Phosphoenolpyruvic acid optical probe, its preparation method and use

Abstract

The present invention relates to phosphoenolpyruvate optical probes. Specifically, the present invention provides phosphoenolpyruvate optical probes 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.

Description

[Technical Field]

[0001] This application claims priority to Chinese Application No. 202211465765.3, filed on November 22, 2022, the contents of which are incorporated herein by reference for all purposes. The present invention relates to the technical field of optical probes, and in particular to phosphoenolpyruvate optical probes and their preparation methods and uses. [Background technology]

[0002] Phosphoenolpyruvate (PEP), a high-energy intermediate in the glycolytic pathway, is an important metabolite linking glycolysis and gluconeogenesis. The high-energy phosphate bond in PEP allows it to act as a phosphate donor in bacterial, plant, and mammalian cells, and it plays an important role in maintaining intracellular ATP content, glucose homeostasis, regulating glucose metabolism, tumor immunity, and cytoprotection.

[0003] Currently, the most commonly used methods for detecting PEP include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) (Non-Patent Document 1), isotope labeling (Non-Patent Document 2), and enzyme activity detection. While HPLC and LC-MS can efficiently and accurately determine changes in PEP content in samples, the extraction of intracellular metabolites requires cumbersome experimental procedures and long cycle times. Isotopic labeling and enzyme activity detection cannot monitor changes in PEP in living cells in real time. Therefore, the development of genetically encoded fluorescent probes that can monitor dynamic changes in PEP in living cells in situ is urgently needed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Ho PC et al. Cell, 2015, 162(6), 1217-1228 [Non-patent document 2] Heiden MGV et al.,Science,2010,329(5998),1492-1499 Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide probes and methods for the real-time localization, high-throughput, quantitative detection of phosphoenolpyruvate inside and outside cells. [Means for solving the problem]

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions: In a first aspect of the present invention, there is provided the following phosphoenolpyruvate binding protein mutants: (a) having the sequence set forth in SEQ ID NO: 1 and having mutations, including amino acid modifications, substitutions or deletions, at one, two, three, four, five, six, seven, eight or more positions selected from the following: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320; (b) a sequence having at least 70% sequence identity with the sequence of (a) and having the mutations described in (1) and retaining the ability to bind phosphoenolpyruvate.

[0007] In one or more embodiments, the phosphoenolpyruvate binding protein mutant has the sequence set forth in SEQ ID NO:1 and has: (I) mutations at one, two, three, four or more sites selected from the following: I253, Q254, R255, G256, G257, and / or (II) mutations at one, two, three, four, five or more sites selected from the following: R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320.

[0008] In one or more embodiments, the mutation site described in (I) includes Q254. Preferably, the mutation site described in (I) includes sites from any one or more groups selected from the following: (I.1) I253, Q254, (I.2) I253, Q254, R255, G256, (I.3) Q254, R255, G256, (I.4) I253, Q254, G256, G257.

[0009] In one or more embodiments, the mutation site described in (II) includes sites from any one or more groups selected from the following: (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.

[0010] In one or more embodiments, the phosphoenolpyruvate binding protein mutant has the sequence set forth in SEQ ID NO:1 and has: (I) mutations at the following sites: I253, Q254, G256, and G257, and (II) mutations at any one or more groups of sites selected from the following: (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, and (II.14) V54 and R72.

[0011] In one or more embodiments, the phosphoenolpyruvate binding protein mutant has the sequence set forth in SEQ ID NO: 1 and has mutations at the following sites: (II.15) V54, 1253, Q254, R255, G256, (II.16) N59, 1253, Q254, R255, G256, (II.17) V54, 1253, Q254, G256, G257, (II.18) N59, 1253, Q254, G256, G257, (II.19) N59, 1253, Q254, G256, G257, (II.20) N59, 1253, Q254, G256, G257, (II.21) N59, 1253, Q254, R255, G256, (II.22) N59, 1253, Q254, G256, G257, (II.23) N59, 1253, Q254, G256, G257, (II.24) N59, 1253, Q254, G256, G257, (II.25) N59, 1253, Q254, G256, G257, (II.26) N59, 1253, Q254, R255, G256, (II.27) N59, 1253, Q254, R255, G256, (II.28) N59, 1253, Q254, G256, G257, (II.29) N59, 1253, Q254, R255, G256, (II.30) I.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.

[0012] In one or more embodiments, I253 is mutated to V, N, F, C, E, G, S, K, H, D, P, L, R, T, or Q.

[0013] In one or more embodiments, Q254 is mutated to L, V, A, R, P, M, K, or I.

[0014] In one or more embodiments, R255 is mutated to L,I,F.

[0015] In one or more embodiments, G256 is mutated to H, F, Y, or V.

[0016] In one or more embodiments, R21 is mutated to K.

[0017] In one or more embodiments, V54 is mutated to G, K, Q.

[0018] In one or more embodiments, R55 is mutated to S.

[0019] In one or more embodiments, V57 is mutated to M.

[0020] In one or more embodiments, A58 is mutated to G.

[0021] In one or more embodiments, N59 is mutated to D, H, or E.

[0022] In one or more embodiments, I60 is mutated to W.

[0023] In one or more embodiments, R72 is mutated to A.

[0024] In one or more embodiments, R212 is mutated to I, E, M.

[0025] In one or more embodiments, G213 is mutated to T.

[0026] In one or more embodiments, K215 is mutated to H, D, E.

[0027] In one or more embodiments, S216 is mutated to D, L, Y, I, M, F, or Q.

[0028] In one or more embodiments, L320 is mutated to Y, N, or F.

[0029] In one or more embodiments, the mutations include one or more selected from the following: I253K, Q254L, R255L and / or G256F.

[0030] In one or more embodiments, the mutations include mutations from any group selected from the following: (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) I253V, Q254A, R255F, G256Y, 53C, Q254L, R255L, G256F, (10)I253E, Q254L, R255L, G256F, (11)I253G, Q2 54L, R255L, G256F, (12)I253I, Q254L, R255L, G256F, (13)I253S, Q254L, R25 5L, G256F, (14) I253G, Q254L, R255L, G256F, (15) I253K, Q254L, R255L, G25 6F, (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)I25 3Q, Q254M, R255F, G256Y, (26)I253T, Q254P, R255F, G256Y, (27)Q254L, R255 F, G256F, (28) I253S, Q254K, R255F, G256F, (29) Q254P, R255F, G256F, (30) I253E, Q254V, R255F, G256F, (31)Q254L, R255F, G256F, (32)I253T, Q254L, R 255F, G256F, (33) Q254L, R255F, G256F, (34) I253D, Q254L, G256V, G257L, (3 5)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)S21 6Y, (58)S216I, (59)S216M, (60)S216F, (61)S216Q, (62)L320Y, (63)L320N, (64)L320F, (65)R72A, (66)V54G, R72 A, (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)L320 F, I253Q, Q254L, G256V, G257L, (79)S216L, I253Q, Q254L, G256V, G257L, (80)S216Y, I253Q, Q254L, G256V, G257L. ,

[0031] A first aspect of the present invention 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, and the phosphoenolpyruvate-sensitive polypeptide is divided into a first portion and a second portion by the optically active polypeptide.

[0032] In one or more embodiments, the phosphoenolpyruvate-sensitive polypeptide has: (1) a sequence set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto and retaining phosphoenolpyruvate binding activity; (2) Functional mutants of the sequence shown in SEQ ID NO: 1, wherein the functional mutants have mutations within 5 (preferably 3, more preferably 2) amino acids linked to the optically active polypeptide and / or have mutations 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, (3) A sequence having at least 70% sequence identity with the sequence described in (2), having the mutation described in (2), and retaining phosphoenolpyruvate sensitivity.

[0033] In one or more embodiments, (2) has the sequence set forth in SEQ ID NO:1 and has mutations at one, two, three, four, five or more sites selected from the following: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320, wherein the mutations include amino acid modifications, substitutions or deletions.

[0034] In one or more embodiments, (2) is the sequence of a phosphoenolpyruvate binding protein mutant described in any one of the embodiments of the first aspect herein.

[0035] In one or more embodiments, the optically active polypeptide is located within or replaces residues 68-71, 168-173, and / or 253-257 of the phosphoenolpyruvate-sensitive polypeptide, where the numbers correspond to the full length of the phosphoenolpyruvate-sensitive polypeptide.

[0036] In one or more embodiments, 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 / 1 More preferably, the optically active polypeptide is located at 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.

[0037] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional mutant thereof, wherein the functional mutant of the fluorescent protein has a mutation within three (preferably two) amino acids linked to the optically active polypeptide.

[0038] In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has a sequence set forth in any one of SEQ ID NOs: 2-9, preferably SEQ ID NO: 2, 6, 7, or 9.

[0039] In one or more embodiments, the functional mutant of the fluorescent protein has a sequence set forth in any one of SEQ ID NOs:2-9 and has a mutation at the position corresponding to amino acid 1 of SEQ ID NO:2, preferably to S, G, N, T, P, A, E, or H. Alternatively or additionally, the functional mutant of the fluorescent protein has a sequence set forth in any one of SEQ ID NOs:2-9 and has a mutation at the position corresponding to amino acid 246 of SEQ ID NO:2, preferably to F, C, W, or H.

[0040] In one or more embodiments, the functional mutant fluorescent protein has a sequence set forth in any one of SEQ ID NOs:2 to 9, and has mutations at positions corresponding to amino acids 1 and 246 of SEQ ID NO:2 selected from any one of the following groups: 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; 1T and 246F.

[0041] In one or more embodiments, the fluorescent protein is The amino acid sequence of the phosphoenolpyruvate-sensitive polypeptide is selected from the group consisting of: 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, and Y1T and N246F. The optically active polypeptide is located at one or more sites selected from the following sites of the phosphoenolpyruvate-sensitive polypeptide: 69 / 71, 170 / 171, 171 / 172, 254 / 255, 254 / 256 and 255 / 257 sites.

[0042] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO:6 or is a mutant thereof having a mutation set forth in any one of the following groups at the amino acid corresponding to amino acid position 1 or 246 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 sites selected from the following of the phosphoenolpyruvate-sensitive polypeptide: sites 171 / 172, 254 / 255 and 254 / 256.

[0043] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO:7 or is a mutant thereof having a mutation set forth in any one of the following groups at the amino acid corresponding to amino acid position 1 or 246 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 sites selected from the following of the phosphoenolpyruvate-sensitive polypeptide: sites 170 / 171, 171 / 172, 254 / 255 and 254 / 256.

