffer, a culture medium, and a phosphoenolpyruvate standard.
  • Another aspect of the present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions where the cell expresses the optical probe, and isolating the optical probe or fusion polypeptide.
  • the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the optical probe or fusion polypeptide described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; 3) isolating the optical probe or fusion polypeptide.
  • Another aspect of the present invention provides a method for detecting phosphoenolpyruvate in a sample, comprising: contacting the optical probe or fusion polypeptide or host cell described herein with the sample, and detecting changes in the optically active polypeptide.
  • the detection can be performed in vivo, in vitro, in a subcellular or in situ manner.
  • the sample is, for example, blood.
  • the present invention also provides a method for quantifying phosphoenolpyruvate in a sample, comprising: contacting the optical probe or fusion polypeptide or host cell described in the present invention with the sample, detecting the optical change of the optically active polypeptide, and quantifying the phosphoenolpyruvate in the sample based on the optical change of the optically active polypeptide.
  • Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion polypeptide described herein or a host cell with a candidate compound in a system containing phosphoenolpyruvate, detecting the optical change of the optically active polypeptide, and screening the compound based on the optical change of the optically active polypeptide.
  • the method can screen compounds with high throughput.
  • a host cell described herein is contacted with a candidate compound in a system containing phosphoenolpyruvate, and a change in the optics of the optically active polypeptide indicates whether the candidate compound is capable of modulating phosphoenolpyruvate uptake by the cell.
  • Another aspect of the present invention provides a method for localizing phosphoenolpyruvate inside and/or outside a cell, comprising: contacting a system containing phosphoenolpyruvate with the optical probe or the host cell, and detecting optical changes of the optically active polypeptide.
  • the system is a solution system, a cellular system, or a subcellular system.
  • Another aspect of the present invention provides the use of the phosphoenolpyruvate optical probe or fusion polypeptide or host cell described herein in detecting phosphoenolpyruvate in a sample, screening compounds or phosphoenolpyruvate intracellular/extracellular localization.
  • the localization is real-time localization.
  • the phosphoenolpyruvate optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, has good specificity, and can be expressed in cells by genetic manipulation methods, and can be used for real-time positioning, high-throughput, and quantitative detection of phosphoenolpyruvate inside and outside cells, eliminating the time-consuming sample processing steps.
  • FIG1 is an SDS-PAGE image of an exemplary phosphoenolpyruvate optical probe
  • FIG2 is a graph showing the fluorescence spectral properties of an exemplary phosphoenolpyruvate optical probe
  • FIG3 is a titration curve of an exemplary phosphoenolpyruvate optical probe to different concentrations of phosphoenolpyruvate;
  • FIG4 is a summary of the specific detection of the other three substrates and sugar metabolism intermediates and analogs by an exemplary phosphoenolpyruvate optical probe
  • FIG5 is a photograph of the subcellular organelle localization of an exemplary phosphoenolpyruvate optical probe in mammalian cells
  • FIG6 is a schematic diagram of an exemplary phosphoenolpyruvate optical probe for dynamically monitoring the phosphoenolpyruvate concentration in the cytosol of mammalian cells;
  • FIG7 is a dot plot of an exemplary phosphoenolpyruvate optical probe for high-throughput compound screening at the living cell level
  • FIG. 8 is a bar graph showing quantification of phosphoenolpyruvate in mouse and human blood using an exemplary phosphoenolpyruvate optical probe.
  • the term "about" when giving a value or range means that the value or range is within 20%, within 10%, and within 5% of the given value or range.
  • compositions “comprising”, “including” and equivalent forms thereof include the meanings of "containing” as well as “consisting of”, for example, a composition “comprising” X may consist of X alone or may contain other substances, such as X+Y.
  • phosphoenolpyruvate sensitive polypeptide refers to a polypeptide that responds to phosphoenolpyruvate, including any response of a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide.
  • Responses include small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and changes in the primary, secondary or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential and/or conformation.
  • Conformation is the three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule containing side groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include transitions from ⁇ -helix to ⁇ -sheet or from ⁇ -sheet to ⁇ -helix. It is understood that as long as the fluorescence of the fluorescent protein portion is changed, the detectable change does not need to be a conformational change.
  • the phosphoenolpyruvate sensitive polypeptide described herein may also include its functional variants.
  • phosphoenolpyruvate sensitive polypeptides include, but are not limited to, variants that can interact with phosphoenolpyruvate to undergo the same or similar changes as the parent phosphoenolpyruvate sensitive polypeptide.
  • the phosphoenolpyruvate sensitive polypeptide of the present invention includes but is not limited to phosphofructokinase TtPFK derived from the extreme thermophilic bacterium Thermus thermophilus or a variant having more than 90% homology thereto.
  • Phosphoenolpyruvate binding protein can sense changes in phosphoenolpyruvate concentration, and the spatial conformation of phosphoenolpyruvate binding protein will also change during the dynamic change of phosphoenolpyruvate concentration.
  • Truncated variants of TtPFK can also be used in the present invention.
  • optical probe refers to a phosphoenolpyruvate-sensitive polypeptide fused to an optically active polypeptide.
  • the inventors have discovered that the conformational changes produced by phosphoenolpyruvate-sensitive polypeptides, such as phosphoenolpyruvate binding proteins, specifically binding to physiological concentrations of phosphoenolpyruvate will cause conformational changes in optically active polypeptides (such as fluorescent proteins), thereby causing changes in the optical properties of the optically active polypeptides.
  • optically active polypeptides such as fluorescent proteins
  • an optically active polypeptide e.g., a fluorescent protein
  • a protein-based "optically active polypeptide” is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used to detect the response of the optical probe of the present invention.
  • the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or the emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the amplitude of excitation at two different wavelengths, the emission amplitude ratio at two different wavelengths, the excited state lifetime or the fluorescence anisotropy.
  • the measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays.
  • the measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a specific wavelength or the integral of the fluorescence over the emission spectrum.
  • the protein substrate is selected to have fluorescent properties that are easily distinguished in the unactivated and activated conformational states.
  • the optically active polypeptides described herein may also include functional variants thereof.
  • Functional variants of optically active polypeptides include, but are not limited to, variants that may undergo the same or similar fluorescent property changes as the parent optically active polypeptide.
  • fluorescent protein refers to a protein that emits fluorescence under the irradiation of excitation light. Fluorescent proteins are used as a basic detection method in the field of biological sciences, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the circular rearranged blue fluorescent protein (cpBFP), circular rearranged green fluorescent protein (cpGFP), and circular rearranged yellow fluorescent protein (cpYFP) derived from mutations of the protein; there is also the red fluorescent protein RFP commonly used in the field of technology, and the circular rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate, etc.
  • GFP green fluorescent protein GFP commonly used in the field of biotechnology
  • cpBFP circular rearranged blue fluorescent protein
  • cpGFP circular rearranged green fluorescent protein
  • cpYFP circular rearranged yellow fluorescent protein
  • cpYFP is shown in SEQ ID NO: 2
  • cpmOrange is shown in SEQ ID NO: 3
  • cpmKate is shown in SEQ ID NO: 4 or 8
  • mCherry is shown in SEQ ID NO: 5
  • cpGFP is shown in SEQ ID NO: 6
  • cpBFP is shown in SEQ ID NO: 7
  • cpmApple is shown in SEQ ID NO: 9.
  • the fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with mutations at position 1 and/or at amino acid position 246 corresponding to SEQ ID NO: 2.
  • the mutation at position 1 is preferably mutated to S, G, N, T, P, A, E or H.
  • the mutation at position 246 corresponding to SEQ ID NO: 2 is preferably mutated to F, C, W or H.
  • the functional variant of the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9 and has a mutation selected from any of the following groups at amino acid position 1 and amino acid position 246 corresponding to SEQ ID NO: 2: 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F.
  • 1S indicates that the amino acid at position 1 is mutated to S
  • 246F indicates that the amino acid at position 246 is mutated to F, and so on.
  • the optically active polypeptide is located in the NC direction in residues 68-71, 168-173 and/or 253-257 of the phosphoenolpyruvate-sensitive polypeptide or replaces the residues therein, and the numbering corresponds to the full length of the phosphoenolpyruvate-sensitive polypeptide.
  • the two numbers in the site represented by the "X/Y" format are consecutive integers, it means that the optically active polypeptide is located between the amino acids described by the numbers.
  • the insertion site 254/255 indicates that the optically active polypeptide is located between amino acids 254 and 255 of the phosphoenolpyruvate-sensitive polypeptide.
  • the optically active polypeptide set forth in SEQ ID NO: 2, 6, 7 or 9 is located at any one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide set forth in SEQ ID NO: 1: 68/69, 68/70, 69/70, 68/71, 69/71, 70/71, 168/169, 168/170, 169/170, 168/171, 169/171, 170/171, 168/172, 169/172, 1 70/172, 171/172, 168/173, 169/173, 170/173, 171/173, 172/173, 253/254, 253/255, 254/255, 253/256, 254/256, 255/256, 253/257, 254/257, 255/257 and/or 256/257.
  • variants having the same function of the polypeptide or protein but different sequences.
  • variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and/or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and/or amino terminal.
  • variants may also include polypeptides or proteins having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% with the polypeptide or protein.
  • amino acid residues are changed without changing the overall configuration and function of a polypeptide or protein, i.e., a function conservative mutation.
  • amino acids with similar or similar properties are substituted, the function of a polypeptide or protein is usually not changed.
  • amino acids with similar properties often refer to amino acid families with similar side chains, which are clearly defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acids with non-polar side chains e.g., alanine, valine, leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan
  • amino acids with ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • amino acids for another example, adding one or more amino acids to the amino terminus and/or carboxyl terminus will generally not change the function of a polypeptide or protein.
  • Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art.
  • Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids.
  • Linker refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the present invention.
  • the number of amino acids at the amino terminal of the connecting region of the phosphoenolpyruvate-sensitive polypeptide and the optically active polypeptide in the present invention is selected from 0 to 3
  • the number of amino acids at the carboxyl terminal is selected from 0 to 2; when When the recombinant optical probe is connected to the functional protein as a basic unit, it can be fused to the amino acid or carboxyl end of the recombinant optical probe.
  • the linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
  • a phosphoenolpyruvate binding protein variant having a mutation at a site selected from the following exhibits a different binding activity with phosphoenolpyruvate: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320 of SEQ ID NO: 1.
  • the amino acid mutation includes modification, substitution or deletion of an amino acid.
  • the present invention provides phosphoenolpyruvate binding protein variants having these mutations and optical probes comprising such phosphoenolpyruvate binding protein variants as phosphoenolpyruvate sensitive polypeptides. Therefore, in one or more embodiments, the phosphoenolpyruvate sensitive polypeptide in the optical probe is a phosphoenolpyruvate binding protein variant as described in any embodiment of the present invention, and the fluorescent protein in the optical probe is shown in SEQ ID NO: 2-9 or its functional variant.
  • the phosphoenolpyruvate-sensitive polypeptide in the optical probe is as shown in SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2
  • the optically active polypeptide is located at the 254/255 or 254/256 position of the phosphoenolpyruvate-sensitive polypeptide
  • the mutation of the optical probe is as shown in any row in Tables 5 and 6.
  • identity means that two or more sequences or subsequences are identical or that a certain percentage of amino acid residues or nucleotides in a specified region are identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection.
  • BLAST and BLAST 2.0 algorithms 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.
  • fusion proteins promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins
  • it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.
  • the terms “functional fragment”, “derivative” and “analog” refer to a protein or polypeptide that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., phosphoenolpyruvate binding protein or fluorescent protein). White.
  • the functional variants, derivatives or analogs of the polypeptide or protein (e.g., phosphoenolpyruvate binding protein or fluorescent protein) of the present invention may be (i) a protein with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein with a substitution group in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g., a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to this protein sequence (e.g., a secretory sequence or a sequence used to purify the protein or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment).
  • a protein with one or more conservative or non-conservative amino acid residues
  • analogs also include analogs with residues different from natural L-amino acids (e.g., D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (e.g., ⁇ , ⁇ -amino acids).
  • L-amino acids e.g., D-amino acids
  • non-naturally occurring or synthetic amino acids e.g., ⁇ , ⁇ -amino acids
  • Modifications (usually without changing the primary structure) include: chemical derivatization of proteins in vivo or in vitro such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or optimize their solubility.
  • glycosylation such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or
  • the fusion polypeptide of the present invention comprises the optical probe described herein and other polypeptides.
  • the optical probe described herein further comprises other polypeptides fused thereto.
  • the other polypeptides described herein do not affect the properties of the optical probe.
  • the other polypeptides may be located at the N-terminus and/or C-terminus of the optical probe.
  • the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting.
  • a linker may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.
  • tags for purification or tags for immunoblotting include 6*histidine (6*His), glutathione S-transferase (GST), Flag.
  • the present invention includes nucleic acid molecules encoding the phosphoenolpyruvate-sensitive polypeptide or optical probe described in the present invention.
  • nucleic acid or “nucleotide” or “polynucleotide” or “nucleic acid sequence” used in the present invention can be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • variant used herein can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants.
  • an allelic variant is a replacement form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change The function of the protein encoded by it.
  • the nucleic acid of the present invention may comprise a nucleotide sequence having a sequence identity of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% with the nucleic acid sequence.
  • nucleic acid fragments that hybridize with the above sequences.
  • a "nucleic acid fragment” has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).
  • the full-length sequence of the optical probe or fusion protein of the present invention or its fragments can usually be obtained by PCR amplification, artificial synthesis or recombination.
  • the steps and reagents used in conventional PCR, synthesis and recombination are known in the art.
  • mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.
  • the present invention also relates to nucleic acid constructs comprising 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 the polypeptide or protein.
  • the nucleic acid constructs can be manipulated prior to insertion into a vector depending on the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the nucleic acid construct is a vector.
  • the vector can 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, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence, and are operably connected to the nucleic acid sequence of the present invention or its complementary sequence.
  • expression control sequence used herein refers to an element that can be operably connected to the target gene to regulate the transcription, translation and expression of the target gene, which can be a replication origin, a promoter, a marker gene or a translation control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc. The selection of the expression control sequence depends on the host cell used.
  • operable connection refers to the connection of the target nucleotide sequence with the regulatory sequence in a manner that allows the expression of the nucleotide sequence.
  • Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequence of the present invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters include: lac or trp promoters of Escherichia coli; ⁇ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can use a commercially available pCDF vector without other special requirements.
  • BamHI and EcoRI are used to respectively encode the nucleotide sequence encoding the optical probe and the expression vector. Double enzyme digestion is performed, and then the two enzyme digestion products are connected to obtain a recombinant expression vector.
  • the present invention does not specifically limit the specific steps and parameters of enzyme digestion and connection, and conventional steps and parameters in the art can be used.
  • the vector is transformed into a host cell to produce a protein or peptide including a fusion protein.