[0044] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO:10 or is a mutant thereof having a mutation set forth in any one of the following groups at the amino acid corresponding to amino acid position 1 or 246 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 sites selected from the following of the phosphoenolpyruvate-sensitive polypeptide: sites 170 / 171, 171 / 172, 254 / 255 and 254 / 256.

[0045] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers described in the present invention may be any amino acid sequence of any length. In one embodiment, the linkers flanking the optically active polypeptide comprise a linker of 5 amino acids or less, for example, a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linkers flanking the optically active polypeptide comprise amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as shown below: a first portion B1 of the phosphoenolpyruvate-sensitive polypeptide, Y, an optically active polypeptide A, and a second portion B2 of the phosphoenolpyruvate-sensitive polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker.

[0046] In one embodiment, the optical probes of the present invention further comprise a localization sequence, for example, to localize the probe to a particular organelle in a cell.

[0047] In one or more embodiments, in the optical probe, the phosphoenolpyruvate-sensitive polypeptide is set forth in SEQ ID NO: 1, and the optically active polypeptide is set forth in SEQ ID NO: 2 to 9 (preferably SEQ ID NO: 2, 6, 7, 9), or a mutant thereof having one or more mutations selected from the following at the amino acid corresponding to amino acid position 1 or 246 of SEQ ID NO: 2: 1S, 1G, 1N, 1T, 1P, 1A, 1E, 1H, 246F, 246C, 246W, or 246H. The optically active polypeptide is located in one or more of the following positions of the phosphoenolpyruvate-sensitive polypeptide: 68 / 69, 68 / 70, 69 / 70, 68 / 71, 69 / 71, 70 / 7 1, 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 and / or 256 / 257. 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.

[0048] In one or more embodiments, in the optical probe, the phosphoenolpyruvate-sensitive polypeptide is set forth 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, G 256Y, 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 and / or L320F, the optically active polypeptide is The optically active polypeptide is represented by SEQ ID NOs: 2 to 9 (preferably SEQ ID NOs: 2, 6, 7, 9), or a mutant thereof having one or more mutations selected from the following at the amino acid corresponding to amino acid position 1 or 246 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 positions 254 / 255 or 254 / 256 of the phosphoenolpyruvate-sensitive polypeptide. Preferably, the mutation in the phosphoenolpyruvate-sensitive polypeptide includes a mutation 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, Q25 4L, 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、Q 254L、R255L、G256F、(70)V54K、I253K、Q254L、R255L、G256F、(71)V 54Q, I253K, Q254L, R255L, G256F, (72)N59H, I253K, Q254L, R255L, G256F, (73)V54Q, I253Q, Q254L, G256V, G257L, (74)N59D, I253Q, Q2 54L、G256V、G257L、(75)R212M、I253Q、Q254L、G256V、G257L、(76)G 213T、I253Q、Q254L、G256V、G257L、(77)K215E、I253Q、Q254L、G256 V、G257L、(78)L320F、I253Q、Q254L、G256V、G257L、(79)S216L、I25 3Q、Q254L、G256V、G257L、(80)S216Y、I253Q、Q254L、G256V、G257L。、

[0049] In one or more embodiments, in the optical probe, the phosphoenolpyruvate-sensitive polypeptide is represented by SEQ ID NO: 1, and the optically active polypeptide is represented by SEQ ID NO: 2 to 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, and 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 the optically active polypeptide, or (3) Q254L of the phosphoenolpyruvate-sensitive polypeptide. , R255L, G256H, and optically active polypeptide 1S; (4) phosphoenolpyruvate-sensitive polypeptide Q254V, R255I, G256H, and optically active polypeptide 246W; (5) phosphoenolpyruvate-sensitive polypeptide I253N, Q254L, R255L, G256F, and optically active polypeptide 1S, 246F; (6) phosphoenolpyruvate-sensitive polypeptide I253F, Q254L, R255L, G256F, and optically active polypeptide 1G; 246F, (7) phosphoenolpyruvate-sensitive polypeptide I253V, Q254A, R255F, G256Y, and optically active polypeptide 246F, (8) phosphoenolpyruvate-sensitive polypeptide I253V, Q254L, R255F, G256Y, and optically active polypeptide 1H, 246F, (9) phosphoenolpyruvate-sensitive polypeptide I253C, Q254L, R255L, G256F, and optically active polypeptide 1N, (10) phosphoenolpyruvate-sensitive polypeptide (11) phosphoenolpyruvate-sensitive polypeptide I253G, Q254L, R255L, G256F and optically active polypeptide 1T; (12) phosphoenolpyruvate-sensitive polypeptide I253I, Q254L, R255L, G256F and optically active polypeptide 1S; (13) phosphoenolpyruvate-sensitive polypeptide I253S, Q254L, R255L;G256F, and optically active polypeptide 1S, (14) phosphoenolpyruvate-sensitive polypeptide I253G, Q254L, R255L, G256F, and optically active polypeptide 1P, (15) phosphoenolpyruvate-sensitive polypeptide I253K, Q254L, R255L, G256F, and optically active polypeptide 1S, (16) phosphoenolpyruvate-sensitive polypeptide I253H, Q254L, R255L, G256F, and optically active polypeptide 1N, (17) phosphoenolpyruvate-sensitive polypeptide I253D, Q254L, R255L, G256F, and optically active polypeptide 1S, (18) phosphoenolpyruvate-sensitive polypeptide Q254L, R255L, G256F, and optically active polypeptide 1S, (19) phosphoenolpyruvate-sensitive polypeptide I253P, Q254L, R255L, G256F, and optically active polypeptide 1T, (20) phosphoenolpyruvate-sensitive polypeptide Q254L, R255L, G256F, and optically active polypeptide 1T, (21) phosphoenolpyruvate-sensitive polypeptide I253L, Q254R, R255L, G256F, (22) phosphoenolpyruvate-sensitive polypeptide I253R, Q254L, R255L, G256F and optically active polypeptide 1S, (23) phosphoenolpyruvate-sensitive polypeptide I253T, Q254L, R255F, G256Y and optically active polypeptide 1T, (24) phosphoenolpyruvate-sensitive polypeptide I253Q, Q254P, R255F, G256Y, (25) phosphoenolpyruvate-sensitive polypeptide I253Q, Q254M, R255F, G256Y, and optically active polypeptide 1P, (26) phosphoenolpyruvate-sensitive polypeptide I253T, Q254P, R255F, G256Y, and optically active polypeptide 1A, (27) phosphoenolpyruvate-sensitive polypeptide Q254L, R255F, G256F, and optically active polypeptide 1N, (28) phosphoenolpyruvate-sensitive polypeptide I253S, Q254K, R255F, G256F, and optically active polypeptide 1E, (29) phosphoenolpyruvate-sensitive polypeptide Q254P, R255F, G256F,and optically active polypeptide 246C, (30) phosphoenolpyruvate-sensitive polypeptide I253E, Q254V, R255F, G256F, and optically active polypeptide 1A, 246C, (31) phosphoenolpyruvate-sensitive polypeptide Q254L, R255F, G256F, and optically active polypeptide 1G, 246C, (32) phosphoenolpyruvate-sensitive polypeptide I253T, Q254L, R255F, G256F, and optically active polypeptide 1S, 246C, (33) phosphoenolpyruvate-sensitive polypeptide Q254L, R255F, G256F of the phosphoenolpyruvate-sensitive polypeptide, and 1T, 246C of the optically active polypeptide, (34) R21K of the phosphoenolpyruvate-sensitive polypeptide, (35) V54K of the phosphoenolpyruvate-sensitive polypeptide, (36) V54Q of the phosphoenolpyruvate-sensitive polypeptide, (37) V54G of the phosphoenolpyruvate-sensitive polypeptide, (38) R55S of the phosphoenolpyruvate-sensitive polypeptide, (39) V57M of the phosphoenolpyruvate-sensitive polypeptide, (40) (41) A58G of phosphoenolpyruvate-sensitive polypeptide, (42) N59D of phosphoenolpyruvate-sensitive polypeptide, (43) N59H of phosphoenolpyruvate-sensitive polypeptide, (44) N59E of phosphoenolpyruvate-sensitive polypeptide, (45) R212I of phosphoenolpyruvate-sensitive polypeptide, (46) R212E of phosphoenolpyruvate-sensitive polypeptide, (47) R212M of phosphoenolpyruvate-sensitive polypeptide, (48) R212I of phosphoenolpyruvate-sensitive polypeptide, (49) R212M of phosphoenolpyruvate-sensitive polypeptide, (50) R212I of phosphoenolpyruvate-sensitive polypeptide, (51) R212E of phosphoenolpyruvate-sensitive polypeptide, (52) R212M of phosphoenolpyruvate-sensitive polypeptide, (53) R212I of phosphoenolpyruvate-sensitive polypeptide, (54) R212M of phosphoenolpyruvate-sensitive polypeptide, (55) R212I of phosphoenolpyruvate-sensitive polypeptide, (56) R212E of phosphoenolpyruvate-sensitive polypeptide, (57) R212M of phosphoenolpyruvate-sensitive polypeptide, (58) R212I of phosphoenolpyruvate-sensitive polypeptide, (59) R212M of phosphoenolpyruvate-sensitive polypeptide, (60) R212M of phosphoenolpyruvate-sensitive polypeptide, (61) R212M of phosphoenolpyruvate-sensitive polypeptide, (62) R212M of phosphoenolpyruvate-sensitive polypeptide, (63) R212M of phosphoenolpyruvate-sensitive polypeptide, (64) R212M G213T of pyruvate-sensitive polypeptide, (49) K215E of phosphoenolpyruvate-sensitive polypeptide, (50) K215H of phosphoenolpyruvate-sensitive polypeptide, (51) K215D of phosphoenolpyruvate-sensitive polypeptide, (52) S216D of phosphoenolpyruvate-sensitive polypeptide, (53) S216L of phosphoenolpyruvate-sensitive polypeptide, (54) S216Y of phosphoenolpyruvate-sensitive polypeptide, (55) S216I of phosphoenolpyruvate-sensitive polypeptide,(56) S216M of phosphoenolpyruvate-sensitive polypeptide, (57) S216F of phosphoenolpyruvate-sensitive polypeptide, (58) S216Q of phosphoenolpyruvate-sensitive polypeptide, (59) L320Y of phosphoenolpyruvate-sensitive polypeptide, (60) L320N of phosphoenolpyruvate-sensitive polypeptide, (61) L320F of phosphoenolpyruvate-sensitive polypeptide, (62) R72A of phosphoenolpyruvate-sensitive polypeptide, (63) V54G and R72A of phosphoenolpyruvate-sensitive polypeptide, (64) V54K, I253G, Q254L, R255L, and G256F of phosphoenolpyruvate-sensitive polypeptide, and optically active polypeptide 1P, (65) phosphoenolpyruvate-sensitive polypeptide (66) phosphoenolpyruvate-sensitive polypeptide N59H, I253G, Q254L, R255L, G256F, and optically active polypeptide 1P; (67) phosphoenolpyruvate-sensitive polypeptide V54K, I253K, Q254L, R255L, G256F, and optically active polypeptide 1S; (68) phosphoenolpyruvate-sensitive polypeptide V54Q, I253K, Q254L, R255L, G256F, and optically active polypeptide 1S; (69) phosphoenolpyruvate-sensitive polypeptide N59H, I253K, Q254L, R255L, G256F, and optically active polypeptide 1S. ,