  • This transfer process can be performed using conventional techniques known to those skilled in the art such as transformation or transfection.
  • the host cell described in the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is a site for recombinant gene amplification.
  • the ideal recipient cell should meet the two conditions of easy acquisition and proliferation.
  • the "host cell” of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells.
  • the host cell is preferably a variety of cells that are conducive to the expression or fermentation production of gene products, and such cells are well known and commonly used in the art. Specifically, it can be bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cells, or animal cells of Bowes melanoma cells, etc.
  • the exemplary host cell used in the embodiment of the present invention is the Escherichia coli BL21-DE3 strain. Those of ordinary skill in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.
  • the method of transferring to the host cell described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfer or electroporation.
  • the host is a prokaryotic organism such as Escherichia coli
  • the method is preferably a CaCl2 method or a MgCl2 method, and the steps used are well known in the art.
  • the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the host cell into which the expression vector is transferred is cultured for amplification and expression to separate and obtain the phosphoenolpyruvic acid optical probe.
  • the host cell amplification and expression culture can be carried out by conventional methods.
  • the culture medium used in the culture can be various conventional culture media.
  • the culture is carried out under conditions suitable for the growth of the host cell.
  • the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties.
  • the present invention does not specifically limit the method for separating the phosphoenolpyruvate fluorescent protein, and the conventional separation method of the fusion protein in the art can be used.
  • optical probe is separated using affinity chromatography using a His tag.
  • the present invention also provides the use of the phosphoenolpyruvate optical probe in real-time positioning, quantitative detection and high-throughput compound screening of phosphoenolpyruvate.
  • the optical probe is preferably connected to the signal peptide in different parts of the cell, transferred into the cell, and the real-time positioning of phosphoenolpyruvate is performed by detecting the strength of the fluorescence signal in the cell; the corresponding phosphoenolpyruvate is quantitatively detected by combining the change of the fluorescence signal with the standard drop curve of phosphoenolpyruvate.
  • the change of the fluorescence signal is displayed by, for example, the ratio of the standardized fluorescence signal.
  • the ratio is the ratio of the ratio of the 485-nanometer fluorescence signal to the 420-nanometer fluorescence signal of the sample to the corresponding ratio of the control.
  • the standard drop curve of phosphoenolpyruvate described in the present invention is drawn based on the fluorescence signal of the phosphoenolpyruvate optical probe under different concentrations of phosphoenolpyruvate.
  • the phosphoenolpyruvate optical probe of the present invention is directly transferred into the cell, and in the process of real-time positioning and quantitative detection of phosphoenolpyruvate, time-consuming sample processing is not required, which is more accurate.
  • the phosphoenolpyruvate optical probe of the present invention When the phosphoenolpyruvate optical probe of the present invention is used for high-throughput compound screening, different compounds are added to the cell culture medium, and the change of the phosphoenolpyruvate content is measured, so as to screen out the compounds that affect the change of the phosphoenolpyruvate content.
  • the application of the phosphoenolpyruvate optical probe described in the present invention in the real-time positioning and quantitative detection of phosphoenolpyruvate and high-throughput compound screening is non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.
  • the present invention also provides a detection kit comprising the optical probe, nucleic acid molecule, nucleic acid construct and/or cell described herein.
  • the kit also contains other reagents required for detecting phosphoenolpyruvate.
  • the other reagents are well known in the art, such as buffer, cell culture medium, phosphoenolpyruvate standard.
  • Exemplary buffers are, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.
  • Item 1 A phosphoenolpyruvate binding protein variant, which:
  • (b) is a sequence having at least 70% sequence identity with the sequence of (a) and having the mutation described in (1) and retaining the ability to bind to phosphoenolpyruvate,
  • the mutation in (a) comprises a mutation at a site selected from any one or more of the following groups:
  • Item 2 A phosphoenolpyruvate optical probe, comprising a phosphoenolpyruvate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the phosphoenolpyruvate-sensitive polypeptide, and
  • Phosphoenolpyruvate-sensitive polypeptide has:
  • (2) is the sequence of the phosphoenolpyruvate binding protein variant described in item 1.
  • Item 3 An optical probe as described in Item 2, wherein the optically active polypeptide is located at residues 68-71, 168-173 and/or 253-257 of the phosphoenolpyruvate-sensitive polypeptide or replaces residues therein.
  • Item 4 The optical probe as described in Item 2, wherein:
  • the optically active polypeptide is located at any one or more of the following sites of the phosphoenolpyruvate-sensitive polypeptide: 68/69, 68/70, 69/70, 68/71, 69/71, 70/71, 168/169, 168/170, 169/170, 168/171, 169/171, 170/171, 168/172, 169/172, 1 70/172, 171/172, 168/173, 169/173, 170/173, 171/173, 172/173, 253/254, 253/255, 254/255, 253/256, 254/256, 255/256, 253/257, 254/257, 255/257 and/or 256/257.
  • optically active polypeptide is located at any one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide: 69/71, 170/171, 171/172, 254/255, 254/256 and 255/257.
  • optically active polypeptide is a fluorescent protein or a functional variant thereof
  • the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9,
  • the functional variant of the fluorescent protein has a mutation within 3 amino acids at the junction with the optically active polypeptide; preferably, the functional variant of the fluorescent protein has a mutation at the first amino acid corresponding to SEQ ID NO: 2, and the mutation is preferably selected from Y1S, Y1G, Y1N, Y1T, Y1P, Y1A, Y1E, Y1H,
  • the functional variant of the fluorescent protein has a mutation at the amino acid position 246 corresponding to SEQ ID NO: 2, and the mutation is preferably selected from N246F, N246C, N246W or N246H.
  • the phosphoenolpyruvate-sensitive polypeptide is as shown in SEQ ID NO: 1, and has one or more of the following mutations: I253V, I253N, I253F, I253C, I253E, I253G, I253S, I253K, I253H, I253D, I253P, I253L, I253R, I253T, I253Q, Q254L, Q254V, Q254A, Q254R, Q254P, Q254M, Q254K, Q254I, R255L, R255I, R255F, G256H, G256F, G256Y, G256V, G257L, R21K, V54G, V54K, V54Q, R55S, V57M, A58G, N59D, N59H, N59E, I60W, R72A, R212I, R212E, R212M, G2
  • a nucleic acid molecule comprising:
  • the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.
  • Item 7 A host cell, wherein:
  • a detection kit comprising:
  • the detection kit optionally further comprises other reagents required for detecting phosphoenolpyruvate using an optical probe.
  • the detection kit further comprises one or more reagents selected from the following: a buffer, a culture medium, and a phosphoenolpyruvate standard.
  • Item 9 A method for preparing the optical probe described in any one of Items 2-4, comprising: providing the host cell described in Item 7, culturing the host cell under conditions where the optical probe is expressed, and isolating the optical probe.
  • optical probe described in any one of item 10, items 2 to 4, the nucleic acid sequence described in item 5, the nucleic acid construct described in item 6 or the host cell described in item 7 in detecting phosphoenolpyruvate in a sample, screening compounds, and localizing phosphoenolpyruvate inside or outside cells,
  • Detecting phosphoenolpyruvate in a sample comprises the steps of contacting the optical probe or the host cell with the sample, detecting an optical change of the optically active polypeptide, and detecting phosphoenolpyruvate in the sample according to the optical change of the optically active polypeptide.
  • the screening compound comprises the steps of: contacting the optical probe or host cell with a candidate compound in a system containing phosphoenolpyruvate, detecting an optical change of an optically active polypeptide, and screening the compound according to the optical change of the optically active polypeptide; preferably, the screening compound comprises the steps of: contacting the host cell with a candidate compound in a system containing phosphoenolpyruvate, and the optical change of the optically active polypeptide indicates whether the candidate compound regulates the uptake of phosphoenolpyruvate by the cell,
  • the intracellular/extracellular localization of phosphoenolpyruvate comprises the steps of: contacting a system containing phosphoenolpyruvate with the optical probe or the host cell, and detecting an optical change of an optically active polypeptide.
  • the system is a solution system, a cell system or a subcellular system.
  • the embodiments mainly use conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods, etc. These methods are well known to ordinary technicians in the field, for example: "Molecular Biology Laboratory Reference Manual” by Jane Roskams et al., “Molecular Cloning Laboratory Guide” (3rd edition, August 2002, Science Press, Beijing) by J. Sambrook, D.W. Russell, translated by Huang Peitang et al.; “Animal Cell Culture: Basic Technical Guide” (5th edition) by Frasheny et al., translated by Zhang Jingbo, Xu Cunshuan et al.; "Concise Cell Biology Laboratory Guide” by J.S. Bonifacion, M. Dassault et al., translated by Zhang Jingbo et al.
  • the pCDF-cpYFP and pCDF-phosphoenolpyruvate binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and verified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. and BGI. The expression plasmids constructed in the examples were sequenced, and the sequence determination was completed by BGI and Jie Li Sequencing.
  • the Taq DNA polymerase used in each example was purchased from Dongsheng Bio, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., primeSTAR DNA polymerase was purchased from TaKaRa, The three polymerases are all provided with corresponding polymerase buffer and dNTPs when purchased. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffers were provided when purchased. The transfection reagent Lip2000Kit was purchased from Invitrogen.
  • the DNA purification kit used in the examples was purchased from BBI (Canada), and the common plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the cloned strain Mach1 was purchased from Invitrogen.
  • the nickel column affinity chromatography column and desalting column fillers were both from GE healthcare.
  • the main instruments used in the examples include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shenneng Gaming Company), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell culture incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).
  • Target fragment amplification PCR 1.
  • This method is mainly used for gene fragment amplification and colony PCR identification of positive clones.
  • the reaction system of PCR amplification is as follows: template sequence 0.5-1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 10 ⁇ pfu buffer 5 ⁇ L, pfu DNA polymerase 0.5 ⁇ L, dNTP (10mM) 1 ⁇ L, sterilized ultrapure water (ddH2O) 41.5-42 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification procedure is as follows: 95°C denaturation for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600bp/min)), 72°C extension for 10 minutes.
  • the long-fragment amplification used in the present invention is mainly a reverse PCR amplification vector, which is a technique used to obtain site-directed mutations in the following embodiments.
  • Reverse PCR primers are designed at the mutation site, and the 5' end of one of the primers contains the mutated nucleotide sequence.
  • the amplified product contains the corresponding mutation site.
  • the long-fragment amplification PCR reaction system is as follows: 1 ⁇ L of template sequence (10pg-1ng), 0.5 ⁇ L of forward primer (25 ⁇ M), 0.5 ⁇ L of reverse primer (25 ⁇ M), 10 ⁇ L of 5 ⁇ PrimerSTAR buffer, 0.5 ⁇ L of PrimerSTAR DNA polymerase, 4 ⁇ L of dNTP (2.5mM), 33.5 ⁇ L of sterile ultrapure water (ddH2O), and a total volume of 50 ⁇ L.
  • the PCR amplification procedure is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50-68°C for 5-15 seconds, 72°C for or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000bp/min)), extension at 72°C for 10 minutes.
  • the system for double enzyme digestion of plasmid vector is as follows: 20 ⁇ L of plasmid vector (about 1.5 ⁇ g), 5 ⁇ L of 10 ⁇ buffer, 11-2 ⁇ L of restriction enzyme, 21-2 ⁇ L of restriction enzyme, and the total volume is made up to 50 ⁇ L with sterile ultrapure water.
  • the reaction conditions are 37°C and 1-7 hours.
  • the ends of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, the 5' end base of the PCR product needs to be subjected to a phosphate group addition reaction. Only DNA molecules with phosphate groups at the end can undergo a ligation reaction.
  • the phosphorylation reaction system is as follows: 5-8 ⁇ L of PCR product fragment DNA sequence, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 polynucleotide kinase (T4 PNK), 0-3 ⁇ L of sterile ultrapure water, and a total volume of 10 ⁇ L.
  • the reaction conditions are 37°C, and inactivation at 72°C for 20 minutes after 30 minutes-2 hours.
  • connection methods between different fragments and vectors are different. Three connection methods are used in the present invention:
  • the principle of this method is that the flat-end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid.
  • the homologous recombination connection system is as follows: 4 ⁇ L of DNA fragment treated with T4 PNK, 4 ⁇ L of linearized vector fragment, 1 ⁇ L of PEG4000, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, a total of 10 ⁇ L.
  • the reaction conditions are 22°C, 30 minutes.
  • DNA fragments cut by restriction endonucleases usually produce protruding sticky ends, so they can be connected to vector fragments containing complementary sticky ends to form recombinant plasmids.
  • the ligation reaction system is as follows: 1-7 ⁇ L of PCR product DNA after restriction digestion, 0.5-7 ⁇ L of plasmid after restriction digestion, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 ⁇ L. Reaction conditions 16°C, 4-8 hours.
  • the 5'-end phosphorylated DNA fragment was connected to the 3' and 5' ends of the linearized vector by self-circularization ligation to obtain a recombinant plasmid.
  • the self-circularization ligation reaction system is as follows: phosphorylation reaction system 10 ⁇ L, T4 ligase (5U/ ⁇ L) 0.5 ⁇ L, total volume 10.5 ⁇ L. Reaction conditions 16°C, 4-16 hours.
  • Resuspension buffer CaCl 2 (100mM), MgCl 2 (70mM), NaAc (40mM)
  • Storage buffer 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10 ⁇ CaCl 2 (1M), 1 mL 10 ⁇ MgCl 2 (700 mM), 1 mL 10 ⁇ NaAc (400 mM), 4.6 mL ddH 2 O
  • the supernatant was purified by a self-assembled nickel column affinity chromatography to obtain the protein.
  • the protein after nickel column affinity chromatography was then passed through a self-assembled desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4).
  • the probe was diluted with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a final concentration of 0.2-5 ⁇ M protein solution.
  • assay buffer 100 mM HEPES, 100 mM NaCl, pH 7.4
  • Phosphoenolpyruvate was prepared as a stock solution at a final concentration of 50 mM in 100 mM HEPES (100 mM HEPES, 100 mM NaCl, pH 7.4).
  • the pCDNA3.1+-based phosphoenolpyruvate 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 and 5% CO 2. Fluorescence detection was performed after the exogenous gene was fully expressed for 24 to 36 hours.
  • the attached HEK293 cells were rinsed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and microplate reader.
  • the TtPFK gene in the extreme thermophilic bacteria gene was amplified by PCR.
  • the PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI.
  • the pCDF vector was double-digested at the same time.
  • T4 DNA ligase the product was used to transform DH5 ⁇ .
  • the transformed DH5 ⁇ was coated on LB plates (streptomycin 100ug/mL) and cultured at 37°C overnight.
  • the grown DH5 ⁇ transformants were subjected to plasmid extraction and PCR identification. The positive plasmid was sequenced correctly for subsequent plasmid construction.
  • Example 2 Expression and detection of cpYFP optical probes at different insertion sites
  • PCR was used to generate the DNA fragment of cpYFP, and the homologous sequence of the cpYFP terminal was introduced through the 5' end of the primer.