[0050] In one or more embodiments, in the optical probe, the phosphoenolpyruvate-sensitive polypeptide is set forth in SEQ ID NO:1 and the optically active polypeptide is set forth in SEQ ID NO:2-9 (preferably, SEQ ID NO:3).Nos. 2, 6, 7, and 9), the optically active polypeptide is located at the 254 / 256 position of the phosphoenolpyruvate-sensitive polypeptide, and the optical probe has the following mutations: (1) I253D, Q254L, G256V, and G257L of the phosphoenolpyruvate-sensitive polypeptide, and 1T and 246F of the optically active polypeptide; (2) I253S, Q254L, G256V, and G257L of the phosphoenolpyruvate-sensitive polypeptide, and 1T and 246F of the optically active polypeptide. 46F, (3) phosphoenolpyruvate-sensitive polypeptide I253Q, Q254L, G256V, G257L and optically active polypeptide 1T, 246F, (4) phosphoenolpyruvate-sensitive polypeptide V54Q, I253Q, Q254L, G256V, G257L and optically active polypeptide 1T, 246F, (5) phosphoenolpyruvate-sensitive polypeptide N59D, I253Q, Q254L, G256V, G257L and optically active polypeptide 1T, 246F, (6) Phosphoenolpyruvate-sensitive polypeptide R212M, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F; (7) Phosphoenolpyruvate-sensitive polypeptide G213T, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F; (8) Phosphoenolpyruvate-sensitive polypeptide K215E, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F; (9) ) Phosphoenolpyruvate-sensitive polypeptide L320F, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F; (10) Phosphoenolpyruvate-sensitive polypeptide S216L, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F; (11) Phosphoenolpyruvate-sensitive polypeptide S216Y, I253Q, Q254L, G256V, G257L, and optically active polypeptide 1T, 246F.

[0051] The present invention further provides a fusion polypeptide comprising an optical probe described in any one of the embodiments herein and another polypeptide. In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to various organelles or subcellular compartments, a tag for purification, or a tag for immunoblotting.

[0052] The present invention further provides a nucleic acid molecule comprising: (a) a coding sequence for a protein mutant, optical probe, or fusion polypeptide described in any one of the embodiments herein, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b), wherein the fragment is a primer.

[0053] The present invention further relates to mutants of the above-described nucleic acid molecules, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and mutants of the protein mutants, optical probes or fusion polypeptides of the invention, or their complementary sequences.

[0054] The present invention further provides a nucleic acid construct comprising a nucleic acid molecule described herein, wherein the nucleic acid sequence encodes a protein mutant, optical probe, or fusion polypeptide described in any one embodiment of the present invention.

[0055] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0056] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0057] In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0058] Another aspect of the present invention further provides a host cell, the host cell (1) expressing an optical probe or fusion polypeptide described in any one of the embodiments of the present invention, (2) comprising a nucleic acid molecule described in any one of the embodiments of the present invention, or (3) comprising a nucleic acid construct described in any one of the embodiments of the present invention. The host cell is preferably Escherichia coli.

[0059] Another aspect of the present invention further provides a kit for detecting phosphoenolpyruvate, the kit comprising an optical probe or a fusion polypeptide or polynucleotide described herein or an optical probe prepared by the methods described herein.

[0060] In one or more embodiments, the kit further comprises one or more reagents selected from the following: a buffer, a medium, and a phosphoenolpyruvate standard.

[0061] Another aspect of the present invention provides a method for preparing an optical probe described herein, the method comprising: providing a host cell that expresses an optical probe or fusion polypeptide described herein, culturing the host cell under conditions such that the optical probe or fusion polypeptide is expressed, and isolating the optical probe or fusion polypeptide.

[0062] In one or more embodiments, the method comprises the following steps: 1) incorporating a nucleic acid molecule encoding an optical probe or fusion polypeptide described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector; and 3) isolating the optical probe or fusion polypeptide described herein.

[0063] Another aspect of the present invention further provides a method for detecting phosphoenolpyruvate in a sample, the method comprising: contacting the sample with an optical probe or fusion polypeptide or host cell described herein and detecting a change in the optically active polypeptide. The detection may be performed in vivo, ex vivo, intracellularly, or in situ. The sample may be, for example, blood.

[0064] Another aspect of the present specification further provides a method for quantifying phosphoenolpyruvate in a sample, the method comprising: contacting an optical probe or fusion polypeptide or host cell described herein with a sample; detecting an optical change in the optically active polypeptide; and quantifying the amount of phosphoenolpyruvate in the sample based on the optical change in the optically active polypeptide.

[0065] Another aspect of the present invention further provides a method for screening compounds (e.g., pharmaceuticals), the method comprising: contacting an optical probe or fusion polypeptide or host cell described herein with a candidate compound in a system containing phosphoenolpyruvate, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide. The method allows for high-throughput screening of compounds.

[0066] In one or more embodiments, a host cell described herein is contacted with a candidate compound in a system comprising phosphoenolpyruvate, and an optical change in the optically active polypeptide indicates whether the candidate compound can modulate the cellular uptake of phosphoenolpyruvate.

[0067] Another aspect of the present invention further provides a method for localizing phosphoenolpyruvate intracellularly and / or extracellularly, the method comprising: contacting the optical probe or the host cell with a system containing phosphoenolpyruvate, and detecting an optical change in the optically active polypeptide.

[0068] In one or more embodiments, the system is a solution system, a cellular system, or an intracellular system.

[0069] Another aspect of the present invention further provides the use of a phosphoenolpyruvate optical probe or fusion polypeptide or host cell described herein in detecting phosphoenolpyruvate in a sample, screening for compounds, or localizing intracellular and / or extracellular phosphoenolpyruvate, which in one or more embodiments is real-time localization. [Effects of the Invention]

[0070] Benefits of the present invention: The phosphoenolpyruvate optical probe provided by the present invention is easy to mature, exhibits large dynamic changes in fluorescence, has excellent specificity, and can be expressed intracellularly using genetic engineering methods. It can localize and detect phosphoenolpyruvate inside and outside of cells in real time, with high throughput and quantitative detection, eliminating the need for time-consuming sample processing procedures. Experimental results show that the phosphoenolpyruvate optical probe provided by the present application has a maximum response to phosphoenolpyruvate that is more than five times that of the control. It can also detect phosphoenolpyruvate locally, qualitatively, and quantitatively in intracellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus, enabling high-throughput compound screening and quantitative detection of phosphoenolpyruvate in blood. [Brief explanation of the drawings]

[0071] The present invention will be further described below with reference to the drawings and examples. [Figure 1] FIG. 1 is an SDS-PAGE image of an exemplary phosphoenolpyruvate optical probe. [Figure 2] FIG. 2 is a characteristic image of the fluorescence spectrum of an exemplary phosphoenolpyruvate optical probe. [Figure 3A] FIG. 3 is a titration curve of an exemplary phosphoenolpyruvate optical probe against different concentrations of phosphoenolpyruvate. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above. [Figure 3L] Same as above. [Figure 3M] Same as above. [Figure 3N] Same as above. [Figure 4] FIG. 4 outlines the specific detection of the remaining three substrates and glycometabolic intermediates and analogs by an exemplary phosphoenolpyruvate optical probe. [Figure 5] FIG. 5 is a photograph of the localization of an exemplary phosphoenolpyruvate optical probe to intracellular organelles in mammalian cells. [Figure 6] FIG. 6 is a schematic diagram of the dynamic monitoring of phosphoenolpyruvate concentrations in the cytoplasm of mammalian cells using an exemplary phosphoenolpyruvate optical probe. [Figure 7] FIG. 7 is a dot plot showing high-throughput compound screening at the live cell level with an exemplary phosphoenolpyruvate optical probe. [Figure 8] FIG. 8 is a bar graph showing the quantification of phosphoenolpyruvate in mouse and human blood with an exemplary phosphoenolpyruvate optical probe. DETAILED DESCRIPTION OF THE INVENTION

[0072] As used herein, the term "about" when referring to a numerical value or range means that the numerical value or range is within 20%, within 10%, and within 5% of the stated numerical value or range.

[0073] As used herein, the terms "comprise," "have," and cognate terms include "contain" and "consist of," e.g., a composition "comprising" X may consist solely of X, or may also include other substances, e.g., X+Y.

[0074] As used herein, the term "phosphoenolpyruvate-sensitive polypeptide" refers to a polypeptide that responds to phosphoenolpyruvate, and the response refers to any response in a chemical, biological, electrical, or physiological parameter of the polypeptide related to the interaction of the sensitive polypeptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the polypeptide, and changes in the primary, secondary, or tertiary structure of the polypeptide (including changes in protonation, electrochemical potential, and / or conformation). "Conformation" refers to the three-dimensional arrangement of a molecule's primary, secondary, and tertiary structure, including side groups within the molecule; a change in the three-dimensional structure of a molecule also changes its conformation. Examples of conformational changes include a transition from alpha helix to beta sheet or from beta sheet to alpha helix. It should be understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety changes. The phosphoenolpyruvate-sensitive polypeptides described herein also include functional mutants thereof. Functional mutants of phosphoenolpyruvate-sensitive polypeptides include, but are not limited to, mutants that can interact with phosphoenolpyruvate and cause the same or similar changes as the parent phosphoenolpyruvate-sensitive polypeptide.

[0075] The phosphoenolpyruvate-sensitive polypeptides described in the present invention include, but are not limited to, phosphofructokinase TtPFK derived from Thermus thermophilus, or mutants having 90% or more homology thereto. Phosphoenolpyruvate-binding proteins can sense changes in phosphoenolpyruvate concentration, and as the phosphoenolpyruvate concentration dynamically changes, the spatial conformation of the phosphoenolpyruvate-binding protein also changes. Truncated mutants of TtPFK may also be used in the present invention.

[0076] As used herein, the term "optical probe" refers to a phosphoenolpyruvate-sensitive polypeptide fused to an optically active polypeptide. The inventors have discovered that the conformational change induced by specific binding of a phosphoenolpyruvate-sensitive polypeptide, such as a phosphoenolpyruvate-binding protein, to physiological concentrations of phosphoenolpyruvate induces a conformational change in the optically active polypeptide (e.g., a fluorescent protein), thereby causing a change in the optical properties of the optically active polypeptide. The presence and / or levels of phosphoenolpyruvate can be detected and / or analyzed by plotting a standard curve using the fluorescence of the fluorescent protein measured at different phosphoenolpyruvate concentrations. When describing the optical probes of the present invention (e.g., when describing insertion or mutation sites), all amino acid residue numbers refer to SEQ ID NO:1.

[0077] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operably inserted into a phosphoenolpyruvate-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is one of the optical properties of an optically active polypeptide and can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation or emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the excitation amplitude at two different wavelengths, the ratio of the emission amplitudes at two different wavelengths, the excited-state lifetime, or the fluorescence anisotropy. A measurable difference in any one of these properties between the active and inactive states is sufficient for the fluorescent protein substrates of the present invention to be used in activity assays. The measurable difference can be determined by determining the amount of a quantitative fluorescence property, such as the amount of fluorescence at a specific wavelength or the integral of fluorescence over the entire emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that allow easy distinction between the inactive and activated conformational states. The optically active polypeptides described herein may further include functional mutants thereof, including, but not limited to, mutants that can produce the same or similar changes in fluorescence properties as the parent optically active polypeptide.

[0078] The term " fluorescent protein " used herein refers to the protein that emits fluorescence when irradiated with excitation light.Fluorescent protein is the basic detection method in the field of biological science, and includes, for example, the green fluorescent protein GFP that is commonly used in the field of biotechnology, and the circular rearrangement blue fluorescent protein (cpBFP), circular rearrangement green fluorescent protein (cpGFP), circular rearrangement yellow fluorescent protein (cpYFP) etc. that are derived by mutation of this protein; Also includes the red fluorescent protein RFP that is commonly used in this field, and the circular rearrangement protein (cpmApple, cpmOrange, cpmKate etc.) that are derived from this protein. Illustratively, cpYFP is represented by SEQ ID NO:2, cpmOrange is represented by SEQ ID NO:3, cpmKate is represented by SEQ ID NO:4 or 8, mCherry is represented by SEQ ID NO:5, cpGFP is represented by SEQ ID NO:6, cpBFP is represented by SEQ ID NO:7, and cpmApple is represented by SEQ ID NO:9.

[0079] The fluorescent protein in the optical probe also includes functional mutants having mutations, including, but not limited to, fluorescent proteins having mutations at position 1 and / or at the position corresponding to amino acid position 246 of SEQ ID NO:2. The mutation at position 1 is preferably S, G, N, T, P, A, E, or H. The mutation at position 246 of SEQ ID NO:2 is preferably F, C, W, or H. In some embodiments, the functional mutant fluorescent protein has a sequence set forth in any one of SEQ ID NOs:2 to 9 and has mutations at positions corresponding to amino acid positions 1 and 246 of SEQ ID NO:2 selected from the following group: 1S and 246F; 1G and 246F; 1H and 246F; 1A and 246C; 1G and 246C; 1S and 246C; 1T and 246C; or 1T and 246F. 1S indicates that the mutation of the amino acid at position 1 is S, and 246F indicates that the mutation of the amino acid at position 246 is F, and so on.

[0080] In the optical probes of the present invention, the optically active polypeptide is located in or replaces residues 68-71, 168-173, and / or 253-257 in the N-C direction of the phosphoenolpyruvate-sensitive polypeptide, where the numbers correspond to the full length of the phosphoenolpyruvate-sensitive polypeptide. As used herein, when the two numbers in an "X / Y" format are consecutive integers, this means that the optically active polypeptide is located between the amino acids indicated by those numbers. For example, insertion site 254 / 255 indicates that the optically active polypeptide is located between amino acids 254 and 255 of the phosphoenolpyruvate-sensitive polypeptide. When the two numbers in an "X / Y" format are not consecutive integers, this means that the optically active polypeptide replaces the amino acid between the amino acids indicated by those numbers. For example, insertion site 254 / 256 indicates that the optically active polypeptide replaces amino acid 255 of the phosphoenolpyruvate-sensitive polypeptide. In exemplary embodiments, 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 sites 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, 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 and / or 256 / 257.

[0081] The terms "variant" or "mutant" as used herein when referring to a polypeptide or protein include mutants that have the same function as the polypeptide or protein but differ in sequence. Mutant polypeptides or proteins may include homologous sequences, conservative mutants, allelic variants, naturally occurring mutants, and induced mutants. These mutants include, but are not limited to, sequences obtained by deleting, inserting, and / or substituting one or more amino acids (generally 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5) from the sequence of the polypeptide or protein, and adding one or more amino acids (generally up to 20, preferably up to 10, and more preferably up to 5) to the carboxyl and / or amino termini. These mutants may further include polypeptides or proteins having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the aforementioned polypeptides or proteins. Without intending to be bound by theory, changes in amino acid residues that do not change the overall configuration and function of a polypeptide or protein are referred to as function-conservative mutations. For example, in the art, substitution with an amino acid with similar properties generally does not change the function of a polypeptide or protein. In the art, it is well-defined that amino acids with similar properties refer to a family of amino acids with similar side chains.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, the addition of one or more amino acids, for example, at the amino and / or carboxyl termini, generally does not alter the function of a polypeptide or protein. Conservative amino acid substitutions for commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with those of genetically encoded amino acids.

[0082] The term "linker" or "connecting region" refers to an amino acid or nucleotide sequence that connects two parts of a polypeptide, protein, or nucleic acid of the present invention. Illustratively, in the present invention, the number of amino acids at the amino terminus of the connecting region between the phosphoenolpyruvate-sensitive polypeptide and the optically active polypeptide is selected to be 0 to 3, and the number of amino acids at the carboxyl terminus is selected to be 0 to 2; when the recombinant optical probe is connected to a functional protein as a basic unit, the linker sequence can be fused to the amino terminus or carboxyl terminus of the recombinant optical probe. The linker sequence is a short peptide chain composed of one or more flexible amino acids, such as Y.

[0083] The present inventors have found that phosphoenolpyruvate-binding protein mutants having mutations at sites selected from the following exhibit binding activity different from that of phosphoenolpyruvate: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, and L320 of SEQ ID NO: 1. The above amino acid mutations include amino acid modifications, substitutions, or deletions.

[0084] The present invention provides phosphoenolpyruvate-binding protein mutants having these mutations, and optical probes comprising such phosphoenolpyruvate-binding protein mutants as phosphoenolpyruvate-sensitive polypeptides. Thus, in one or more embodiments, the phosphoenolpyruvate-sensitive polypeptide in the optical probe is a phosphoenolpyruvate-binding protein mutant described in any one of the embodiments herein, and the fluorescent protein in the optical probe is set forth in SEQ ID NOs: 2 to 9 or a functional mutant thereof.

[0085] In some specific embodiments, the phosphoenolpyruvate-sensitive polypeptide in the optical probe is set forth in SEQ ID NO:1, the optically active polypeptide is set forth in SEQ ID NO:2, the optically active polypeptide is located at positions 254 / 255 or 254 / 256 of the phosphoenolpyruvate-sensitive polypeptide, and the mutations in the optical probe are set forth in any row of Tables 5 and 6.

[0086] With respect to two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences being identical, or having a certain percentage of amino acid residues or nucleotides identical over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical), when the two or more sequences or subsequences are compared and aligned for maximum correspondence using methods known in the art, such as sequence comparison algorithms, over a comparison window or designated region. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0087] It is well known to those skilled in the art that gene cloning procedures often require the design of appropriate restriction sites, which inevitably introduce one or more extraneous residues at the termini of the expressed polypeptide or protein, but which do not affect the activity of the target polypeptide or protein. For example, to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of host cells, or facilitate the purification of a recombinant protein, it is often necessary to add several amino acids to the N-terminus, C-terminus, or other appropriate region of the recombinant protein, including, but not limited to, an appropriate linker peptide, a signal peptide, a leader peptide, a terminal extension, a tag such as glutathione S-transferase (GST), maltose E-binding protein, protein A, 6His, or Flag, or a protease site for factor Xa, thrombin, or enterokinase.

[0088] As used herein, the terms "functional fragment," "derivative," and "analog" refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., phosphoenolpyruvate-binding protein or fluorescent protein). Functional mutants, derivatives, or analogs of the polypeptides or proteins (e.g., phosphoenolpyruvate-binding protein or fluorescent protein) of the present invention may be (i) proteins in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) have been substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins with substitutions at one or more amino acid residues; or (iii) proteins formed by fusing the mature protein with another compound (e.g., a compound that extends the half-life of the protein, e.g., polyethylene glycol); or (iv) proteins formed by fusing additional amino acid sequences to the protein sequence (e.g., a secretory sequence, a sequence for purifying the protein, a protein protease sequence, or a fusion protein formed with an antigenic IgG fragment). Based on the teachings herein, these functional mutants, derivatives, and analogs are well known to those skilled in the art. The analogs also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids) or non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the phosphoenolpyruvate-sensitive polypeptides of the present invention are not limited to the exemplary proteins, mutants, derivatives, and analogs listed above. Modifications (which generally do not alter primary structure) include chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include proteins produced by glycosylation, e.g., glycosylation modifications during protein synthesis and processing or in further processing steps. This modification can be achieved by exposing the protein to a glycosylating enzyme (e.g., a mammalian glycosylating or deglycosylating enzyme).Modified forms also include sequences with phosphorylated amino acid residues (e.g., tyrosine phosphate, serine phosphate, threonine phosphate), as well as proteins modified to improve their proteolytic resistance or optimize their solubility.

[0089] The fusion polypeptides of the present invention comprise an optical probe described herein and another polypeptide. In some embodiments, the optical probe described herein further comprises another polypeptide fused thereto. The other polypeptide described herein does not affect the properties of the optical probe. The other polypeptide may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to various organelles or subcellular compartments, a tag for purification, or a tag for immunoblotting. A linker may be present between the optical probe and the other polypeptide in the fusion polypeptide described herein.