  • PCR amplification was used to generate the pCDF phosphoenolpyruvate binding protein linearization vector, whose 5' and 3' ends respectively carried the sequences that were completely consistent with the two ends of cpYFP (15bp to 20bp).
  • the pCDF-TtPFK and cpYFP fragments were homologously recombined under the action of Hieff Clone Enzyme.
  • the product was transformed into DH5 ⁇ , and the transformed DH5 ⁇ was spread on LB plates (streptomycin 100ug/mL) and cultured at 37°C overnight.
  • the positive clones identified by PCR were extracted and sequenced. Sequencing was completed by Jie Li Sequencing Company.
  • the recombinant plasmid was transformed into BL21 (DE3) to induce expression, and the protein was purified.
  • the size of the protein was around 63Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the TtPFK-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-TtPFK-cpYFP. The results are shown in Figure 1.
  • the supernatant of Escherichia coli expressing the TtPFK-cpYFP fusion protein was used for phosphoenolpyruvate response screening, and the detection signal of the fusion fluorescent protein containing 1 mM phosphoenolpyruvate was divided by the detection signal of the fusion fluorescent protein without phosphoenolpyruvate.
  • the detection results show that the optical probes whose supernatant expressing the TtPFK-cpYFP fusion protein responded to phosphoenolpyruvate more than 1.3 times are 69/71, 170/171, 171/172, 254/255, 254/256 and 255/257 sites are inserted, and the response to phosphoenolpyruvate is not affected by the other TtPFK binding substrates at sites 254/255 and 254/256.
  • Example 3 Expression and detection of cpGFP optical probes at different insertion sites
  • cpYFP was replaced with cpGFP to construct a phosphoenolpyruvate green fluorescent protein fluorescent probe.
  • Table 2 the detection results show that the supernatant expressing the TtPFK-cpGFP fusion protein has an optical probe that responds to phosphoenolpyruvate more than 1.3 times, including 171/172, 254/255 and 254/256 sites or the corresponding amino acid sites of their family proteins, and the response to phosphoenolpyruvate is not affected by the other TtPFK binding substrates at 254/255 and 254/256 sites.
  • Example 4 Expression and detection of cpBFP optical probes at different insertion sites
  • cpYFP was replaced with cpBFP to construct a phosphoenolpyruvate blue fluorescent protein fluorescent probe.
  • Table 3 the detection results show that the supernatant expressing the TtPFK-cpBFP fusion protein has an optical probe that responds to phosphoenolpyruvate more than 1.3 times, including 170/171, 171/172, 254/255 and 254/256 sites or the corresponding amino acid sites of their family proteins, and the response to phosphoenolpyruvate is not affected by the other TtPFK binding substrates at 254/255 and 254/256 sites.
  • Example 5 Expression and detection of cpmApple optical probes at different insertion sites
  • cpYFP was replaced with cpmApple to construct a phosphoenolpyruvate red fluorescent protein fluorescent probe.
  • Table 4 the detection results show that the supernatant expressing the TtPFK-cpmApple fusion protein has an optical probe that responds to phosphoenolpyruvate more than 1.3 times, including 170/171, 171/172, 254/255 and 254/256 sites or the corresponding amino acid sites of their family proteins, and the response to phosphoenolpyruvate is not affected by the other TtPFK binding substrates at 254/255 and 254/256 sites.
  • Example 2 For the optical probe obtained in Example 2 that responds to phosphoenolpyruvate more than 1.3 times and is not affected by the other binding substrates of TtPFK, that is, the two optical probes inserted at the 254/255 and 254/256 sites of TtPFK are linearized by inverse PCR, the sequence of the linker mutation site is introduced into the primer, and the obtained PCR product is homologously recombined under the action of Hieff Clone Enzyme to establish a mutation library.
  • amino acid sequence and nucleic acid sequence of 254/255-TtPFK-I253K/Q254L/R255L/G256F-cpYFP-Y1S are shown in SEQ ID NO: 12 and 13, respectively.
  • the mutant library recombinant plasmid was transformed into BL21 (DE3) to induce expression, and the probe protein was purified for phosphoenolpyruvate response screening.
  • the detection signal of the fusion fluorescent protein containing 2mM phosphoenolpyruvate was divided by the detection signal of the fusion fluorescent protein without phosphoenolpyruvate. The detection results showed that the optical probes with a response to phosphoenolpyruvate greater than or equal to 2 times are shown in Table 5.
  • Example 7 Expression and detection of cpYFP optical probes incorporating pocket mutations
  • the affinity and substrate specificity of the phosphoenolpyruvic acid optical probe to phosphoenolpyruvic acid were adjusted by amino acid mutations at positions R163, R172 and R246.
  • the probe was linearized by inverse PCR, the sequence of the mutation site was introduced into the primer, and the obtained PCR product was homologously recombined to obtain a plasmid containing the above site mutations.
  • the mutant plasmid was transformed into BL21 (DE3) to induce expression, and the mutant probe protein was purified to perform a phosphoenolpyruvic acid response test, and the detection signal of the fusion fluorescent protein containing 2mM hypophosphoenolpyruvic acid was divided by the detection signal of the fusion fluorescent protein without phosphoenolpyruvic acid.
  • the test results show that the optical probes that respond to phosphoenolpyruvic acid more than or equal to 2 times are shown in Table 6.
  • the two purified phosphoenolpyruvate optical probes described in Example 6 and Example 7 were treated with 0 mM and 5 mM phosphoenolpyruvate for 10 minutes, respectively, and then the fluorescence spectra were detected using a fluorescence spectrophotometer.
  • excitation spectrum was recorded with an excitation range of 370nm to 510nm and an emission wavelength of 530nm, and read every 5nm. The results showed that the probe had two excitation peaks at about 410 and 490nm, as shown in Figure 2.
  • the purified 81 phosphoenolpyruvate probes described in Examples 6 and 7 were subjected to phosphoenolpyruvate detection in a concentration gradient (0-10 mM). After the purified probes were treated for 10 minutes, the changes in the fluorescence intensity at 528 nm emission with excitation at 420 nm and the ratio of the fluorescence intensity at 528 nm emission with excitation at 485 nm were detected. The results are shown in FIG3 .
  • the K d values of the 81 phosphoenolpyruvate optical probes are (binding constants) are 0.8, 1.55, 5.64, 8.5, 11.42, 9.66, 5.56, 31.99, 81.67, 31.05, 68.4, 16.55, 45, 29.51, 30.9, 17.52, 21.83, 44.17, 40.32, 6.54, 20.43, 435.8, 18.16, 16.69, 52.13, 42.67, 12.92, 60.83, 32.61, 80.64, 74.29, 5.96, 20.22, 9.15, 13.38, 8.93, 13.08, 715.7, 7.84, 23.32, 91.13, 2 94.1, 0.4492, 0.36, 0.4957, 37.43, 4.62, 177.7, 59.81, 103.2, 56.47, 16.24, 29.71, 14.28, 75.89, 202.9, 0.2544, 0.2853, 0.24, 0.25, 0.1598, 0.6172, 127.3, 20
  • Example 9 Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles
  • 293 cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, then washed with PBS, placed in HBSS solution, and fluorescence was detected under the FITC channel using an inverted fluorescence microscope. As shown in Figure 5.
  • the phosphoenolpyruvate optical probe can be localized to subcellular organelles including cytoplasm, extracellular membrane, nucleus, endoplasmic reticulum, mitochondria and nuclear exclusion by fusing with different specific localization signal peptides. Different subcellular structures show fluorescence, and the distribution and intensity of fluorescence are different.
  • Example 10 High-throughput compound screening based on optical probes in living cells
  • the transfected 293 cells were rinsed with PBS, placed in HBSS solution (without phosphoenolpyruvate) for 1 hour, and then treated with 10 ⁇ M of the compound for 1 hour. Phosphoenolpyruvate was added dropwise to each sample. The changes in the ratio of the fluorescence intensity at 528nm emission with 420nm excitation and 528nm emission with 485nm excitation were recorded using an ELISA instrument. The samples not treated with any compound were used as controls for standardization. The results are shown in Figure 7. Among the 2000 compounds used, most of the compounds had little effect on the entry of phosphoenolpyruvate into cells. 23 compounds can increase the cell's ability to uptake phosphoenolpyruvate, and another 7 compounds can significantly reduce the cell's uptake of phosphoenolpyruvate.
  • Example 11 Quantitative detection of phosphoenolpyruvate in blood using an optical probe
  • the phosphoenolpyruvate optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has a large dynamic change in fluorescence, has good specificity, and can be expressed in cells through genetic manipulation methods, and can locate and quantitatively detect phosphoenolpyruvate in real time inside and outside cells; and can perform high-throughput compound screening.

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Abstract

本发明涉及磷酸烯醇式丙酮酸光学探针。具体地，本发明提供一种磷酸烯醇式丙酮酸光学探针，其包含磷酸烯醇式丙酮酸敏感多肽和光学活性多肽，其中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的序列内。

Description

一种磷酸烯醇式丙酮酸光学探针及其制备方法和应用
本申请要求2022年11月22日提交的中国申请202211465765.3的优先权，其内容出于所有目的通过引用并入本文。
技术领域
本发明涉及光学探针技术领域，尤其涉及一种磷酸烯醇式丙酮酸光学探针及其制备方法和应用。
背景技术
磷酸烯醇式丙酮酸(Phosphoenolpyruvate，PEP)作为糖酵解途径中的高能中间物质，是连接糖酵解与糖异生途径的关键代谢物。PEP含有的高能磷酸键使其在细菌、植物及哺乳动物细胞中均扮演了磷酸供体的角色，在维持胞内ATP含量、葡萄糖稳态、糖代谢调控、肿瘤免疫和细胞保护方面等均发挥着重要作用。
目前最常用的检测PEP的方法有高效液相色谱法、液相色谱质谱联用法(Ho P C等.Cell，2015，162(6)，1217-1228)、同位素标记法(Heiden M G V等，Science，2010，329(5998)，1492-1499)和酶活检测方法等。高效液相色谱法和液相色谱质谱联用法可以高效并精准地测定样品中PEP的含量变化，但其中涉及细胞内代谢物的提取，实验步骤繁琐且周期较长。同位素标记法和酶活检测法均不能实时监控活细胞内PEP的变化，因此亟需开发出能够原位监测活细胞内PEP动态变化的遗传编码荧光探针。
发明内容
本发明的目的在于提供在细胞内、外实时定位、高通量、定量检测磷酸烯醇式丙酮酸的探针和方法。
为了实现上述发明目的，本发明提供以下技术方案：
本发明第一方面提供一种磷酸烯醇式丙酮酸结合蛋白变体，其：
(a)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个、3个、4个、5个、6个、7个、8个或更多个位点具有突变：I253、Q254、R255、G256、G257、 R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，所述突变包括氨基酸的修饰、取代或缺失，
(b)是与(a)的序列具有至少70％序列相同性并具有(1)所述突变并保留对磷酸烯醇式丙酮酸结合能力的序列。
在一个或多个实施方案中，所述磷酸烯醇式丙酮酸结合蛋白变体具有SEQ ID NO：1所示的序列，并且：(I)在选自以下的1个、2个、3个、4个或更多个位点具有突变：I253、Q254、R255、G256、G257，和/或，(II)在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320。
在一个或多个实施方案中，(I)中所述突变的位点包括Q254。优选地，(I)中所述突变的位点包括选自以下任一组或多组的位点：(I.1)I253、Q254，(I.2)I253、Q254、R255、G256，(I.3)Q254、R255、G256，(I.4)I253、Q254、G256、G257
在一个或多个实施方案中，(II)中所述突变的位点包括选自以下任一组或多组的位点：(II.1)R21，(II.2)V54，(II.3)R55，(II.4)V57，(II.5)A58，(II.6)I60，(II.7)N59，(II.8)R212，(II.9)G213，(II.10)K215，(II.11)S216，(II.12)L320，(II.13)R72，(II.14)V54、R72。
在一个或多个实施方案中，所述磷酸烯醇式丙酮酸结合蛋白变体具有SEQ ID NO：1所示的序列，并且：(I)在以下位点具有突变：I253、Q254、G256和G257，和(II)在选自以下任一组或多组的位点具有突变：(II.1)R21，(II.2)V54，(II.3)R55，(II.4)V57，(II.5)A58，(II.6)I60，(II.7)N59，(II.8)R212，(II.9)G213，(II.10)K215，(II.11)S216，(II.12)L320，(II.13)R72，(II.14)V54、R72。
在一个或多个实施方案中，所述磷酸烯醇式丙酮酸结合蛋白变体具有SEQ ID NO：1所示的序列，并且在以下位点具有突变：(II.15)V54、I253、Q254、R255、G256，(II.16)N59、I253、Q254、R255、G256，(II.17)V54、I253、Q254、G256、G257，(II.18)N59、I253、Q254、G256、G257，(II.19)R212、I253、Q254、G256、G257，(II.20)G213、I253、Q254、G256、G257，(II.21)K215、I253、Q254、G256、G257，(II.22)L320、I253、Q254、G256、G257，(II.23)S216、I253、Q254、G256、G257。
在一个或多个实施方案中，I253突变为V、N、F、C、E、G、S、K、H、D、P、L、R、T或Q。
在一个或多个实施方案中，Q254突变为L、V、A、R、P、M、K、I。
在一个或多个实施方案中，R255突变为L、I、F。
在一个或多个实施方案中，G256突变为H、F、Y、V。
在一个或多个实施方案中，R21突变为K。
在一个或多个实施方案中，V54突变为G、K、Q。
在一个或多个实施方案中，R55突变为S。
在一个或多个实施方案中，V57突变为M。
在一个或多个实施方案中，A58突变为G。
在一个或多个实施方案中，N59突变为D、H或E。
在一个或多个实施方案中，I60突变为W。
在一个或多个实施方案中，R72突变为A。
在一个或多个实施方案中，R212突变为I、E、M。
在一个或多个实施方案中，G213突变为T。
在一个或多个实施方案中，K215突变为H、D、E。
在一个或多个实施方案中，S216突变为D、L、Y、I、M、F或Q。
在一个或多个实施方案中，L320突变为Y、N或F。
在一个或多个实施方案中，所述突变包含选自以下的一个或多个：I253K，Q254L，R255L和/或G256F。
在一个或多个实施方案中，所述突变包含选自以下任一组的突变：(1)I253Y、Q254I，(2)I253V、Q254L、R255L、G256H，(3)Q254L、R255L、G256H，(4)Q254V、R255I、G256H，(5)I253N、Q254L、R255L、G256F，(6)I253F、Q254L、R255L、G256F，(7)I253V、Q254A、R255F、G256Y，(8)I253V、Q254L、R255F、G256Y，(9)I253C、Q254L、R255L、G256F，(10)I253E、Q254L、R255L、G256F，(11)I253G、Q254L、R255L、G256F，(12)I253I、Q254L、R255L、G256F，(13)I253S、Q254L、R255L、G256F，(14)I253G、Q254L、R255L、G256F，(15)I253K、Q254L、R255L、G256F，(16)I253H、Q254L、R255L、G256F，(17)I253D、Q254L、R255L、G256F，(18)Q254L、R255L、G256F，(19)I253P、Q254L、R255L、G256F，(20)Q254L、R255L、G256F，(21)I253L、Q254R、R255L、G256F，(22)I253R、Q254L、R255L、G256F，(23)I253T、Q254L、R255F、G256Y，(24)I253Q、Q254P、R255F、 G256Y，(25)I253Q、Q254M、R255F、G256Y，(26)I253T、Q254P、R255F、G256Y，(27)Q254L、R255F、G256F，(28)I253S、Q254K、R255F、G256F，(29)Q254P、R255F、G256F，(30)I253E、Q254V、R255F、G256F，(31)Q254L、R255F、G256F，(32)I253T、Q254L、R255F、G256F，(33)Q254L、R255F、G256F，(34)I253D、Q254L、G256V、G257L，(35)I253S、Q254L、G256V、G257L，(36)I253Q、Q254L、G256V、G257L，(37)R21K，(38)V54K，(39)V54Q，(40)V54G，(41)R55S，(42)V57M，(43)A58G，(44)I60W，(45)N59D，(46)N59H，(47)N59E，(48)R212I，(49)R212E，(50)R212M，(51)G213T，(52)K215E，(53)K215H，(54)K215D，(55)S216D，(56)S216L，(57)S216Y，(58)S216I，(59)S216M，(60)S216F，(61)S216Q，(62)L320Y，(63)L320N，(64)L320F，(65)R72A，(66)V54G、R72A，(67)V54K、I253G、Q254L、R255L、G256F，(68)V54Q、I253G、Q254L、R255L、G256F，(69)N59H、I253G、Q254L、R255L、G256F，(70)V54K、I253K、Q254L、R255L、G256F，(71)V54Q、I253K、Q254L、R255L、G256F，(72)N59H、I253K、Q254L、R255L、G256F，(73)V54Q、I253Q、Q254L、G256V、G257L，(74)N59D、I253Q、Q254L、G256V、G257L，(75)R212M、I253Q、Q254L、G256V、G257L，(76)G213T、I253Q、Q254L、G256V、G257L，(77)K215E、I253Q、Q254L、G256V、G257L，(78)L320F、I253Q、Q254L、G256V、G257L，(79)S216L、I253Q、Q254L、G256V、G257L，(80)S216Y、I253Q、Q254L、G256V、G257L。
本发明第一方面还提供一种磷酸烯醇式丙酮酸光学探针，包含磷酸烯醇式丙酮酸敏感多肽和光学活性多肽，其中光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的序列内。磷酸烯醇式丙酮酸敏感多肽被光学活性多肽分为第一部分和第二部分。
在一个或多个实施方案中，磷酸烯醇式丙酮酸敏感多肽具有：
(1)SEQ ID NO：1所示的序列，或与它们有至少70％序列相同性并保留对磷酸烯醇式丙酮酸结合活性的序列，
(2)SEQ ID NO：1所示序列的功能变体，该功能变体在与光学活性多肽连接处的5个(优选3个，更优选2个)氨基酸内具有突变，和/或，在在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，
(3)与(2)所述序列具有至少70％序列相同性并具有(2)所述突变并保留对磷酸烯醇式丙酮酸敏感性的序列。
在一个或多个实施方案中，(2)是SEQ ID NO：1所示的序列并且在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：I253、Q254、R255、G256、G257、R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，所述突变包括氨基酸的修饰、取代或缺失。
在一个或多个实施方案中，(2)是本文第一方面任一实施方案所述的磷酸烯醇式丙酮酸结合蛋白变体的序列。
在一个或多个实施方案中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的残基68-71，168-173和/或253-257中或置换其中的残基，编号对应于磷酸烯醇式丙酮酸敏感多肽的全长。
在一个或多个实施方案中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170，169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和/或256/257。更优选地，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：69/71，170/171，171/172，254/255，254/256和255/257。
在一个或多个实施方案中，所述光学活性多肽是荧光蛋白或其功能变体，其中，荧光蛋白的功能变体在与光学活性多肽连接处的3个(优选2个)氨基酸内具有突变。
在一个实施方式中，荧光蛋白选自黄色荧光蛋白、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白、苹果红荧光蛋白。在一个实施方式中，荧光蛋白具有SEQ ID NO：2-9中任一所示的序列，优选SEQ ID NO：2、6、7或9。
在一个或多个实施方案中，荧光蛋白的功能变体具有SEQ ID NO：2-9中任一所示的序列并且在对应于SEQ ID NO：2的第1位氨基酸处具有突变，该突变优选突变为S、G、N、T、P、A、E、H。或者或此外，荧光蛋白的功能变体具有SEQ ID NO：2-9中任一所示的序列并且在对应于SEQ ID NO：2的第246位氨基酸处具有突变，该突变优选突变为F、C、W或H。
在一个或多个实施方案中，荧光蛋白的功能变体具有SEQ ID NO：2-9中任一所示的序列并在对应于SEQ ID NO：2的第1位氨基酸和第246位氨基酸处具有选自以 下任一组所示的突变：1S和246F；1G和246F；1H和246F；1A和246C；1G和246C；1S和246C；1T和246C；1T和246F。
在一个或多个实施方案中，荧光蛋白具有SEQ ID NO：2所示的序列或是其第1位和/或第246位氨基酸处具有以下任一组所示突变的变体：Y1S；Y1G；Y1N；Y1T；Y1P；Y1A；Y1E；Y1H；N246F；N246C；N246W；N246H；Y1S和N246F；Y1G和N246F；Y1H和N246F；Y1A和N246C；Y1G和N246C；Y1S和N246C；Y1T和N246C；Y1T和N246F，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自以下一个或多个位点：69/71，170/171，171/172，254/255，254/256和255/257位点。
在一个或多个实施方案中，荧光蛋白具有SEQ ID NO：6所示的序列或是其在对应于SEQ ID NO：2的第1位或第246位氨基酸的氨基酸处具有以下任一组所示突变的变体：1S；1G；1N；1T；1P；1A；1E；1H；246F；246C；246W；246H；1S和246F；1G和246F；1H和246F；1A和246C；1G和246C；1S和246C；1T和246C；1T和246F，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自以下一个或多个位点：171/172，254/255和254/256位点。
在一个或多个实施方案中，荧光蛋白具有SEQ ID NO：7所示的序列或是其在对应于SEQ ID NO：2的第1位或第246位氨基酸的氨基酸处具有以下任一组所示突变的变体：1S；1G；1N；1T；1P；1A；1E；1H；246F；246C；246W；246H；1S和246F；1G和246F；1H和246F；1A和246C；1G和246C；1S和246C；1T和246C；1T和246F，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自以下一个或多个位点：170/171，171/172，254/255和254/256位点。
在一个或多个实施方案中，荧光蛋白具有SEQ ID NO：10所示的序列或是其在对应于SEQ ID NO：2的第1位或第246位氨基酸的氨基酸处具有以下任一组所示突变的变体：1S；1G；1N；1T；1P；1A；1E；1H；246F；246C；246W；246H；1S和246F；1G和246F；1H和246F；1A和246C；1G和246C；1S和246C；1T和246C；1T和246F，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自以下一个或多个位点：170/171，171/172，254/255和254/256位点。
在一个实施方式中，光学探针还包含侧接所述光学活性多肽的一个或多个接头。本发明所述接头可以是任何长度的任何氨基酸序列。在一个实施方式中，光学活性多肽侧翼包含不超过5个氨基酸的接头，例如0、1、2、3、4个氨基酸的接头。在一个实施方式中，光学活性多肽侧翼的接头包含氨基酸Y。在一个实施方式中，接头Y位于光学活性多肽的N端和/或C端。在一个实施方式中，光学探针如下所示： 磷酸烯醇式丙酮酸敏感多肽的第一部分B1、Y、光学活性多肽A、磷酸烯醇式丙酮酸敏感多肽的第二部分B2。在一个实施方式中，本发明光学探针不包含接头。
在一个实施方式中，本发明光学探针还包含定位序列，用于将探针定位到例如细胞的特定细胞器。