[0090] Subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, plasma membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the purification tag or immunoblotting tag includes hexahistidine (6*His), glutathione S-transferase (GST), and Flag.

[0091] The present invention includes nucleic acid molecules encoding the phosphoenolpyruvate-sensitive polypeptides or optical probes described herein. The terms "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" used herein may refer to either DNA or RNA. DNA includes cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may have a coding strand and a non-coding strand. When referring to nucleic acids, the term "mutant" used herein may refer to naturally occurring allelic mutants or non-naturally occurring mutants. These nucleotide mutants include degenerate mutants, substitution mutants, deletion mutants, and insertion mutants. As known in the art, allelic mutants are substituted forms of nucleic acids that may involve the substitution, deletion, or insertion of one or more nucleotides but do not substantially alter the function of the encoded mutant protein. The nucleic acids of the present invention may comprise nucleotide sequences having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the above nucleic acid sequences. The present invention also relates to nucleic acid fragments that hybridize to the above sequences. As used herein, the length of a "nucleic acid fragment" includes at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR).

[0092] The full-length sequences or fragments of the optical probes or fusion proteins of the present invention can be obtained typically by PCR amplification, artificial synthesis, or recombination. Procedures and reagents used in typical PCR, synthesis, and recombination methods are known in the art. Mutations may also be introduced into the protein sequences of the present invention by methods such as mutagenic PCR or chemical synthesis.

[0093] The present invention also relates to nucleic acid constructs, which comprise the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of a polypeptide or protein. Depending on the expression vector's requirements, the nucleic acid construct may be manipulated before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0094] In one embodiment, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, including plasmids, phagemids, phage derivatives, animal viruses, and cosmids.

[0095] A typical expression vector contains an expression control sequence that can be used to control the expression of a desired nucleic acid sequence and is operably linked to the nucleic acid sequence of the present invention or its complementary sequence. As used herein, "expression control sequence" refers to an element operably linked to a target gene that controls the transcription, translation, and expression of the target gene. It may be an origin of replication, a promoter, a marker gene, or a translation control element (including an enhancer, operator, terminator, ribosome binding site, etc.). The choice of expression control sequence depends on the host cell used. In a recombinant expression vector, "operably linked" means that the desired nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters include the E. coli lac or trp promoter, the λ phage PL promoter, and eukaryotic promoters include the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the long terminal repeats of reverse transcription viruses, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the commercially available pCDF vector may be used as the expression vector, with no other special requirements. For example, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and the digested products are then ligated to obtain a recombinant expression vector. The present invention is not particularly limited by the specific procedures and parameters for enzymatic cleavage and ligation, and conventional procedures and parameters in the art can be used.

[0096] After obtaining a recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide, including a fusion protein. This transfer process can be carried out by conventional techniques, such as transformation or transfection, well known to those skilled in the art. The term "host cell" as used herein refers to a cell that can accept and accommodate a recombinant DNA molecule and serve as a site for amplifying the recombinant gene. An ideal recipient cell must be easily available and easily propagated. The "host cell" of the present invention includes prokaryotic and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells, and mammalian cells. The host cells are preferably cells that are advantageous for the expression or fermentation production of gene products, and such cells are well known and commonly used in the art. Specific examples include bacterial cells of Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells such as Drosophila S2 or Sf9, and animal cells such as CHO, COS, HEK293, HeLa, and Bowes' melanoma cells. An exemplary host cell used in the examples of the present invention is the E. coli strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers and host cells.

[0097] The methods for introducing DNA into host cells described in the present invention are conventional methods in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfection, or electroporation.When the host is a prokaryotic organism such as E. coli, the above-mentioned method is preferably the CaCl2 method or the MgCl2 method, and the procedures used are well known in the art.When the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0098] In the present invention, an expression vector is introduced into a host cell, and then the host cell is amplified, expressed, and cultured to isolate and obtain a phosphoenolpyruvate optical probe. Conventional methods can be used for amplifying, expressing, and culturing the host cell. Depending on the type of host cell used, various conventional media may be used for culturing. The host cell is cultured under conditions suitable for its growth.

[0099] In the present invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be isolated and purified using various separation methods based on its physical, chemical, and other properties. In the present invention, the method for isolating the phosphoenolpyruvate fluorescent protein is not particularly limited, and any conventional fusion protein isolation method known in the art may be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, cell disruption by osmosis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), other liquid chromatography techniques, and combinations of these methods. In one embodiment, the optical probe is isolated using affinity chromatography using a His tag.

[0100] The present invention further provides the use of the phosphoenolpyruvate optical probe in real-time localization, quantitative detection, and high-throughput compound screening of phosphoenolpyruvate. In one aspect, the phosphoenolpyruvate optical probe is preferably linked to a signal peptide at different cellular sites, introduced into the cell, and the intensity of the fluorescent signal in the cell is detected to perform real-time localization of phosphoenolpyruvate; the corresponding phosphoenolpyruvate is quantitatively detected by combining the change in the fluorescent signal with a standard titration curve of phosphoenolpyruvate. The change in the fluorescent signal is represented, for example, by a normalized fluorescent signal ratio. In an embodiment involving cpYFP, ​​the ratio is the ratio between the 485 nm fluorescent signal and the 420 nm fluorescent signal of the sample and the corresponding ratio of the control. The phosphoenolpyruvate standard titration curve described in the present invention is plotted based on the fluorescent signal of the phosphoenolpyruvate optical probe under conditions of different phosphoenolpyruvate concentrations. The phosphoenolpyruvate optical probe described in the present invention can be directly introduced into cells, and the process of real-time localization and quantitative detection of phosphoenolpyruvate does not require time-consuming sample processing, making it more accurate.When the phosphoenolpyruvate optical probe of the present invention is used in high-throughput compound screening, different compounds can be added to cell culture media, changes in phosphoenolpyruvate content can be measured, and compounds that affect the changes in phosphoenolpyruvate content can be screened.The applications of the phosphoenolpyruvate optical probe described in the present invention for real-time localization and quantitative detection of phosphoenolpyruvate and high-throughput compound screening are both for non-diagnostic and therapeutic purposes and do not include the diagnosis and treatment of diseases.

[0101] The present invention further provides a detection kit comprising the optical probe, nucleic acid molecule, nucleic acid construct, and / or cell described herein. The kit may further comprise other reagents necessary for detecting phosphoenolpyruvate. Such other reagents are well known in the art and include, for example, buffers, cell culture media, and phosphoenolpyruvate standards. An exemplary buffer is, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0102] Some specific embodiments Item 1, (a) having the sequence set forth in SEQ ID NO: 1 and having mutations, including amino acid modifications, substitutions or deletions, at one, two, three, four, five or more sites selected from the following: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320; (b) a sequence having at least 70% sequence identity with the sequence of (a), having the mutations described in (1), and retaining the ability to bind phosphoenolpyruvate; a phosphoenolpyruvate binding protein mutant characterized by: Preferably, the mutation described in (a) comprises a mutation at any one or more of the following groups of sites:

[0103] Item 2, 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; Phosphoenolpyruvate-sensitive polypeptides are (1) a sequence set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto and retaining phosphoenolpyruvate binding activity; (2) A functional mutant of the sequence set forth in SEQ ID NO: 1, wherein the functional mutant has a mutation within the 5 amino acids linked to the optically active polypeptide and / or has a mutation at one, two, three, four, five or more positions selected from the following: R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320, or (3) A sequence having at least 70% sequence identity with the sequence described in (2), having the mutation described in (2), and retaining phosphoenolpyruvate sensitivity. and Preferably, (2) is the sequence of the phosphoenolpyruvate binding protein mutant described in item 1. A phosphoenolpyruvate optical probe comprising:

[0104] Item 3: The optical probe according to Item 2, wherein the optically active polypeptide is located at or replaces residues 68 to 71, 168 to 173, and / or 253 to 257 of the phosphoenolpyruvate-sensitive polypeptide.

[0105] Item 4: The optically active polypeptide is located in one or more of the following regions 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, 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 and / or 256 / 257 3. The optical probe according to item 2, characterized in that: More preferably, The optically active polypeptide is located at 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; More preferably, the optically active polypeptide is a fluorescent protein or a functional mutant thereof; Preferably, The fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2 to 9; The functional mutant of the fluorescent protein has a mutation within the three amino acids linked to the optically active polypeptide; preferably, the functional mutant of the fluorescent protein has a mutation at a position corresponding to amino acid position 1 of SEQ ID NO:2, and the mutation is preferably selected from Y1S, Y1G, Y1N, Y1T, Y1P, Y1A, Y1E, and Y1H; The functional mutant of the fluorescent protein has a mutation at the position corresponding to amino acid 246 of SEQ ID NO:2, said mutation preferably being selected from N246F, N246C, N246W or N246H; More preferably, in said optical probe, the phosphoenolpyruvate-sensitive polypeptide is as set forth 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, G213T, K215H, K215D, K215E, S216D, S216L, S216Y, S216I, S216M, S216F, S216Q, L320Y, L320N and / or L320F, the optically active polypeptide is The optically active polypeptide is represented by ID NOs: 2 to 9, or a mutant thereof having one or more of the following mutations: Y1S, Y1G, Y1N, Y1T, Y1P, Y1A, Y1E, Y1H, N246F, N246C, N246W, and is located at the 254 / 255 or 254 / 256 position of the phosphoenolpyruvate-sensitive polypeptide.

[0106] Item 5, (a) a coding sequence of the optical probe according to any one of items 2 to 4; (b) Complementary sequence of (a) A nucleic acid molecule comprising:

[0107] Item 6. A nucleic acid construct comprising the nucleic acid molecule according to Item 5. Preferably, said nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

[0108] Item 7, (1) expressing the optical probe described in any one of items 2 to 4; (2) comprising the nucleic acid molecule described in item 5; or (3) The nucleic acid construct according to item 6 is included. A host cell characterized by:

[0109] Item 8, (1) The optical probe according to any one of items 2 to 4; (2) The nucleic acid sequence described in item 5; (3) A nucleic acid construct according to item 6, or (4) A host cell according to item 7. a detection kit comprising: The detection kit optionally includes other reagents necessary for detecting phosphoenolpyruvate using an optical probe; Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer solution, a medium, and a phosphoenolpyruvate standard.

[0110] Item 9. A method for preparing the optical probe according to any one of Items 2 to 4, comprising providing the host cell according to Item 7, culturing the host cell under conditions in which the optical probe is expressed, and isolating the optical probe.

[0111] Item 10: Use of the optical probe according to any one of items 2 to 4, the nucleic acid sequence according to item 5, the nucleic acid construct according to item 6, or the host cell according to item 7 in detecting phosphoenolpyruvate in a sample, screening compounds, or the intracellular and / or extracellular localization of phosphoenolpyruvate; Preferably, Detecting phosphoenolpyruvate in the sample includes contacting the optical probe or host cell with the sample, detecting an optical change in the optically active polypeptide, and detecting phosphoenolpyruvate in the sample based on the optical change in the optically active polypeptide; The screening of the compound includes contacting the optical probe or host cell with a candidate compound in a system containing phosphoenolpyruvate, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide; preferably, the screening of the compound includes contacting the host cell with a candidate compound in a system containing phosphoenolpyruvate, and the optical change in the optically active polypeptide indicates whether the candidate compound can regulate the cellular uptake of phosphoenolpyruvate; said intracellular and / or extracellular localization of phosphoenolpyruvate comprises contacting said optical probe or said host cell with a system containing phosphoenolpyruvate and detecting an optical change in the optically active polypeptide; More preferably, the system is a solution system, a cell system, or an intracellular system.

[0112] Concentrations, amounts, percentages, and other numerical values ​​may be presented herein in a range format, with the understanding that this range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the values ​​expressly recited at the upper and lower limits of the range, but also all individual values ​​or subranges encompassed within the range. [Example]

[0113] The phosphoenolpyruvate optical probe provided in the present invention will be described in detail below in conjunction with examples, which should not be construed as limiting the protection scope of the present invention.

[0114] I. Experimental Materials and Reagents In the examples, conventional genetic engineering, molecular biology cloning methods, cell culture methods, imaging methods, etc. are mainly used, and these methods are well known to those skilled in the art, such as "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al.; "Molecular Cloning Laboratory Guide" by J. Sambrook, D.W. Russell, and translated by Huang Peitang et al. (3rd ed., August 2002, Science Press, Beijing); "Animal Cell Culture: A Guide to Basic Techniques" by Frasier et al., translated by Zhang Jingbo, Xu Cunshuan et al. (5th ed.); "Concise Cell Biology Laboratory Guide" by J.S. Bonifacion, M. Dassault et al., translated by Zhang Jingbo et al.

[0115] 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 in PCR were synthesized and purified by Shanghai Jierui Bioengineering Technology Co., Ltd. and BGI, and verified to be correct by mass spectrometry. The expression plasmids constructed in the examples were sequenced by BGI and JIELI Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology Co., Ltd., pfu DNA polymerase from Tiangen Biochemical Technology (Beijing) Co., Ltd., and primeSTAR DNA polymerase from TaKaRa. The three polymerases were also provided with the corresponding polymerase buffer and dNTPs at the time of purchase. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligase and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffers were also provided. The transfection reagent Lip2000 kit was purchased from Invitrogen. Compounds such as phosphoenolpyruvate were purchased from Sigma. Unless otherwise noted, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Ameresco. 96-well black detection plates and 384-well black fluorescence detection plates were purchased from Grenier.

[0116] 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 clonal strain Mach1 was purchased from Invitrogen. The nickel affinity chromatography column and desalting column packing were from GE Healthcare.

[0117] The main equipment used in the examples includes a Biotek Synergy 2 multifunction microplate reader (Bio-Tek, USA), an X-15R high-speed refrigerated centrifuge (Beckman, USA), a Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), a PCR amplification device (Biometra, Germany), an ultrasonic homogenizer (Ningbo Xinzhi Company), a nucleic acid electrophoresis device (Shenneng Bocai Company), a fluorescence spectrophotometer (Varian, USA), a CO2 constant-temperature cell culture incubator (SANYO), and an inverted fluorescence microscope (Nikon, Japan).

[0118] II. Molecular Biology and Cell Experimental Methods II.1 Polymerase Chain Reaction (PCR): 1. PCR for target fragment amplification: This method was primarily used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system was as follows: 0.5–1 μL template sequence, 0.5 μL forward primer (25 μM), 0.5 μL reverse primer (25 μM), 5 μL 10× pfu buffer, 0.5 μL pfu DNA polymerase, 1 μL dNTPs (10 mM), and 41.5–42 μL sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification program consisted of denaturation at 95°C for 2–10 minutes, 30 cycles of 94–96°C for 30–45 seconds, 50–65°C for 30–45 seconds, and 72°C for a fixed time (600 bp / min), followed by a 10-minute extension at 72°C.

[0119] 2. PCR for amplification of long fragments (>2500 bp): The long fragment amplification used in this invention primarily involves amplifying vectors using inverse PCR, a technique used to obtain site-specific mutations in the following examples. Reverse PCR primers are designed for the mutation site, with the mutated nucleotide sequence at the 5' end of one of the primers. The amplified product contains the corresponding mutation site. The PCR reaction system for long fragment amplification is as follows: 1 μL (10 pg to 1 ng) of template sequence, 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 10 μL of 5x PrimerSTAR buffer, 0.5 μL of PrimerSTAR DNA polymerase, 4 μL of dNTPs (2.5 mM), and 33.5 μL of sterile ultrapure water (ddHO), for a total volume of 50 μL. The PCR amplification program was denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50–68°C for 5–15 seconds, 72°C for a fixed time (1000 bp / min)), and extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a fixed time (1000 bp / min)), and extension at 72°C for 10 minutes.

[0120] II.2 Endonuclease digestion reaction: The double enzyme digestion system for plasmid vectors was as follows: 20 μL (approximately 1.5 μg) of plasmid vector, 5 μL of 10x buffer, 11-2 μL of restriction enzyme 1, 1-2 μL of restriction enzyme 2, and sterile ultrapure water to a total volume of 50 μL. Reaction conditions were 37°C, 1-7 hours.

[0121] II.3 Phosphorylation of the 5' ends of DNA fragments While plasmids and genomes extracted from microorganisms contain phosphate groups at their termini, PCR products do not. Therefore, a phosphate group addition reaction is required at the 5' end of the PCR product. Only DNA molecules with a phosphate group at their termini can undergo ligation. The phosphorylation reaction system is as follows: 5-8 μL of PCR product fragment DNA sequence, 1 μL of 10x T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0122] II.4 Ligation reaction between target fragment and vector The ligation methods between different fragments and vectors are different, and in the present invention, the following three ligation methods were used. 1. Blunt-end ligation of blunt-ended short fragments and linearized vector The principle of this method is to phosphorylate the blunt-end products obtained by PCR at the 5' end of the DNA fragment using T4PNK, then ligate them with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10x T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. Reaction conditions: 22°C, 30 minutes.

[0123] 2. Ligation of sticky-ended DNA fragments with sticky-ended vector fragments DNA fragments digested with restriction enzymes typically generate protruding sticky ends, which can then be ligated to vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of restriction-digested PCR product fragment DNA, 0.5-7 μL of restriction-digested plasmid, 1 μL of 10x T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. Reaction conditions: 16°C, 4-8 hours.

[0124] 3. Self-circularization and ligation of the 5' phosphorylated DNA fragments containing site-specific mutations introduced by inverse PCR The 5'-phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via self-circularization ligation to obtain the recombinant plasmid. The self-circularization ligation reaction system was as follows: 10 μL of phosphorylation reaction system, 0.5 μL of T4 ligase (5 U / μL), and a total volume of 10.5 μL. Reaction conditions were 16°C, 4 to 16 hours.

[0125] II.5 Preparation and transformation of competent cells Preparation of competent cells: 1. A single colony (such as Mach1) was picked and inoculated into 5 mL of LB medium, and cultured overnight at 37°C in a shaker. 2. 0.5 to 1 mL of the overnight cultured bacterial solution was transferred to 50 mL of LB medium and cultured at 37°C, 220 rpm for 3 to 5 hours until the OD600 reached 0.5. 3. Cells were pre-cooled in an ice bath for 2 hours. The mixture was centrifuged at 4000 rpm for 10 minutes at 4.4°C. 5. The supernatant was discarded, and the cells were resuspended in 5 mL of pre-chilled buffer. After the cells were homogenized, resuspension buffer was added to bring the final volume to 50 mL. 6. Ice bath for 45 minutes The mixture was centrifuged at 4000 rpm for 10 min at 7.4°C, and the bacteria were resuspended in 5 mL of storage buffer pre-chilled on ice. 8. 100 μL of bacterial solution was placed in each EP tube and frozen at -80°C or in liquid nitrogen.

[0126] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM) Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10× CaCl2 (1 M), 1 mL 10× MgCl2 (700 mM), 1 mL 10× NaAc (400 mM), 4.6 mL ddH2O

[0127] Transformation of competent cells: 1. Thaw 100 μL of competent cells in an ice bath. 2. Add an appropriate volume of ligation product, gently mix until homogenous, and place in an ice bath for 30 minutes. The volume of the ligation product added is usually less than 1 / 10 of the volume of the competent cells. 3. The bacterial solution was heat shocked in a 42°C water bath for 90 seconds, then immediately transferred to an ice bath and left for 5 minutes. 4. 500 μL of LB was added, and the mixture was cultured in a thermostatic shaker at 37°C and 200 rpm for 1 hour. 5. The bacterial solution was centrifuged at 4000 rpm for 3 minutes, 200 μL of the supernatant was retained, the bacteria were mixed uniformly by blowing, and the mixture was spread evenly on the surface of an agar plate containing an appropriate antibiotic. The plate was then inverted and placed in a 37°C incubator overnight.