在一个或多个实施方案中，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2-9(优选SEQ ID NO：2、6、7、9)所示或是其在对应于SEQ ID NO：2的第1位或第246位氨基酸的氨基酸处具有选自以下任一个或多个突变的变体：1S、1G、1N、1T、1P、1A、1E、1H、246F、246C、246W或246H，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170，169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和/或256/257。优选地，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：69/71，170/171，171/172，254/255，254/256和255/257。
在一个或多个实施方案中，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，并且具有以下的一个或多个突变：I253V，I253N，I253F，I253C，I253E，I253G，I253S，I253K，I253H，I253D，I253P，I253L，I253R，I253T，I253Q，Q254L，Q254V，Q254A，Q254R，Q254P，Q254M，Q254K，Q254I，R255L，R255I，R255F，G256H，G256F，G256Y，G256V，G257L，R21K，V54G，V54K，V54Q，R55S，V57M，A58G，N59D，N59H，N59E，I60W，R72A，R212I，R212E，R212M，G213T，K215H，K215D，K215E，S216D，S216L，S216Y，S216I，S216M，S216F，S216Q，L320Y，L320N和/或L320F，光学活性多肽如SEQ ID NO：2-9所示(优选SEQ ID NO：2、6、7、9)或是其在对应于SEQ ID NO：2的第1位或第246位氨基酸的氨基酸处具有选自以下任一个或多个突变的变体：1S、1G、1N、1T、1P、1A、1E、1H、246F、246C、246W或246H，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/255或254/256位点。优选地，磷酸烯醇式丙酮酸敏感多肽的突变包含选自以下任一组的突变：(1)I253Y、Q254I，(2)I253V、Q254L、R255L、G256H，(3)Q254L、R255L、G256H，(4)Q254V、R255I、G256H，(5)I253N、Q254L、R255L、G256F，(6)I253F、Q254L、R255L、G256F，(7)I253V、Q254A、R255F、G256Y，(8)I253V、Q254L、R255F、G256Y，(9)I253C、Q254L、R255L、G256F，(10)I253E、 Q254L、R255L、G256F，(11)I253G、Q254L、R255L、G256F，(12)I253I、Q254L、R255L、G256F，(13)I253S、Q254L、R255L、G256F，(14)I253G、Q254L、R255L、G256F，(15)I253K、Q254L、R255L、G256F，(16)I253H、Q254L、R255L、G256F，(17)I253D、Q254L、R255L、G256F，(18)Q254L、R255L、G256F，(19)I253P、Q254L、R255L、G256F，(20)Q254L、R255L、G256F，(21)I253L、Q254R、R255L、G256F，(22)I253R、Q254L、R255L、G256F，(23)I253T、Q254L、R255F、G256Y，(24)I253Q、Q254P、R255F、G256Y，(25)I253Q、Q254M、R255F、G256Y，(26)I253T、Q254P、R255F、G256Y，(27)Q254L、R255F、G256F，(28)I253S、Q254K、R255F、G256F，(29)Q254P、R255F、G256F，(30)I253E、Q254V、R255F、G256F，(31)Q254L、R255F、G256F，(32)I253T、Q254L、R255F、G256F，(33)Q254L、R255F、G256F，(34)I253D、Q254L、G256V、G257L，(35)I253S、Q254L、G256V、G257L，(36)I253Q、Q254L、G256V、G257L，(37)R21K，(38)V54K，(39)V54Q，(40)V54G，(41)R55S，(42)V57M，(43)A58G，(44)I60W，(45)N59D，(46)N59H，(47)N59E，(48)R212I，(49)R212E，(50)R212M，(51)G213T，(52)K215E，(53)K215H，(54)K215D，(55)S216D，(56)S216L，(57)S216Y，(58)S216I，(59)S216M，(60)S216F，(61)S216Q，(62)L320Y，(63)L320N，(64)L320F，(65)R72A，(66)V54G、R72A，(67)V54K、I253G、Q254L、R255L、G256F，(68)V54Q、I253G、Q254L、R255L、G256F，(69)N59H、I253G、Q254L、R255L、G256F，(70)V54K、I253K、Q254L、R255L、G256F，(71)V54Q、I253K、Q254L、R255L、G256F，(72)N59H、I253K、Q254L、R255L、G256F，(73)V54Q、I253Q、Q254L、G256V、G257L，(74)N59D、I253Q、Q254L、G256V、G257L，(75)R212M、I253Q、Q254L、G256V、G257L，(76)G213T、I253Q、Q254L、G256V、G257L，(77)K215E、I253Q、Q254L、G256V、G257L，(78)L320F、I253Q、Q254L、G256V、G257L，(79)S216L、I253Q、Q254L、G256V、G257L，(80)S216Y、I253Q、Q254L、G256V、G257L。
在一个或多个实施方案中，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2-9所示(优选SEQ ID NO：2、6、7、9)，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/255位点，并且，所述光学探针具有如下所示的突变：(1)磷酸烯醇式丙酮酸敏感多肽的I253Y、Q254I，(2)磷酸烯醇式丙酮酸敏感多肽的I253V、Q254L、R255L、G256H，和光 学活性多肽的1S，(3)磷酸烯醇式丙酮酸敏感多肽的Q254L、R255L、G256H，和光学活性多肽的1S，(4)磷酸烯醇式丙酮酸敏感多肽的Q254V、R255I、G256H，和光学活性多肽的246W，(5)磷酸烯醇式丙酮酸敏感多肽的I253N、Q254L、R255L、G256F，和光学活性多肽的1S、246F，(6)磷酸烯醇式丙酮酸敏感多肽的I253F、Q254L、R255L、G256F，和光学活性多肽的1G、246F，(7)磷酸烯醇式丙酮酸敏感多肽的I253V、Q254A、R255F、G256Y，和光学活性多肽的246F，(8)磷酸烯醇式丙酮酸敏感多肽的I253V、Q254L、R255F、G256Y，和光学活性多肽的1H、246F，(9)磷酸烯醇式丙酮酸敏感多肽的I253C、Q254L、R255L、G256F，和光学活性多肽的1N，(10)磷酸烯醇式丙酮酸敏感多肽的I253E、Q254L、R255L、G256F，和光学活性多肽的1S，(11)磷酸烯醇式丙酮酸敏感多肽的I253G、Q254L、R255L、G256F，和光学活性多肽的1T，(12)磷酸烯醇式丙酮酸敏感多肽的I253I、Q254L、R255L、G256F，和光学活性多肽的1S，(13)磷酸烯醇式丙酮酸敏感多肽的I253S、Q254L、R255L、G256F，和光学活性多肽的1S，(14)磷酸烯醇式丙酮酸敏感多肽的I253G、Q254L、R255L、G256F，和光学活性多肽的1P，(15)磷酸烯醇式丙酮酸敏感多肽的I253K、Q254L、R255L、G256F，和光学活性多肽的1S，(16)磷酸烯醇式丙酮酸敏感多肽的I253H、Q254L、R255L、G256F，和光学活性多肽的1N，(17)磷酸烯醇式丙酮酸敏感多肽的I253D、Q254L、R255L、G256F，和光学活性多肽的1S，(18)磷酸烯醇式丙酮酸敏感多肽的Q254L、R255L、G256F，和光学活性多肽的1S，(19)磷酸烯醇式丙酮酸敏感多肽的I253P、Q254L、R255L、G256F，和光学活性多肽的1T，(20)磷酸烯醇式丙酮酸敏感多肽的Q254L、R255L、G256F，和光学活性多肽的1T，(21)磷酸烯醇式丙酮酸敏感多肽的I253L、Q254R、R255L、G256F，(22)磷酸烯醇式丙酮酸敏感多肽的I253R、Q254L、R255L、G256F，和光学活性多肽的1S，(23)磷酸烯醇式丙酮酸敏感多肽的I253T、Q254L、R255F、G256Y，和光学活性多肽的1T，(24)磷酸烯醇式丙酮酸敏感多肽的I253Q、Q254P、R255F、G256Y，(25)磷酸烯醇式丙酮酸敏感多肽的I253Q、Q254M、R255F、G256Y，和光学活性多肽的1P，(26)磷酸烯醇式丙酮酸敏感多肽的I253T、Q254P、R255F、G256Y，和光学活性多肽的1A，(27)磷酸烯醇式丙酮酸敏感多肽的Q254L、R255F、G256F，和光学活性多肽的1N，(28)磷酸烯醇式丙酮酸敏感多肽的I253S、Q254K、R255F、G256F，和光学活性多肽的1E，(29)磷酸烯醇式丙酮酸敏感多肽的Q254P、R255F、G256F，和光学活性多肽的246C，(30)磷酸烯醇式丙酮酸敏感多肽的I253E、Q254V、R255F、G256F，和光学活性多肽的1A、246C，(31)磷酸烯醇式 丙酮酸敏感多肽的Q254L、R255F、G256F，和光学活性多肽的1G、246C，(32)磷酸烯醇式丙酮酸敏感多肽的I253T、Q254L、R255F、G256F，和光学活性多肽的1S、246C，(33)磷酸烯醇式丙酮酸敏感多肽的Q254L、R255F、G256F，和光学活性多肽的1T、246C，(34)磷酸烯醇式丙酮酸敏感多肽的R21K，(35)磷酸烯醇式丙酮酸敏感多肽的V54K，(36)磷酸烯醇式丙酮酸敏感多肽的V54Q，(37)磷酸烯醇式丙酮酸敏感多肽的V54G，(38)磷酸烯醇式丙酮酸敏感多肽的R55S，(39)磷酸烯醇式丙酮酸敏感多肽的V57M，(40)磷酸烯醇式丙酮酸敏感多肽的A58G，(41)磷酸烯醇式丙酮酸敏感多肽的I60W，(42)磷酸烯醇式丙酮酸敏感多肽的N59D，(43)磷酸烯醇式丙酮酸敏感多肽的N59H，(44)磷酸烯醇式丙酮酸敏感多肽的N59E，(45)磷酸烯醇式丙酮酸敏感多肽的R212I，(46)磷酸烯醇式丙酮酸敏感多肽的R212E，(47)磷酸烯醇式丙酮酸敏感多肽的R212M，(48)磷酸烯醇式丙酮酸敏感多肽的G213T，(49)磷酸烯醇式丙酮酸敏感多肽的K215E，(50)磷酸烯醇式丙酮酸敏感多肽的K215H，(51)磷酸烯醇式丙酮酸敏感多肽的K215D，(52)磷酸烯醇式丙酮酸敏感多肽的S216D，(53)磷酸烯醇式丙酮酸敏感多肽的S216L，(54)磷酸烯醇式丙酮酸敏感多肽的S216Y，(55)磷酸烯醇式丙酮酸敏感多肽的S216I，(56)磷酸烯醇式丙酮酸敏感多肽的S216M，(57)磷酸烯醇式丙酮酸敏感多肽的S216F，(58)磷酸烯醇式丙酮酸敏感多肽的S216Q，(59)磷酸烯醇式丙酮酸敏感多肽的L320Y，(60)磷酸烯醇式丙酮酸敏感多肽的L320N，(61)磷酸烯醇式丙酮酸敏感多肽的L320F，(62)磷酸烯醇式丙酮酸敏感多肽的R72A，(63)磷酸烯醇式丙酮酸敏感多肽的V54G、R72A，(64)磷酸烯醇式丙酮酸敏感多肽的V54K、I253G、Q254L、R255L、G256F，和光学活性多肽的1P，(65)磷酸烯醇式丙酮酸敏感多肽的V54Q、I253G、Q254L、R255L、G256F，和光学活性多肽的1P，(66)磷酸烯醇式丙酮酸敏感多肽的N59H、I253G、Q254L、R255L、G256F，和光学活性多肽的1P，(67)磷酸烯醇式丙酮酸敏感多肽的V54K、I253K、Q254L、R255L、G256F，和光学活性多肽的1S，(68)磷酸烯醇式丙酮酸敏感多肽的V54Q、I253K、Q254L、R255L、G256F，和光学活性多肽的1S，(69)磷酸烯醇式丙酮酸敏感多肽的N59H、I253K、Q254L、R255L、G256F，和光学活性多肽的1S。
在一个或多个实施方案中，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2-9所示(优选SEQ ID NO：2、6、7、9)，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/256位点，并且，所述光学探针具有如下所示的突变：(1)磷酸烯醇式丙酮酸敏感多肽的I253D、 Q254L、G256V、G257L，和光学活性多肽的1T、246F，(2)磷酸烯醇式丙酮酸敏感多肽的I253S、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(3)磷酸烯醇式丙酮酸敏感多肽的I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(4)磷酸烯醇式丙酮酸敏感多肽的V54Q、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(5)磷酸烯醇式丙酮酸敏感多肽的N59D、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(6)磷酸烯醇式丙酮酸敏感多肽的R212M、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(7)磷酸烯醇式丙酮酸敏感多肽的G213T、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(8)磷酸烯醇式丙酮酸敏感多肽的K215E、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(9)磷酸烯醇式丙酮酸敏感多肽的L320F、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(10)磷酸烯醇式丙酮酸敏感多肽的S216L、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F，(11)磷酸烯醇式丙酮酸敏感多肽的S216Y、I253Q、Q254L、G256V、G257L，和光学活性多肽的1T、246F。
本发明还提供融合多肽，包含本文任一实施方案所述的光学探针和其它多肽。在一些实施方式中，其他多肽位于所述光学探针的N端和/或C端。在一些实施方式中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。
本发明还提供核酸分子，其包含：(a)本文任一实施方案所述的蛋白变体、光学探针或融合多肽的编码序列，或(b)(a)的互补序列，或(c)(a)或(b)的片段。所述片段是引物。
本发明还涉及上述核酸分子的变体，包括编码本发明蛋白变体、光学探针或融合多肽的片段、类似物、衍生物、可溶性片段和变体的核酸序列或其互补序列。
本发明还提供包含本文所述核酸分子的核酸构建物。该核酸序列编码本发明任一实施方案所述的蛋白变体、光学探针或融合多肽。
在一个或多个实施方案中，所述核酸构建物是克隆载体、表达载体或重组载体。
在一个或多个实施方案中，所述核酸分子与表达控制序列操作性连接。
在一些实施方案中，表达载体选自原核表达载体、真核表达载体和病毒载体。
本发明另一方面还提供一种宿主细胞，所述宿主细胞：(1)表达本发明任一实施方案所述的光学探针或融合多肽；(2)包含本发明任一实施方案所述的核酸分子；或(3)包含本发明任一实施方案所述的核酸构建物。所述宿主细胞优选大肠杆菌。
本发明另一方面还提供磷酸烯醇式丙酮酸检测试剂盒，包括本文所述光学探针或融合多肽或多核苷酸或如本文所述方法制备的光学探针。
在一个或多个实施方案中，所述试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、磷酸烯醇式丙酮酸标准品。
本发明另一方面提供制备本文所述光学探针的方法，包括：提供表达本文所述光学探针或融合多肽的宿主细胞，在所述细胞表达的条件下培养所述宿主细胞，和分离光学探针或融合多肽。
在一个或多个实施方案中，所述方法包括包括以下步骤：1)将编码本文所述光学探针或融合多肽的核酸分子纳入表达载体；2)将表达载体转移到宿主细胞中；2)在适合所述表达载体表达的条件下培养所述宿主细胞，3)分离所述光学探针或融合多肽。
本发明另一方面还提供检测样品中磷酸烯醇式丙酮酸的方法，包括：使本文所述光学探针或融合多肽或宿主细胞与样品接触，和检测光学活性多肽的变化。所述检测可以在体内、体外、亚细胞或原位进行。所述样品例如血液。
本文另一方面还提供定量样品中磷酸烯醇式丙酮酸的方法，包括：使本文所述光学探针或融合多肽或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化定量样品中的磷酸烯醇式丙酮酸。
本发明另一方面还提供筛选化合物(例如药物)的方法，包括：在含磷酸烯醇式丙酮酸的体系中使本文所述光学探针或融合多肽或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物。所述方法可以高通量地筛选化合物。
在一个或多个实施方案中，在含磷酸烯醇式丙酮酸的体系中使本文所述宿主细胞与候选化合物接触，并且光学活性多肽的光学变化指示所述候选化合物是否能调节细胞对磷酸烯醇式丙酮酸的摄取。
本发明另一方面还提供对所述磷酸烯醇式丙酮酸进行细胞内和/或外定位的方法，包括：将含磷酸烯醇式丙酮酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化。
在一个或多个实施方案中，所述体系是溶液体系、细胞体系、亚细胞体系。
本发明另一方面还提供本文所述磷酸烯醇式丙酮酸光学探针或融合多肽或宿主细胞在检测样品中的磷酸烯醇式丙酮酸、筛选化合物或磷酸烯醇式丙酮酸细胞内/外定位中的应用。在一个或多个实施方案中，所述定位是实时定位。
本发明的有益效果：本发明提供的磷酸烯醇式丙酮酸光学探针易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、高通量、定量检测磷酸烯醇式丙酮酸，省去了耗时的处理样品步骤。实验效果表明本申请所提供的磷酸烯醇式丙酮酸光学探针对磷酸烯醇式丙酮酸的最高响应达到对照的5倍以上，并且可以在细胞浆、线粒体、细胞核、内质网、溶酶体和高尔基体等亚细胞结构中对细胞进行定位、定性、定量检测，并且可以进行高通量的化合物筛选以及血液中磷酸烯醇式丙酮酸定量检测。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为示例性磷酸烯醇式丙酮酸光学探针的SDS-PAGE图；