[0128] II.6 Protein expression, purification and fluorescence detection 1. Transform the expression vector (e.g., pCDF-based phosphoenolpyruvate optical probe expression vector) into BL21(DE3) cells and culture overnight by inversion. Transfer the clone from the plate to a 250 mL Erlenmeyer flask, place it in a shaker at 37°C, and culture at 220 rpm until the OD reaches 0.4-0.8. Add 1 / 1000 (v / v) IPTG (1 M) and induce expression at 18°C ​​for 24-36 hours. 2. After the induction of expression was completed, the bacteria were collected by centrifugation at 4000 rpm for 30 minutes, the bacterial pellet was resuspended in 50 mM phosphate buffer, and the bacteria were sonicated until transparent. The mixture was centrifuged at 9600 rpm for 20 minutes at 4°C. 3. The supernatant was centrifuged and passed through a self-made nickel column affinity chromatography column to purify the protein. The protein purified through the nickel column affinity chromatography column was further passed through a self-made desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4). 4. After identifying the purified protein by SDS-PAGE, the probe was diluted to a final concentration of 0.2-5 µM in assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). A stock solution of phosphoenolpyruvate was prepared in assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a final concentration of 50 mM. 100 μL of the 5.1 μM protein solution was incubated at 37°C for 10 minutes, and then titrated with phosphoenolpyruvate. The fluorescence intensity of the protein emitted at 528 nm after excitation at 420 nm and at 528 nm after excitation at 485 nm was measured. Fluorescence excitation and emission measurements of the samples were completed using a multifunctional fluorescence microplate reader. 100 μL of the 6.1 μM protein solution was incubated at 37°C for 10 minutes, phosphoenolpyruvate was added, and the absorption and fluorescence spectra of the protein were measured. The absorption and fluorescence spectra of the sample were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0129] II.7 Transfection into mammalian cells and fluorescence detection 1. 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% CO2. Fluorescence detection was performed 24–36 hours after full expression of the foreign gene. 2. After the induction of expression was completed, the adhered HEK293 cells were rinsed with PBS three times, placed in HBSS solution, and detected by fluorescence microscopy and microplate reader, respectively.

[0130] Example 1: Phosphoenolpyruvate binding protein plasmid The TtPFK gene in Thermus thermophilus was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI. At the same time, the pCDF vector was double-digested. After ligation with T4 DNA ligase, the product was transformed into DH5α. The transformed DH5α was plated on an LB plate (100 μg / mL streptomycin) and grown overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. After the positive plasmid was verified to be correct by sequencing, the next plasmid construction was carried out.

[0131] Example 2: Expression and detection of cpYFP optical probes at different insertion sites In this example, based on pCDF-TtPFK and according to the crystal structure of phosphoenolpyruvate-binding protein, the following sites were selected for inserting cpYFP, ​​and the corresponding pCDF-TtPFK-cpYFP plasmids were obtained: 68 / 69, 68 / 70, 69 / 70, 68 / 71, 69 / 71, 70 / 71, 168 / 169, 168 / 170, 169 / 170, 168 / 1 As an exemplary exhibit, the amino acid and nucleic acid sequences of 254 / 255-TtPFK-cpYFP are set forth in SEQ ID NOs: 10 and 11, respectively.

[0132] PCR was used to generate a DNA fragment of cpYFP, ​​simultaneously introducing a homologous sequence to the cpYFP terminus through the 5' end of the primer. PCR amplification produced the pCDF-phosphoenolpyruvate-binding protein linearized vector, which contained sequences (15-20 bp) at its 5' and 3' ends that perfectly matched those at both ends of cpYFP. The linearized pCDF-TtPFK and cpYFP fragment underwent homologous recombination using Hieff Clone Enzyme. The product was then transformed into DH5α cells, which were then plated onto LB plates (100 μg / mL streptomycin) and grown overnight at 37°C. Plasmids from positive clones identified by PCR were extracted and sequenced. Sequencing was performed by JIELI Sequencing Company.

[0133] After verifying the accuracy by sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis revealed that the protein size was approximately 63 kDa. This size was consistent with the size of the TtPFK-cpYFP fusion protein containing a His-tag purification tag expressed by pCDF-TtPFK-cpYFP. The results are shown in Figure 1.

[0134] A phosphoenolpyruvate response screen was performed using the supernatant of disrupted E. coli cells expressing the TtPFK-cpYFP fusion protein. 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. As shown in Table 1, the results showed that the optical probes inserted into the 69 / 71, 170 / 171, 171 / 172, 254 / 255, 254 / 256, and 255 / 257 sites in the supernatant of disrupted E. coli cells expressing the TtPFK-cpYFP fusion protein responded to phosphoenolpyruvate by 1.3-fold or more. The 254 / 255 and 254 / 256 sites were not affected by other TtPFK-binding substrates.

[0135] [Table 1]

[0136] Example 3: Expression and detection of cpGFP optical probes at different insertion sites Following the method described in Example 2, a phosphoenolpyruvate-binding green fluorescent protein (GPP) probe was constructed by replacing cpYFP with cpGFP. As shown in Table 2, the detection results showed that, among the optical probes in the supernatant of disrupted E. coli cells expressing the TtPFK-cpGFP fusion protein, the optical probes inserted into the 171 / 172, 254 / 255, and 254 / 256 amino acid sites or the amino acid sites corresponding to these family proteins, responded to phosphoenolpyruvate by 1.3-fold or more. The response to phosphoenolpyruvate at the 254 / 255 and 254 / 256 sites was not affected by other TtPFK-binding substrates.

[0137] [Table 2]

[0138] Example 4: Expression and detection of cpBFP optical probes at different insertion sites Following the method described in Example 2, a phosphoenolpyruvate blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpBFP. As shown in Table 3, the detection results showed that, among the optical probes in the supernatant of disrupted E. coli cells expressing the TtPFK-cpBFP fusion protein, the optical probes inserted into the 170 / 171, 171 / 172, 254 / 255, and 254 / 256 amino acid sites or the amino acid sites corresponding to these family proteins, responded to phosphoenolpyruvate by 1.3-fold or more. Among these, the 254 / 255 and 254 / 256 sites were not affected by other TtPFK-binding substrates in their response to phosphoenolpyruvate.

[0139] [Table 3]

[0140] Example 5: Expression and detection of cpmApple optical probe at different insertion sites Following the method described in Example 2, a phosphoenolpyruvate red fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpmApple. As shown in Table 4, the detection results showed that, among the optical probes in the supernatant of disrupted E. coli cells expressing the TtPFK-cpmApple fusion protein, the optical probes inserted into the 170 / 171, 171 / 172, 254 / 255, and 254 / 256 amino acid sites or their corresponding amino acid sites in family proteins, responded 1.3-fold or more to phosphoenolpyruvate. Among these, the 254 / 255 and 254 / 256 sites were not affected by other TtPFK-binding substrates in their response to phosphoenolpyruvate.

[0141] [Table 4]

[0142] Example 6: Expression and detection of linker-mutated cpYFP optical probes Based on the optical probes obtained in Example 2 that reacted 1.3-fold more strongly to phosphoenolpyruvate and were unaffected by other binding substrates of TtPFK (i.e., two optical probes inserted into the 254 / 255 and 254 / 256 sites of TtPFK), the probes were linearized by inverse PCR, linker mutation site sequences were introduced into the primers, and the resulting PCR products were subjected to homologous recombination using Hieff Clone Enzyme to establish a mutant library. As an exemplary demonstration, the amino acid and nucleic acid sequences of 254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S are shown in SEQ ID NOs: 12 and 13, respectively.

[0143] The mutant library recombinant plasmid was transformed into BL21(DE3), expression was induced, the probe protein was purified, and a phosphoenolpyruvate response screening was performed. The detection signal of the fusion fluorescent protein containing 2 mM phosphoenolpyruvate was divided by the detection signal of the fusion fluorescent protein without phosphoenolpyruvate. The detection results show that the optical probes with a response to phosphoenolpyruvate of 2-fold or more are shown in Table 5.

[0144] [Table 5] JPEG2026507391000006.jpg28167

[0145] Example 7: Expression and detection of cpYFP optical probes with binding pocket mutations The optical probes 254 / 255-TtPFK-cpYFP obtained in Example 2 and 254 / 255-TtPFK-I253G / Q254L / R255L / G256F-cpYFP-Y1P, 254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S, and 254 / 256-TtPFK-I253Q / Q254L / G256V / G257L-cpYFP-Y1T / N246F obtained in Example 6 were phosphoenoic The affinity and substrate specificity of the phosphoenolpyruvate optical probe for phosphoenolpyruvate were tuned by amino acid mutations at positions 21, R25, V54, R55, V57, A58, N59, I60, I61, R155, G186, R212, G213, K214, K215, S216, and L320 in the ATP-binding pocket, and at positions R72, R163, R172, and R246 in the ATP-binding pocket. The probe was linearized by inverse PCR, and the sequences of the mutation sites were introduced into primers. The resulting PCR products were then subjected to homologous recombination to obtain plasmids containing the mutations at the above sites. The mutant plasmids were transformed into BL21(DE3) and expression was induced. The mutant probe proteins were purified and screened for phosphoenolpyruvate response. The detection signal of the fusion fluorescent protein containing 2 mM phosphoenolpyruvate was divided by the detection signal of the fusion fluorescent protein without phosphoenolpyruvate. The optical probes with a response to phosphoenolpyruvate of 2-fold or more are shown in Table 6.

[0146] [Table 6] JPEG2026507391000008.jpg67169

[0147] Example 8. Performance of optical probe mutants As an example, the two purified phosphoenolpyruvate optical probes described in Examples 6 and 7 were treated with 0 mM and 5 mM phosphoenolpyruvate, respectively, for 10 minutes, and the fluorescence spectra were detected using a fluorescence spectrophotometer.

[0148] Measurement of excitation spectrum: The excitation spectrum was recorded with an excitation range of 370 nm to 510 nm and an emission wavelength of 530 nm, and readings were taken every 5 nm. The results, as shown in Figure 2, showed that the probe had two excitation peaks at approximately 410 nm and 490 nm.

[0149] Measurement of emission spectra: The excitation wavelengths were fixed at 420 nm and 460 nm, and emission spectra were recorded at 470-600 nm and 490-600 nm, with readings taken every 5 nm. The results are shown in Figure 2.

[0150] The purified 81 phosphoenolpyruvate probes described in Examples 6 and 7 were subjected to phosphoenolpyruvate detection at a concentration gradient (0-10 mM). After 10 minutes of treatment with the purified probes, the change in the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was detected. As shown in Figure 3, the K of the 81 phosphoenolpyruvate optical probes was d The 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, 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 and 2.365 μM.

[0151] We detected the reactivity of 81 phosphoenolpyruvate optical probes with three TtPFK-binding substrates: ATP, ADP, and F6P, and one phosphoenolpyruvate optical probe with glucose, lactate, pyruvate, F6P, FBP, and other intermediates of glycolysis. As shown in Figure 4, the results showed good specificity.

[0152] Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles In this example, different localization signal peptides were fused to the optical probe 254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S, and the optical probe was localized to various organelles.

[0153] Optical probe plasmids fused to different localization signal peptides were transfected into 293 cells for 36 hours, washed with PBS, placed in HBSS solution, and then subjected to fluorescence detection under the FITC channel using an inverted fluorescence microscope. The results are shown in Figure 5. By fusing phosphoenolpyruvate optical probes with localization signal peptides of different specificities, they can be localized to intracellular organelles, such as the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nucleoplasm (excluding nucleoli). Fluorescence was observed in different intracellular structures, with different fluorescence distributions and intensities.

[0154] 293 cells were transfected with the cytoplasmic optical probe plasmid for 36 hours, then washed with PBS and placed in HBSS solution. The change in the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was measured for 30 minutes. The results are shown in Figure 6. 5 mM oxalate was added and detection continued for 30 minutes. The 485 / 420 ratio of the sample with oxalate gradually increased, reaching a maximum of 3.5 times the initial value, while the 485 / 420 ratio of the control group without oxalate remained unchanged at 0.437.

[0155] Example 10: Optical probe-based high-throughput compound screening in living cells In this example, HeLa cells expressing 254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S in the cytoplasm were used for high-throughput compound screening.

[0156] Transfected 293 cells were washed with PBS, placed in HBSS solution (without phosphoenolpyruvate), and treated for 1 hour, followed by treatment with 10 μM compounds for 1 hour. Phosphoenolpyruvate was added dropwise to each sample. Using a microplate reader, the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was recorded. Normalization was performed using a sample untreated with any compound as a control. The results are shown in Figure 7. Of the 2,000 compounds used, the majority had little effect on the cellular uptake of phosphoenolpyruvate. Twenty-three compounds were able to promote cellular uptake of phosphoenolpyruvate, and seven compounds were able to significantly decrease cellular uptake of phosphoenolpyruvate.

[0157] Example 11: Quantitative detection of phosphoenolpyruvate in blood using an optical probe In this example, phosphoenolpyruvate in mouse and human blood supernatants was analyzed using purified 254 / 255-TtPFK-S216I-cpYFP, ​​which has a Kd of approximately 0.24 μM.

[0158] After mixing 254 / 255-TtPFK-S216I-cpYFP with diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was measured using a microplate reader. The results are shown in Figure 8. The phosphoenolpyruvate content in mouse blood was approximately 91 μM, and the phosphoenolpyruvate content in human blood was approximately 18 μM.

[0159] As can be seen from the above examples, the phosphoenolpyruvate optical probe provided by the present invention has a relatively small protein, is easy to mature, exhibits large dynamic changes in fluorescence, and has excellent specificity. It can be expressed intracellularly using genetic engineering methods, and can localize and detect phosphoenolpyruvate inside and outside the cells in real time in a high-throughput and quantitative manner, and also enables high-throughput compound screening.

[0160] Other embodiments Although various embodiments have been described herein, it should be understood that the appended claims also encompass various modifications that may occur to those skilled in the art upon reading this specification without departing from the concept and scope of the present invention.

[0161] Sequences herein 1>TtPFK (1-322 full length) MKRIGVFTSGGDAPGMNAAIRAVVRQAHALGVEVIGIRRGYAGMIQGEMVPLGVRDVANIIQRGGTILLTARSQEFLTEEGRAKAYAKLQAAGIEGLVAIGGDGTFRGALFLVEEHGMPVVGVPGTIDNDLYGTDYTIGFDTAVNTALEAIDRIRDTAASHERVFFIEVMGRHAGFIALDVGLAGGAEVIAVPEEVDPKAVAEVLEASQRRGKKSSIVVVAEGAYPGGAAGLLAAIREHLQVEARVTVLGHIQRGGSPTAKDRILASRLGAPAVEALVGGASGVMVGEVEGEVDLTPLKEAVERRKDINRALLRLLSQVLAL 2>cpYFP YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN 3>cpmOrange VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA 4>cpmKate MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDMALKLVGGGHLICNLKTTYRSKK 5>mCherry MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK 6>cpGFP NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN 7>cpBFP NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN 8>mKate MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN 9>cpmApple VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA 10>254 / 255-TtPFK-cpYFP MKRIGVFTSGGDAPGMNAAIRAVVRQAHALGVEVIGIRRGYAGMIQGEVMPLGVRDVANIIQRGGTILLTARSQEFLTEEGRAKAYAKLQAAGIEGLVAIGGDGTFRGALFLVEEHGMPVVGVPGTIDNDLYGTDYTIGFDT AVNTALEAIDRIRDTAASHERVFFIEVMGRHAGFIALDVGLAGGAEVIAVPEEPVDPKAVAEVLEASQRRGKKSSIVVVAEGAYPGGAAGLLAAIRHLQVEARVTVLGHIQYNSDNVYIMADKQKNGIKANFKIRHNVEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKC FARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNRGGSPTAKDRILASRLGAPAVEALVGGASGVMVGEVEGEVDLTPLKEAVERKDINRALLRLSQVLAL 11>254 / 255-TtPFK-cpYFP nucleic acid sequence 12>254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S MKRIGVFTSGGDAPGMNAAIRAVVRQAHALGVEVIGIRRGYAGMIQGEVMPLGVRDVANIIQRGGTILLTARSQEFLTEEGRAKAYAKLQAAGIEGLVAIGGDGTFRGALFLVEEHGMPVVGVPGTIDNDLYGTDYTIGFDT AVNTALEAIDRIRDTAASHERVFFIEVMGRHAGFIALDVGLAGGAEVIAVPEEPVDPKAVAEVLEASQRRGKKSSIVVVAEGAYPGGAAGLLAAIRHLQVEARVTVLGHKLNSDNVYIMADKQKNGIKANFKIRHNVEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKC FARYPDMHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLFGSPTAKDRILASRLGAPAVEALVGGASGVMVGEVEGEVDLTPLKEAVERKDINRALLRLSQVLAL 13>254 / 255-TtPFK-I253K / Q254L / R255L / G256F-cpYFP-Y1S nucleic acid sequence

Claims

1. (a) having the sequence set forth in SEQ ID NO: 1 and having mutations, including amino acid modifications, substitutions or deletions, at one, two, three, four, five or more sites selected from the following: I253, Q254, R255, G256, G257, R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320; (b) a sequence having at least 70% sequence identity with the sequence of (a), and having the mutations described in (1), and retaining the ability to bind phosphoenolpyruvate; a phosphoenolpyruvate binding protein mutant characterized by: Preferably, the mutation described in (a) comprises a mutation at any one or more of the following groups of sites:

2. a phosphoenolpyruvate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the phosphoenolpyruvate-sensitive polypeptide; Phosphoenolpyruvate-sensitive polypeptides are (1) a sequence set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto and retaining phosphoenolpyruvate binding activity; (2) A functional mutant of the sequence set forth in SEQ ID NO: 1, wherein the functional mutant has a mutation within the 5 amino acids linked to the optically active polypeptide and / or has a mutation at one, two, three, four, five or more positions selected from the following: R21, V54, R55, V57, A58, N59, I60, R72, R212, G213, K215, S216, L320, or (3) A sequence having at least 70% sequence identity with the sequence described in (2), having the mutation described in (2), and retaining phosphoenolpyruvate sensitivity. and Preferably, (2) is the sequence of a phosphoenolpyruvate binding protein mutant as defined in claim 1. A phosphoenolpyruvate optical probe comprising:

3. 3. The optical probe of claim 2, wherein the optically active polypeptide is located at or replaces residues 68-71, 168-173 and / or 253-257 of the phosphoenolpyruvate-sensitive polypeptide.

4. The optically active polypeptide is located at 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 / 1 72, 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 and / or 256 / 257 3. An optical probe according to claim 2; More preferably, The optically active polypeptide is located at 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; More preferably, the optically active polypeptide is a fluorescent protein or a functional mutant thereof; Preferably, The fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2 to 9; The functional mutant of the fluorescent protein has a mutation within the three amino acids linked to the optically active polypeptide; preferably, the functional mutant of the fluorescent protein has a mutation at the position corresponding to the amino acid at position 1 of SEQ ID NO: 2, and the mutation is preferably selected from Y1S, Y1G, Y1N, Y1T, Y1P, Y1A, Y1E, and Y1H; The functional mutant of the fluorescent protein has a mutation at the position corresponding to amino acid 246 of SEQ ID NO: 2, and said mutation is preferably selected from N246F, N246C, N246W or N246H; More preferably, in said optical probe, the phosphoenolpyruvate-sensitive polypeptide is set forth 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, G213T, K215H, K215D, K215E, S216D, S216L, S216Y, S216I, S216M, S216F, S216Q, L320Y, L320N and / or L320F, the optically active polypeptide is The optically active polypeptide is represented by ID NOs: 2 to 9, or a mutant thereof having one or more of the following mutations: Y1S, Y1G, Y1N, Y1T, Y1P, Y1A, Y1E, Y1H, N246F, N246C, N246W, and is located at positions 254 / 255 or 254 / 256 of the phosphoenolpyruvate-sensitive polypeptide.

5. (a) a coding sequence for an optical probe according to any one of claims 2 to 4; (b) the complementary sequence of (a) A nucleic acid molecule comprising:

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

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

8. (1) An optical probe according to any one of claims 2 to 4, (2) A nucleic acid sequence according to claim 5, (3) A nucleic acid construct according to claim 6, or (4) The host cell according to claim 7. a detection kit comprising: The detection kit optionally includes other reagents necessary for detecting phosphoenolpyruvate using an optical probe; Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer, a medium, and a phosphoenolpyruvate standard.

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

10. Use of an optical probe according to any one of claims 2 to 4, a nucleic acid sequence according to claim 5, a nucleic acid construct according to claim 6 or a host cell according to claim 7 in the detection of phosphoenolpyruvate in a sample, in the screening of compounds or in the intracellular and / or extracellular localization of phosphoenolpyruvate; Preferably, Detecting phosphoenolpyruvate in the sample includes contacting the optical probe or the host cell with the sample, detecting an optical change in the optically active polypeptide, and detecting phosphoenolpyruvate in the sample based on the optical change in the optically active polypeptide; The screening of the compound comprises contacting the optical probe or host cell with a candidate compound in a system comprising phosphoenolpyruvate, detecting an optical change in the optically active polypeptide, and screening the candidate compound based on the optical change in the optically active polypeptide; preferably, the screening of the compound comprises contacting the host cell with a candidate compound in a system comprising phosphoenolpyruvate, and the optical change in the optically active polypeptide indicates whether the candidate compound can regulate the cellular uptake of phosphoenolpyruvate; said intracellular and / or extracellular localization of phosphoenolpyruvate comprises contacting said optical probe or said host cell with a system containing phosphoenolpyruvate and detecting an optical change in the optically active polypeptide; More preferably, the system is a solution system, a cell system, or an intracellular system.

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