图2为示例性磷酸烯醇式丙酮酸光学探针的荧光光谱性质图；
图3为示例性磷酸烯醇式丙酮酸光学探针对不同浓度磷酸烯醇式丙酮酸的滴定曲线；
图4为示例性磷酸烯醇式丙酮酸光学探针对其余三种底物和糖代谢中间物及相似物的特异性检测汇总；
图5为示例性磷酸烯醇式丙酮酸光学探针在哺乳动物细胞中的亚细胞器定位照片；
图6为对示例性磷酸烯醇式丙酮酸光学探针在哺乳动物细胞中对胞浆中的磷酸烯醇式丙酮酸浓度进行动态监测的示意图；
图7为示例性磷酸烯醇式丙酮酸光学探针在活细胞水平进行高通量化合物筛选的点图；
图8为示例性磷酸烯醇式丙酮酸光学探针对小鼠和人血液中的磷酸烯醇式丙酮酸进行定量的柱状图。
具体实施方式
在给出数值或范围时，本文所用术语“约”指该数值或范围在给定数值或范围的20％以内、10％以内和5％以内。
本文所用术语“包含”、“包括”和其等同形式包括“含有”以及“由......组成”的含义，例如“包含”X的组合物可仅由X组成或可含有其它物质，例如X+Y。
本文所用术语“磷酸烯醇式丙酮酸敏感多肽”指对磷酸烯醇式丙酮酸产生响应的多肽，所述响应包括与敏感多肽的相互作用相关的多肽的化学，生物学，电学或生理学参数的任何响应。响应包括小的变化，例如，多肽的氨基酸或肽片段的方向的变化以及例如多肽的一级，二级或三级结构的变化，包括例如质子化，电化学势和/或构象的变化。“构象”是分子中包含侧基的分子的一级，二级和三级结构的三维排列；当分子的三维结构发生变化时，构象发生变化。构象变化的实例包括从α-螺旋转变为β-折叠或从β-折叠转变为α-螺旋。可以理解的是，只要荧光蛋白部分的荧光被改变，可检测到的改变不需要是构象改变。本文所述磷酸烯醇式丙酮酸敏感多肽还可包括其功能变体。磷酸烯醇式丙酮酸敏感多肽的功能变体包括但不限于可以与磷酸烯醇式丙酮酸相互作用从而发生与亲本磷酸烯醇式丙酮酸敏感多肽相同或相似变化的变体。
本发明所述磷酸烯醇式丙酮酸敏感多肽包括但不限于源自极端嗜热菌Thermus thermophilus的磷酸果糖激酶TtPFK或与其有90％以上同源性的变体。磷酸烯醇式丙酮酸结合蛋白可以感应磷酸烯醇式丙酮酸浓度的变化，在磷酸烯醇式丙酮酸浓度动态变化的过程中磷酸烯醇式丙酮酸结合蛋白的空间构象也会发生改变。TtPFK的截短变体亦可用于本发明。
本文所用术语“光学探针”是指与光学活性多肽融合的磷酸烯醇式丙酮酸敏感多肽。发明人发现，磷酸烯醇式丙酮酸敏感多肽例如磷酸烯醇式丙酮酸结合蛋白专一性地对生理浓度的磷酸烯醇式丙酮酸结合后所产生的构象变化会引起光学活性多肽(例如荧光蛋白)的构象变化，进而导致光学活性多肽的光学性质发生改变。借助不同磷酸烯醇式丙酮酸浓度下测定的荧光蛋白的荧光绘制标准曲线，可以检测并分析磷酸烯醇式丙酮酸的存在和/或水平。当描述本发明光学探针时(例如描述插入位点或突变位点时)，提及氨基酸残基编号均参考SEQ ID NO：1。
在本发明的光学探针中，光学活性多肽(例如荧光蛋白)可操作地插入磷酸烯醇式丙酮酸敏感多肽中。基于蛋白质的“光学活性多肽”是具有发射荧光能力的多肽。荧光是光学活性多肽的一种光学性质，其可用作检测本发明的光学探针的响应 性的手段。如本文所用，术语“荧光性质”是指适当激发波长下的摩尔消光系数，荧光量子效率，激发光谱或发射光谱的形状，激发波长最大值和发射波长最大值，两个不同波长激发的振幅，两个不同波长的发射振幅比，激发态寿命或荧光各向异性。活性和无活性状态之间的这些性质中的任何一个的可测量的差异足以用于本发明的荧光蛋白底物在活性测定中的效用。可测量的差异可通过确定任何定量荧光性质的量来确定，例如，特定波长处的荧光量或荧光在发射光谱上的积分。优选地，选择蛋白质底物以具有在未激活和活化的构象状态下容易区分的荧光特性。本文所述光学活性多肽还可包括其功能变体。光学活性多肽的功能变体包括但不限于可以发生与亲本光学活性多肽相同或相似荧光性质变化的变体。
本文所用术语“荧光蛋白”指在激发光照射下发出荧光的蛋白质。荧光蛋白作为生物科学领域的基础检测手段，例如生物技术领域常用的绿色荧光蛋白GFP及由该蛋白突变衍生出的环状重排的蓝色荧光蛋白(cpBFP)、环状重排的绿色荧光蛋白(cpGFP)、环状重排的黄色荧光蛋白(cpYFP)等；还有本技术领域常用的红色荧光蛋白RFP，及由该蛋白衍生出来的环状重排的蛋白，如cpmApple，cpmOrange，cpmKate等。示例性地，cpYFP如SEQ ID NO：2所示，cpmOrange如SEQ ID NO：3所示，cpmKate如SEQ ID NO：4或8所示，mCherry如SEQ ID NO：5所示，cpGFP如SEQ ID NO：6所示，cpBFP如SEQ ID NO：7所示，cpmApple如SEQ ID NO：9所示。
光学探针中的荧光蛋白也包括具有突变的功能变体，包括但不限于在第1位和/或对应于SEQ ID NO：2的第246位氨基酸具有突变的荧光蛋白。第1位的突变优选突变为S、G、N、T、P、A、E或H。对应于SEQ ID NO：2的第246位的突变优选突变为F、C、W或H。在一些实施方案中，荧光蛋白的功能变体具有SEQ ID NO：2-9中任一所示的序列并在对应于SEQ ID NO：2的第1位氨基酸和第246位氨基酸处具有选自以下任一组所示的突变：1S和246F；1G和246F；1H和246F；1A和246C；1G和246C；1S和246C；1T和246C；1T和246F。1S表示第1位氨基酸突变为S，246F表示第246位氨基酸突变为F，以此类推。
在本发明的光学探针中，光学活性多肽以N-C方向位于N-C方向的磷酸烯醇式丙酮酸敏感多肽的残基68-71，168-173和/或253-257中或置换其中的残基，编号对应于磷酸烯醇式丙酮酸敏感多肽的全长。本文中，如果以“X/Y”形式表示的位点中的两个数字是连续的整数，则表示光学活性多肽位于该数字所述的氨基酸之间。例如插入位点254/255表示光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的氨基酸254与255之间。如果以“X/Y”形式表示的位点中的两个数字不是连续的整数，则 表示光学活性多肽置换该数字所示氨基酸之间的氨基酸。例如插入位点254/256表示光学活性多肽置换磷酸烯醇式丙酮酸敏感多肽的氨基酸255。在示例性实施方式中，SEQ ID NO：2、6、7或9所示的光学活性多肽位于SEQ ID NO：1所示的磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170，169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和/或256/257。
提到某多肽或蛋白时，本发明所用术语“变体”或“突变体”包括具有所述多肽或蛋白相同功能、但序列不同的变体。多肽或蛋白的变体可包括：同源序列、保守性变体、等位变体、天然突变体、诱导突变体。这些变体包括但并不限于：在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个，较佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸，以及在其羧基末端和/或氨基末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸获得的序列。这些变体还可包含与所述多肽或蛋白的序列相同性为至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的多肽或蛋白。不希望受理论限制，氨基酸残基发生改变而不改变多肽或蛋白质的总体构型和功能，即功能保守突变。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变多肽或蛋白的功能。在本领域中，性能相似的氨基酸往往指具有相似侧链的氨基酸家族，在本领域已有明确定义。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、精氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如，在氨基末端和/或羧基末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。对于许多常见已知非遗传性编码氨基酸的保守氨基酸取代本领域已知。其他非编码氨基酸的保守取代可基于其物理性质与遗传上编码的氨基酸的性质的比较来确定。
“接头”或“连接区”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核苷酸序列。示例性地，本发明中磷酸烯醇式丙酮酸敏感多肽与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-3个，羧基端的氨基酸数目选择的是0-2个；当 重组光学探针作为基本单元与功能蛋白连接时，可以融合在重组光学探针的氨基酸或羧基端。接头序列可为一个或多个柔性氨基酸组成的短肽链，如Y。
发明人发现，在选自以下的位点具有突变的磷酸烯醇式丙酮酸结合蛋白变体表现出与磷酸烯醇式丙酮酸不同的结合活性：SEQ ID NO：1的I253，Q254，R255，G256，G257，R21，V54，R55，V57，A58，N59，I60，R72，R212，G213，K215，S216，L320。所述氨基酸突变包括氨基酸的修饰、取代或缺失。
本发明提供具有这些突变的磷酸烯醇式丙酮酸结合蛋白变体以及包含此类磷酸烯醇式丙酮酸结合蛋白变体作为磷酸烯醇式丙酮酸敏感多肽的光学探针。因此，在一个或多个实施方案中，光学探针中的磷酸烯醇式丙酮酸敏感多肽是本文任一实施方案所述的磷酸烯醇式丙酮酸结合蛋白变体，光学探针中的荧光蛋白如SEQ ID NO：2-9或其功能变体所示。
在一些具体实施方案中，光学探针中的磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/255或254/256位点，光学探针的突变如表5和表6中任一行所示。
在两种或多种多肽或核酸分子序列中，术语“相同性”或“相同性百分数”指在比较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对和目测检查来比较和比对最大对应性时，两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如，60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别可参见Altschul等(1977)Nucleic Acids Res.25：3389和Altschul等(1990)J.Mol.Biol.215：403。
本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基，而这并不影响目的多肽或蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如磷酸烯醇式丙酮酸结合蛋白或荧光蛋白)相同的生物学功能或活性的蛋 白。本发明的多肽或蛋白(例如磷酸烯醇式丙酮酸结合蛋白或荧光蛋白)的功能变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白，或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物，例如聚乙二醇)融合所形成的蛋白，或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如分泌序列或用来纯化此蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些功能变体、衍生物和类似物属于本领域熟练技术人员公知的范围。所述类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的磷酸烯醇式丙酮酸敏感多肽并不限于上述列举的代表性蛋白、变体、衍生物和类似物。修饰(通常不改变一级结构)形式包括：体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的蛋白。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。
本发明融合多肽包含本文所述光学探针和其它多肽。在一些实施方案中，本文所述光学探针还包含与之融合的其它多肽。本文所述其他多肽不影响光学探针的性质。其他多肽可位于所述光学探针的N端和/或C端。在一些实施方案中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。本文所述融合多肽中的光学探针和其它多肽之间可具有接头。
本文所述亚细胞器包括细胞浆、线粒体、细胞核、内质网、细胞膜、高尔基体、溶酶体和过氧化物酶体等。在一些实施方案中，用于纯化的标签或者用于免疫印迹的标签包括6组氨酸(6*His)、谷胱甘肽硫转移酶(GST)、Flag。
本发明包含编码本发明所述磷酸烯醇式丙酮酸敏感多肽或光学探针的核酸分子。本发明所用术语“核酸”或“核苷酸”或“多核苷酸”或“核酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。提到核酸时，本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并变体、取代变体、缺失变体和插入变体。如本领域所知的，等位变体是一个核酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变 其编码的蛋白的功能。本发明核酸可包含与所述核酸序列的序列相同性为至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)。
本发明光学探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、人工合成法或重组法获得。本领域知晓常规PCR、合成法、重组法的步骤和所用试剂。此外，可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。
本发明也涉及核酸构建物，该核酸构建物含有本文所述的多核苷酸，以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式被操作以保证所述多肽或蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体、表达载体、或同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。
典型的表达载体包含可用于调节期望核酸序列表达的表达控制序列，与本发明所述的核酸序列或其互补序列操作性连接。本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件，可以是复制起点、启动子、标记基因或翻译控制元件，包括增强子、操纵子、终止子、核糖体结合位点等，表达控制序列的选择取决于所用的宿主细胞。在重组表达载体中，“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。在一个实施方案中，表达载体可采用市售的pCDF载体，无其他特殊要求。示例性地，采用BamHI和EcoRI分别对编码所述光学探针的核苷酸序列和表达载体 进行双酶切，然后将二者的酶切产物连接得到重组表达载体。本发明对酶切和连接的具体步骤和参数没有特殊限定，采用本领域常规的步骤和参数即可。
在获得重组表达载体后，将该载体转化到宿主细胞中，以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。本发明所述的宿主细胞是指能够接收和容纳重组DNA分子的细胞，是重组基因扩增的场所，理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞，具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞，此类细胞已为本领域熟知并常用。具体的可为大肠杆菌，链霉菌属，鼠伤寒沙门氏菌的细菌细胞，真菌细胞如酵母，植物细胞，果蝇S2或Sf9的昆虫细胞，CHO、COS、HEK293、HeLa细胞、或Bowes黑素瘤细胞的动物细胞等。在本发明实施例中所用的示例性宿主细胞为大肠杆菌BL21-DE3菌株。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明所述的转移到宿主细胞的方法为本领域常规的方法，包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时，所述方法优选的为CaCl2法或MgCl2法处理，所用的步骤为本领域公知。当宿主细胞是真核细胞时，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
本发明在将表达载体转入宿主细胞后，对转入表达载体的宿主细胞进行扩增表达培养，分离得到磷酸烯醇式丙酮酸光学探针。所述宿主细胞扩增表达培养采用常规的方法即可。根据所用的宿主细胞种类，培养中所用的培养基可以是各种常规培养基。在适于宿主细胞生长的条件下进行培养。
在本发明中，光学探针在细胞内、细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。本发明对分离所述磷酸烯醇式丙酮酸荧光蛋白的方法没有特殊限定，采用本领域常规的融合蛋白的分离方法即可。这些方法是本领域技术人员所熟知的，包括但并不限于：常规的复性处理、盐析方法、离心、渗透破菌、超声处理、超离心、分子筛层析、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。在一个实施方案中，利用His标签的亲和层析法进行光学探针的分离。
本发明还提供了所述磷酸烯醇式丙酮酸光学探针在磷酸烯醇式丙酮酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面，所述的磷酸烯醇式丙酮酸 光学探针优选与细胞不同部位的信号肽连接，转入到细胞中，通过检测细胞中荧光信号的强弱，进行磷酸烯醇式丙酮酸的实时定位；通过磷酸烯醇式丙酮酸标准滴加曲线结合荧光信号的变化进行相应磷酸烯醇式丙酮酸的定量检测。荧光信号的变化通过例如标准化后的荧光信号比值展示，在涉及cpYFP的实施方案中，所述比值是样品的485纳米荧光信号与420纳米荧光信号之比与对照的相应之比的比值。本发明所述的磷酸烯醇式丙酮酸标准滴加曲线是根据磷酸烯醇式丙酮酸光学探针在不同浓度磷酸烯醇式丙酮酸的情况下的荧光信号绘制而成。本发明所述磷酸烯醇式丙酮酸光学探针直接转入细胞中，在磷酸烯醇式丙酮酸实时定位和定量检测过程中，不需要耗时的样品处理过程，更加准确。本发明磷酸烯醇式丙酮酸光学探针在进行高通量化合物筛选时，将不同的化合物添加到细胞培养液中，测定磷酸烯醇式丙酮酸含量的变化，从而筛选出对磷酸烯醇式丙酮酸含量变化有影响的化合物。在本发明中所述的磷酸烯醇式丙酮酸光学探针在磷酸烯醇式丙酮酸实时定位、定量检测以及高通量化合物筛选中的应用，均是非诊断和治疗目的，不涉及疾病的诊断和治疗。
本发明还提供包括本文所述光学探针、核酸分子、核酸构建物和/或细胞的检测试剂盒。所述试剂盒还包含检测磷酸烯醇式丙酮酸所需的其他试剂。所述其他试剂本领域周知，例如缓冲液、细胞培养基、磷酸烯醇式丙酮酸标品。示例性缓冲液例如100mM HEPES和100mM NaCl，pH 7.4。
部分具体实施方案
项目1、一种磷酸烯醇式丙酮酸结合蛋白变体，其：
(a)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：I253、Q254、R255、G256、G257、R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，所述突变包括氨基酸的修饰、取代或缺失，
(b)是与(a)的序列具有至少70％序列相同性并具有(1)所述突变并保留对磷酸烯醇式丙酮酸结合能力的序列，
优选地，(a)中所述突变包括选自以下任一组或多组的位点处的突变：
项目2、一种磷酸烯醇式丙酮酸光学探针，包含磷酸烯醇式丙酮酸敏感多肽和光学活性多肽，其中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的序列内，并且
磷酸烯醇式丙酮酸敏感多肽具有：
(1)SEQ ID NO：1所示的序列，或与它们有至少70％序列相同性并保留对磷酸烯醇式丙酮酸结合活性的序列，
(2)SEQ ID NO：1所示序列的功能变体，该功能变体在与光学活性多肽连接处的5个氨基酸内具有突变，和/或，在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，或
(3)与(2)所述序列具有至少70％序列相同性并具有(2)所述突变并保留对磷酸烯醇式丙酮酸敏感性的序列，
优选地，(2)是项目1中所述的磷酸烯醇式丙酮酸结合蛋白变体的序列。
项目3.如项目2所述的光学探针，其中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的残基68-71，168-173和/或253-257中或置换其中的残基。
项目4.如项目2所述的光学探针，其中，
光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170，169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和/或256/257。
进一步优选地，
光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：69/71，170/171，171/172，254/255，254/256和255/257。
进一步优选地，其特征在于，光学活性多肽是荧光蛋白或其功能变体，
优选地，
荧光蛋白具有SEQ ID NO：2-9中任一所示的序列，
荧光蛋白的功能变体在与光学活性多肽连接处的3个氨基酸内具有突变；优选地，荧光蛋白的功能变体在对应于SEQ ID NO：2的第1位氨基酸处具有突变，所述突变优选选自Y1S、Y1G、Y1N、Y1T、Y1P、Y1A、Y1E、Y1H，
荧光蛋白的功能变体在对应于SEQ ID NO：2的第246位氨基酸处具有突变，所述突变优选选自N246F、N246C、N246W或N246H。
进一步优选地，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，并且具有以下的一个或多个突变：I253V，I253N，I253F，I253C，I253E，I253G，I253S，I253K，I253H，I253D，I253P，I253L，I253R，I253T，I253Q，Q254L，Q254V，Q254A，Q254R，Q254P，Q254M，Q254K，Q254I，R255L， R255I，R255F，G256H，G256F，G256Y，G256V，G257L，R21K，V54G，V54K，V54Q，R55S，V57M，A58G，N59D，N59H，N59E，I60W，R72A，R212I，R212E，R212M，G213T，K215H，K215D，K215E，S216D，S216L，S216Y，S216I，S216M，S216F，S216Q，L320Y，L320N和/或L320F，光学活性多肽如SEQ ID NO：2-9所示或是其具有以下的一个或多个突变的变体：Y1S、Y1G、Y1N、Y1T、Y1P、Y1A、Y1E、Y1H、N246F、N246C、N246W，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/255或254/256位点。
项目5、一种核酸分子，其包含：
(a)项目2-4中任一项所述的光学探针的编码序列，
(b)(a)的互补序列。
项目6、包含项目5所述的核酸分子的核酸构建物，
优选地，所述核酸构建物是克隆载体、表达载体或重组载体。
项目7、一种宿主细胞，所述宿主细胞：
(1)表达项目2-4中任一项所述的光学探针；
(2)包含项目5所述的核酸分子；或
(3)包含项目6所述的核酸构建物。
项目8.一种检测试剂盒，其包含：
(1)项目2-4中任一项所述的光学探针，
(2)项目5所述的核酸序列，
(3)项目6所述的核酸构建物，或
(4)项目7所述的宿主细胞，
所述检测试剂盒任选还包含利用光学探针检测磷酸烯醇式丙酮酸所需的其他试剂，
优选地，所述检测试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、磷酸烯醇式丙酮酸标准品。
项目9、制备项目2-4中任一项所述的光学探针的方法，包括：提供项目7所述的宿主细胞，在所述光学探针表达的条件下培养所述宿主细胞，和分离光学探针。
项目10、项目2-4中任一项所述的光学探针、项目5所述的核酸序列、项目6所述的核酸构建物或项目7所述的宿主细胞在检测样品中的磷酸烯醇式丙酮酸、筛选化合物、磷酸烯醇式丙酮酸细胞内/外定位中的应用，
优选地，
检测样品中的磷酸烯醇式丙酮酸包括步骤：使所述光学探针或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化检测样品中的磷酸烯醇式丙酮酸，
所述筛选化合物包括步骤：在含磷酸烯醇式丙酮酸的体系中使所述光学探针或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物；优选地，所述筛选化合物包括步骤：在含磷酸烯醇式丙酮酸的体系中使所述宿主细胞与候选化合物接触，并且光学活性多肽的光学变化指示所述候选化合物是否调节细胞对磷酸烯醇式丙酮酸的摄取，
所述磷酸烯醇式丙酮酸细胞内/外定位包括步骤：将含磷酸烯醇式丙酮酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化，
更优选地，所述体系是溶液体系、细胞体系或亚细胞体系。
在本文中，浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解，使用这种范围形式只是为了方便和简洁，应该被弹性地解读为包括范围上下限所明确提及的数值，还应包括该范围内包括的所有单个数值或子范围。
实施例
下面结合实施例对本发明提供的磷酸烯醇式丙酮酸光学探针进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
I.实验材料和试剂
实施例中主要采用常规的基因工程分子生物学克隆方法和细胞培养以及成像方法等，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实验参考手册》，J.萨姆布鲁克，D.W.拉塞尔著，黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)；费雷谢尼等的《动物细胞培养：基本技术指南》(第五版)，章静波，徐存拴等译；J.S.博尼费斯农，M.达索等的《精编细胞生物学实验指南》，章静波等译。
实施例中所用的基于pCDF-cpYFP，pCDF-磷酸烯醇式丙酮酸结合蛋白质粒由华东理工大学蛋白质实验室构建，pCDF质粒载体购自Invitrogen公司。所有用于PCR的引物均由上海捷瑞生物工程技术有限公司和华大基因合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由华大基因公司和杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物，pfu DNA聚合酶购自天根生化科技(北京)有限公司，primeSTAR DNA聚合酶购自TaKaRa公司， 三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTPs。BamHI、BglII、HindIII、NdeI、XhoI、EcoRI、SpeI等限制性内切酶、T4连接酶、T4磷酸化酶(T4 PNK)购自Fermentas公司，购买时附带有相对应的缓冲液等。转染试剂Lip2000Kit购于Invitrogen公司。磷酸烯醇式丙酮酸等化合物均购自Sigma公司。除非特别声明，无机盐类等化学试剂均购自Sigma-Aldrich公司。HEPES盐，氨苄青霉素(Amp)和嘌呤霉素购自Ameresco公司。96孔检测黑板、384孔荧光检测黑板购自Grenier公司。
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大)，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株Mach1购自Invitrogen公司。镍柱亲和层析柱和脱盐柱填料均来自GE healthcare公司。
实施例中用到的主要仪器包括：Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，超声破碎仪(宁波新芝公司)，核酸电泳仪(申能博彩公司)，荧光分光光度计(美国Varian公司)，CO2恒温细胞培养箱(SANYO)，倒置荧光显微镜(日本尼康公司)。
II.分子生物学方法和细胞实验方法
II.1聚合酶链式反应(PCR)：
1.目的片段扩增PCR：
该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆。所述PCR扩增的反应体系如下：模板序列0.5-1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，10×pfu缓冲液5μL，pfu DNA聚合酶0.5μL，dNTP(10mM)1μL，灭菌超纯水(ddH2O)41.5-42μL，总体积50μL。PCR扩增程序如下：95℃变性2-10分钟，30轮循环(94-96℃持续30-45秒，50-65℃持续30-45秒，72℃持续一定时间(600bp/min))，72℃延伸10分钟。
2.长片段(＞2500bp)扩增PCR：
本发明中使用的长片段扩增，主要是反向PCR扩增载体，在下述实施例中用于获得定点突变的一种技术。在变异部位设计反向PCR引物，其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物就含有相应的突变位点。长片段扩增PCR反应体系如下：模板序列(10pg-1ng)1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，5×PrimerSTAR缓冲液10μL，PrimerSTAR DNA聚合酶0.5μL，dNTP(2.5mM)4μL，灭菌超纯水(ddH2O)33.5μL，总体积50μL。PCR扩增程序如下：95℃变性5分钟，30轮循环(98℃持续10秒，50-68℃持续5-15秒，72℃持 续一定时间(1000bp/min))，72℃延伸10分钟；或者95℃变性5分钟，30轮循环(98℃持续10秒，68℃持续一定时间(1000bp/min))，72℃延伸10分钟。
II.2核酸内切酶酶切反应：
对质粒载体进行双酶切的体系如下：质粒载体20μL(约1.5μg)，10×缓冲液5μL，限制性内切酶11-2μL，限制性内切酶21-2μL，用灭菌超纯水补至总体积50μL。反应条件37℃，1-7小时。
II.3 DNA片段5’端磷酸化反应
从微生物中抽提出的质粒或者基因组末端都含有磷酸基团，而PCR产物没有，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应。磷酸化反应体系如下：PCR产物片段DNA序列5-8μL，10×T4连接酶缓冲液1μL，T4多聚核苷酸激酶(T4 PNK)1μL，灭菌超纯水0-3μL，总体积10μL。反应条件37℃，30分钟-2小时后72℃灭活20分钟。
II.4目的片段和载体的连接反应
不同的片段和载体之间的连接方法有所差异，本发明中使用了三种连接方法
1.平末端短片段和线性化载体的平末端连接
该方法的原理是PCR获得的平末端产物在T4 PNK作用下对DNA片段的5’末端进行磷酸化反应后，与线性化的载体在PEG4000和T4 DNA连接酶的作用下连接获得重组质粒。同源重组连接体系如下：T4 PNK处理的DNA片段4μL，线性化载体片段4μL，PEG4000 1μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，总计10μL。反应条件22℃，30分钟。
2.含有粘性末端的DNA片段和含有粘性末端载体片段的连接
通过限制性内切酶切割的DNA片段通常会产生突出的粘性末端，因此可以和含有序列互补的粘性末端载体片段连接，形成重组质粒。连接反应体系如下：酶切后的PCR产物片段DNA 1-7μL，酶切后的质粒0.5-7μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，灭菌超纯水补至总体积10μL。反应条件16℃，4-8小时。
3.反向PCR引入定点突变后5’端磷酸化的DNA片段产物自身环化的连接反应
将5’端磷酸化的DNA片段通过自身环化连接反应将线性化载体的3’端和5’端连接反应得到重组质粒。自身环化连接反应体系如下：磷酸化反应体系10μL，T4连接酶(5U/μL)0.5μL，总体积10.5μL。反应条件16℃，4-16小时。
II.5感受态细胞的制备与转化
感受态细胞的制备：
1.挑取单菌落(如Mach1)接种于5mL LB培养基中，37℃摇床过夜。
2.取0.5-1mL过夜培养的菌液转种到50mL LB培养基中，37℃，220rpm培养3至5小时，直到OD600达到0.5。
3.冰浴预冷细胞2小时。
4. 4℃，4000rpm离心10分钟。
5.弃上清，用5mL预冷的缓冲液重悬细胞，待均匀后再加入重悬缓冲液至终体积为50mL。
6.冰浴45分钟。
7. 4℃ 4000rpm离心10分钟，用5mL冰预冷的储存缓冲液重悬细菌。
8.每个EP管中放100μL菌液，-80℃或液氮冻存。
重悬缓冲液：CaCl2(100mM)、MgCl2(70mM)、NaAc(40mM)
储存缓冲液：0.5mL DMSO、1.9mL 80％甘油、1mL 10×CaCl2(1M)、1mL 10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2O
感受态细胞的转化：
1.取100μL感受态细胞于冰浴上融化。
2.加入适当体积的连接产物，轻轻吹打混匀，冰浴30分钟。通常加入的连接产物的体积少于感受态细胞体积的1/10。
3.将菌液放入42℃水浴中热激90秒，迅速转移至冰浴中放置5分钟。
4.加入500μL LB，于37℃恒温摇床上200rpm培养1小时。
5.将菌液4000rpm离心3分钟，留200μL上清将菌体吹匀，均匀涂布于含适当抗生素的琼脂平板表面，平板于37℃恒温培养箱内倒置过夜。
II.6蛋白质的表达，纯化和荧光检测
1.将表达载体(例如以pCDF为基础的磷酸烯醇式丙酮酸光学探针表达载体)转化到BL21(DE3)细胞中，倒置培养过夜，从平板上挑取克隆到250ml锥形瓶中，置于37℃摇床，220rpm培养至OD＝0.4-0.8，加入1/1000(v/v)的IPTG(1M)，18℃诱导表达24-36小时。
2.诱导表达完成后，4000rpm，30分钟离心收菌，加入50mM的磷酸盐缓冲液重悬菌体沉淀，超声破碎至菌体澄清。9600rpm，4℃离心20分钟。
3.离心上清通过自装的镍柱亲和层析柱纯化获得蛋白，镍柱亲和层析后的蛋白再通过自装的脱盐柱获得溶解在100mM HEPES缓冲液(pH 7.4)中的蛋白。
4.纯化的蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mM HEPES，100mM NaCl，pH 7.4)稀释探针成终浓度为0.2-5μM的蛋白溶液。用测定缓冲液 (100mM HEPES，100mM NaCl，pH 7.4)将磷酸烯醇式丙酮酸配制成终浓度为50mM的储液。
5.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入磷酸烯醇式丙酮酸滴定，测定蛋白的420nm光激发后528nm发射和485nm光激发后528nm发射的荧光强度。对样品的荧光激发、发射测定利用多功能荧光酶标仪完成。
6.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入磷酸烯醇式丙酮酸，测定蛋白的吸收光谱和荧光光谱。对样品的吸收光谱和荧光光谱的测定通过分光光度计和荧光分光光度计完成。
II.7哺乳动物细胞的转染和荧光检测
1.将pCDNA3.1+为基础的磷酸烯醇式丙酮酸光学探针质粒通过转染试剂Lipofectamine2000(Invitrogen)转染到HEK293中，置于37℃，5％CO2的细胞培养箱中培养。待外源基因充分表达24～36h后进行荧光检测。
2.诱导表达完成后，将贴壁的HEK293细胞，用PBS冲洗三次，置于HBSS溶液中分别进行荧光显微镜和酶标仪检测。
实施例1：磷酸烯醇式丙酮酸结合蛋白质粒
通过PCR扩增极端嗜热菌基因中的TtPFK基因，PCR产物凝胶电泳后回收后用HindIII和XhoI酶切，同时对pCDF载体进行相应的双酶切。用T4 DNA连接酶连接后，用产物转化DH5α，转化的DH5α涂布于LB平板(链霉素100ug/mL)，置于37℃培养过夜。将生长DH5α转化子进行质粒抽提后，进行PCR鉴定。阳性质粒经过测序正确后进行后续的质粒构建。
实施例2：不同插入位点的cpYFP光学探针的表达和检测
本实施例中，以pCDF-TtPFK为基础根据磷酸烯醇式丙酮酸结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应pCDF-TtPFK-cpYFP质粒：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170，169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和256/257。仅作为示例性展示，254/255-TtPFK-cpYFP的氨基酸序列和核酸序列分别如SEQ ID NO：10和11所示。
利用PCR产生cpYFP的DNA片段，同时通过引物5’端引入cpYFP末端同源序列，PCR扩增产生pCDF磷酸烯醇式丙酮酸结合蛋白线性化载体，其5’和3’最末端分别带有和cpYFP两末端对应的完全一致的序列(15bp～20bp)。将线性化 的pCDF-TtPFK和cpYFP片段在Hieff Clone Enzyme的作用下发生同源重组。产物转化DH5α，转化的DH5α涂布于LB平板(链霉素100ug/mL)，置于37℃培养过夜。PCR鉴定的阳性克隆抽质粒后测序。由杰李测序公司完成测序。
经过测序正确后，将重组质粒转化到BL21(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在63Kda附近。该大小符合pCDF-TtPFK-cpYFP表达出的含His-tag纯化标签的TtPFK-cpYFP融合蛋白质的大小。结果如图1所示。
用表达TtPFK-cpYFP融合蛋白质的大肠杆菌的破碎上清进行磷酸烯醇式丙酮酸响应筛选，将含有1mM磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号除以无磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号。如表1所示，检测结果显示表达有TtPFK-cpYFP融合蛋白质的破碎上清对磷酸烯醇式丙酮酸响应超过1.3倍的光学探针有69/71，170/171，171/172，254/255，254/256和255/257位点实施插入的光学探针，其中对磷酸烯醇式丙酮酸的响应不受其余TtPFK结合底物影响的有254/255和254/256位点。
表1
实施例3：不同插入位点的cpGFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpGFP，构建磷酸烯醇式丙酮酸绿色荧光蛋白荧光探针。如表2所示，检测结果显示表达有TtPFK-cpGFP融合蛋白质的破碎上清对磷酸烯醇式丙酮酸响应超过1.3倍的光学探针有171/172，254/255和254/256位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针，其中对磷酸烯醇式丙酮酸的响应不受其余TtPFK结合底物影响的有254/255和254/256位点。
表2
实施例4：不同插入位点的cpBFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpBFP，构建磷酸烯醇式丙酮酸蓝色荧光蛋白荧光探针。如表3所示，检测结果显示表达有TtPFK-cpBFP融合蛋白质的破碎上清对磷酸烯醇式丙酮酸响应超过1.3倍的光学探针有170/171，171/172，254/255和254/256位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针，其中对磷酸烯醇式丙酮酸的响应不受其余TtPFK结合底物影响的有254/255和254/256位点。
表3
实施例5：不同插入位点的cpmApple光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpmApple，构建磷酸烯醇式丙酮酸红色荧光蛋白荧光探针。如表4所示，检测结果显示表达有TtPFK-cpmApple融合蛋白质的破碎上清对磷酸烯醇式丙酮酸响应超过1.3倍的光学探针有170/171，171/172，254/255和254/256位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针，其中对磷酸烯醇式丙酮酸的响应不受其余TtPFK结合底物影响的有254/255和254/256位点。
表4
实施例6：linker突变的cpYFP光学探针的表达和检测
对于实施例2中所得的对磷酸烯醇式丙酮酸响应超过1.3倍的且不受TtPFK其余结合底物影响的光学探针，即在TtPFK的254/255、254/256位点实施插入的2种光学探针基础上通过反向PCR线性化探针，引物中引入linker突变位点的序列，对得到的PCR产物在Hieff Clone Enzyme的作用下进行同源重组，建立突变库。仅作为示例性展示，254/255-TtPFK-I253K/Q254L/R255L/G256F-cpYFP-Y1S的氨基酸序列和核酸序列分别如SEQ ID NO：12和13所示。
将突变库重组质粒转化到BL21(DE3)中诱导表达，纯化探针蛋白进行磷酸烯醇式丙酮酸响应筛选，将含有2mM磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号除以无磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号。检测结果显示对磷酸烯醇式丙酮酸响应超过或等于2倍的光学探针如表5所示。
表5
实施例7：结合口袋突变的cpYFP光学探针的表达和检测
对于实施例2中所得的254/255-TtPFK-cpYFP和实施例6中所得的254/255-TtPFK-I253G/Q254L/R255L/G256F-cpYFP-Y1P，254/255-TtPFK-I253K/Q254L/R255L/G256F-cpYFP-Y1S和254/256-TtPFK-I253Q/Q254L/G256V/G257L-cpYFP-Y1T/N246F的光学探针，进行磷酸烯醇式丙酮酸结合口袋处的R21，R25，V54，R55，V57，A58，N59，I60，I61，R155，G186，R212，G213，K214，K215，S216和L320位氨基酸突变，ATP结合口袋处的R72， R163，R172和R246位氨基酸突变来调整磷酸烯醇式丙酮酸光学探针对磷酸烯醇式丙酮酸的亲和力和底物特异性。通过反向PCR线性化探针，引物中引入突变位点的序列，对得到的PCR产物进行同源重组，得到含有以上位点突变的质粒。将突变质粒转化到BL21(DE3)中诱导表达，纯化突变体探针蛋白进行磷酸烯醇式丙酮酸响应测试，将含有2mM次磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号除以无磷酸烯醇式丙酮酸的融合荧光蛋白质的检测信号。检测结果显示对磷酸烯醇式丙酮酸响应超过或等于2倍的光学探针如表6所示。
表6

实施例8.光学探针突变体的性能
示例性地，将纯化的实施例6和实施例7所述的2个磷酸烯醇式丙酮酸光学探针分别进行0mM和5mM磷酸烯醇式丙酮酸处理10分钟后，使用荧光分光光度计进行荧光谱的检测。
对激发光谱的测定：以370nm至510nm的激发范围和530nm的发射波长记录激发光谱，每5nm读取一次。结果显示，探针在约410和490nm处有两个激发峰，如图2所示。
对发射光谱的测定：固定激发波长分别为420nm和460nm，记录470-600nm和490-600nm的发射光谱，每5nm读取一次。结果如图2所示。
将纯化的实施例6和实施例7所述的81个磷酸烯醇式丙酮酸探针进行浓度梯度(0～10mM)的磷酸烯醇式丙酮酸检测。对纯化的探针处理10分钟后，检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图3所示，81个磷酸烯醇式丙酮酸光学探针的Kd(结合常数)分别为0.8、1.55、5.64、8.5、11.42、9.66、5.56、31.99、81.67、31.05、68.4、16.55、45、29.51、30.9、17.52、21.83、44.17、40.32、6.54、20.43、435.8、18.16、16.69、52.13、42.67、12.92、60.83、32.61、80.64、74.29、5.96、20.22、9.15、13.38、8.93、13.08、715.7、7.84、23.32、91.13、294.1、0.4492、0.36、0.4957、37.43、4.62、177.7、59.81、103.2、56.47、16.24、29.71、14.28、75.89、202.9、0.2544、0.2853、0.24、0.25、0.1598、0.6172、127.3、200.4、70.17、10.26、788.8、267.3、769.5、178.3、129.4、327.5、87.67、281.3、374、714.2、199.3、249、1773、1.393和2.365μM。
将81个磷酸烯醇式丙酮酸光学探针与3种TtPFK结合底物ATP、ADP和F6P进行反应性检测，同时将1个磷酸烯醇式丙酮酸光学探针与葡萄糖、乳酸、丙酮酸、F6P和FBP等糖代谢中间物进行反应性检测，结果表明其具有很好的特异性，如图4所示。
实施例9：光学探针的亚细胞器定位和光学探针在亚细胞器内的性能
本实施例中，使用不同的定位信号肽与光学探针254/255-TtPFK-I253K/Q254L/R255L/G256F-cpYFP-Y1S融合，将光学探针定位到不同的细胞器中。
用融合不同定位信号肽的光学探针质粒转染293细胞36小时后，使用PBS冲洗，置于HBSS溶液中使用倒置荧光显微镜进行FITC通道下进行荧光检测。结果如 图5所示。磷酸烯醇式丙酮酸光学探针通过与不同的特异定位信号肽融合能够定位到包括细胞浆、细胞外膜、细胞核、内质网、线粒体和核排阻等亚细胞器中。不同的亚细胞结构中都显示有荧光，并且荧光的分布和强度各不相同。
用胞浆表达的光学探针质粒转染293细胞36小时后，使用PBS冲洗，置于HBSS溶液中，检测30min时间段内420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图6所示，添加5mM Oxalate，继续检测30分钟。添加Oxalate的样品的485/420逐渐增加，最高可以达到初始值的3.5倍，而不添加Oxalate的对照组的485/420为0.437保持不变。
实施例10：在活细胞中基于光学探针进行高通量化合物筛选
本实施例中，我们使用胞浆表达的254/255-TtPFK-I253K/Q254L/R255L/G256F-cpYFP-Y1S的HeLa细胞进行高通量化合物筛选。
经转染的293细胞使用PBS冲洗，置于HBSS溶液中(无磷酸烯醇式丙酮酸)处理1小时，然后使用10μM的化合物处理1小时。各样品中分别滴加磷酸烯醇式丙酮酸。使用酶标仪记录420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值变化。以未用任何化合物处理的样品作为对照进行标准化。结果如图7所示。在使用的2000种化合物中，绝大部分的化合物对磷酸烯醇式丙酮酸进入细胞影响极小。有23种化合物能够提高细胞对磷酸烯醇式丙酮酸的摄取能力，另外有7种化合物能够明显降低细胞对磷酸烯醇式丙酮酸的摄取。
实施例11：光学探针定量检测血液中的磷酸烯醇式丙酮酸
在本实施中，使用纯化的Kd为0.24μM的254/255-TtPFK-S216I-cpYFP对小鼠和人的血液上清中的磷酸烯醇式丙酮酸进行分析。
将254/255-TtPFK-S216I-cpYFP与稀释的血液上清混合处理10分钟后，使用酶标仪检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值。结果如图8所示，小鼠血液中的磷酸烯醇式丙酮酸含量在91μM左右，人血液中的磷酸烯醇式丙酮酸含量在18μM左右。
由以上实施例可知，本发明提供的磷酸烯醇式丙酮酸光学探针，蛋白分子量相对较小且易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、定量检测磷酸烯醇式丙酮酸；并且能够进行高通量的化合物筛选。
其它实施方式
本说明书描述了许多实施方式。然而应理解，本领域技术人员通过阅读本说明书获知的不背离本发明的构思和范围的各种改进，也应包括在所附权利要求书的范围内。
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Claims (10)

  1. 一种磷酸烯醇式丙酮酸结合蛋白变体，其：
    (a)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：I253、Q254、R255、G256、G257、R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，所述突变包括氨基酸的修饰、取代或缺失，
    (b)是与(a)的序列具有至少70％序列相同性并具有(1)所述突变并保留对磷酸烯醇式丙酮酸结合能力的序列，
    优选地，(a)中所述突变包括选自以下任一组或多组的位点处的突变：
  2. 一种磷酸烯醇式丙酮酸光学探针，包含磷酸烯醇式丙酮酸敏感多肽和光学活性多肽，其中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的序列内，并且
    磷酸烯醇式丙酮酸敏感多肽具有：
    (1)SEQ ID NO：1所示的序列，或与它们有至少70％序列相同性并保留对磷酸烯醇式丙酮酸结合活性的序列，
    (2)SEQ ID NO：1所示序列的功能变体，该功能变体在与光学活性多肽连接处的5个氨基酸内具有突变，和/或，在选自以下的1个、2个、3个、4个、5个或更多个位点具有突变：R21、V54、R55、V57、A58、N59、I60、R72、R212、G213、K215、S216、L320，或
    (3)与(2)所述序列具有至少70％序列相同性并具有(2)所述突变并保留对磷酸烯醇式丙酮酸敏感性的序列，
    优选地，(2)是权利要求1中所述的磷酸烯醇式丙酮酸结合蛋白变体的序列。
  3. 如权利要求2所述的光学探针，其中，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的残基68-71，168-173和/或253-257中或置换其中的残基。
  4. 如权利要求2所述的光学探针，其中，
    光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：68/69，68/70，69/70，68/71，69/71，70/71，168/169，168/170， 169/170，168/171，169/171，170/171，168/172，169/172，170/172，171/172，168/173，169/173，170/173，171/173，172/173，253/254，253/255，254/255，253/256，254/256，255/256，253/257，254/257，255/257和/或256/257。
    进一步优选地，
    光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的选自下述位点中的任一个或多个：69/71，170/171，171/172，254/255，254/256和255/257。
    进一步优选地，其特征在于，光学活性多肽是荧光蛋白或其功能变体，
    优选地，
    荧光蛋白具有SEQ ID NO：2-9中任一所示的序列，
    荧光蛋白的功能变体在与光学活性多肽连接处的3个氨基酸内具有突变；优选地，荧光蛋白的功能变体在对应于SEQ ID NO：2的第1位氨基酸处具有突变，所述突变优选选自Y1S、Y1G、Y1N、Y1T、Y1P、Y1A、Y1E、Y1H，
    荧光蛋白的功能变体在对应于SEQ ID NO：2的第246位氨基酸处具有突变，所述突变优选选自N246F、N246C、N246W或N246H。
    进一步优选地，所述光学探针中，磷酸烯醇式丙酮酸敏感多肽如SEQ ID NO：1所示，并且具有以下的一个或多个突变：I253V，I253N，I253F，I253C，I253E，I253G，I253S，I253K，I253H，I253D，I253P，I253L，I253R，I253T，I253Q，Q254L，Q254V，Q254A，Q254R，Q254P，Q254M，Q254K，Q254I，R255L，R255I，R255F，G256H，G256F，G256Y，G256V，G257L，R21K，V54G，V54K，V54Q，R55S，V57M，A58G，N59D，N59H，N59E，I60W，R72A，R212I，R212E，R212M，G213T，K215H，K215D，K215E，S216D，S216L，S216Y，S216I，S216M，S216F，S216Q，L320Y，L320N和/或L320F，光学活性多肽如SEQ ID NO：2-9所示或是其具有以下的一个或多个突变的变体：Y1S、Y1G、Y1N、Y1T、Y1P、Y1A、Y1E、Y1H、N246F、N246C、N246W，光学活性多肽位于磷酸烯醇式丙酮酸敏感多肽的254/255或254/256位点。
  5. 一种核酸分子，其包含：
    (a)权利要求2-4中任一项所述的光学探针的编码序列，
    (b)(a)的互补序列。
  6. 包含权利要求5所述的核酸分子的核酸构建物，
    优选地，所述核酸构建物是克隆载体、表达载体或重组载体。
  7. 一种宿主细胞，所述宿主细胞：
    (1)表达权利要求2-4中任一项所述的光学探针；
    (2)包含权利要求5所述的核酸分子；或
    (3)包含权利要求6所述的核酸构建物。
  8. 一种检测试剂盒，其包含：
    (1)权利要求2-4中任一项所述的光学探针，
    (2)权利要求5所述的核酸序列，
    (3)权利要求6所述的核酸构建物，或
    (4)权利要求7所述的宿主细胞，
    所述检测试剂盒任选还包含利用光学探针检测磷酸烯醇式丙酮酸所需的其他试剂，
    优选地，所述检测试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、磷酸烯醇式丙酮酸标准品。
  9. 制备权利要求2-4中任一项所述的光学探针的方法，包括：提供权利要求7所述的宿主细胞，在所述光学探针表达的条件下培养所述宿主细胞，和分离光学探针。
  10. 权利要求2-4中任一项所述的光学探针、权利要求5所述的核酸序列、权利要求6所述的核酸构建物或权利要求7所述的宿主细胞在检测样品中的磷酸烯醇式丙酮酸、筛选化合物、磷酸烯醇式丙酮酸细胞内/外定位中的应用，
    优选地，
    检测样品中的磷酸烯醇式丙酮酸包括步骤：使所述光学探针或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化检测样品中的磷酸烯醇式丙酮酸，
    所述筛选化合物包括步骤：在含磷酸烯醇式丙酮酸的体系中使所述光学探针或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物；优选地，所述筛选化合物包括步骤：在含磷酸烯醇式丙酮酸的体系中使所述宿主细胞与候选化合物接触，并且光 学活性多肽的光学变化指示所述候选化合物是否调节细胞对磷酸烯醇式丙酮酸的摄取，
    所述磷酸烯醇式丙酮酸细胞内/外定位包括步骤：将含磷酸烯醇式丙酮酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化，
    更优选地，所述体系是溶液体系、细胞体系或亚细胞体系。
PCT/CN2023/133332 2022-11-22 2023-11-22 一种磷酸烯醇式丙酮酸光学探针及其制备方法和应用 Ceased WO2024109819A1 (zh)

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