CN119490571A

CN119490571A - A glyoxylic acid optical probe and its preparation method and application

Info

Publication number: CN119490571A
Application number: CN202311035167.7A
Authority: CN
Inventors: 杨弋; 赵玉政; 李写; 刘雅倩; 陈晓倩
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2025-02-21

Abstract

本发明涉及一种乙醛酸光学探针及其制备方法和应用。一方面，本发明涉及一种光学探针，包含乙醛酸敏感多肽或其功能变体和光学活性多肽或其功能变体，其中光学活性多肽或其功能变体位于乙醛酸敏感多肽或其功能变体的序列内。本发明也涉及上述探针的制备方法及其在检测乙醛酸中的应用。The present invention relates to a glyoxylic acid optical probe and a preparation method and application thereof. On the one hand, the present invention relates to an optical probe comprising a glyoxylic acid-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located within the sequence of the glyoxylic acid-sensitive polypeptide or a functional variant thereof. The present invention also relates to a preparation method of the above-mentioned probe and its application in detecting glyoxylic acid.

Description

Glyoxylate optical probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of optical probes, in particular to a glyoxylate optical probe and a preparation method and application thereof.

Background

Glyoxylic acid (Glyoxylate) is an organic compound containing 2 carbon atoms, having both aldehyde and carboxylic acid groups. In the organism, the glyoxylic acid metabolic disorder can cause oxalic acid accumulation to induce homooxalic urine, so that the method has important significance for rapid, sensitive and specific measurement of glyoxylic acid.

TAPAS PATRAS et al found that with increasing glyoxylate concentration, growth of HT-29 and HCT-116 cells was significantly inhibited by MTT and colony formation experiments. In vitro experiment results show that the glyoxylate with the super-physiological concentration has an anti-proliferation effect on human colon cancer cells through the mediation of oxidative stress, and the glyoxylate can be regarded as a novel colon cancer treatment strategy. Patel et al demonstrate that glyoxylic acid can inhibit calcitriol receptor-vitamin D response element interactions in vitro. Glyoxylic acid also blocks calcitriol-induced expression of chloramphenicol acetyl transferase activity in transiently transfected JEG cells and inhibits rat intestinal 24-hydroxylase activity. These findings indicate that glyoxylic acid can inhibit the binding of VDR to VDRE, which may alter the biological role of calcitriol in renal failure. Victoria j. Et al use metabolite profiles to describe human plasma in diabetic and non-diabetic patients, with elevated levels of reactive glyoxylate prior to measuring the high levels of blood glucose typical of diabetes. Elevated plasma glyoxylate levels were also observed in the C57BLKS/J-Lepr ^db/db-/- mouse model, supporting the results of glyoxylate associated with the diabetic phenotype. Glyoxylate as a novel marker for diabetes contributes to the further development of improved novel diagnostic and therapeutic methods.

The most commonly used method for detecting glyoxylic acid is a high-performance liquid phase method at present, but the detection and analysis method needs professional analysis instruments and equipment, has complicated steps, is only suitable for in-vitro detection, and cannot monitor the glyoxylic acid concentration change in living cells in real time. Therefore, development of a new detection method is needed to realize the real-time positioning, quantification and high-throughput detection of glyoxylic acid in cells and outside cells, which is simple, convenient and rapid and has high specificity.

Disclosure of Invention

The invention aims to provide a probe and a method for detecting glyoxylic acid in real time in a cell and outside the cell in a high-throughput and quantitative manner.

In order to achieve the above object, the present invention provides the following technical solutions:

the first aspect of the present invention provides a glyoxylate binding protein variant which:

(1) Having the sequence shown in SEQ ID NO. 1 and having a mutation at 1, 2 or 3,4, 5, 6 or 7 positions E115, N116, I118, G119, T59, S139, S141 comprising a modification, substitution or deletion of an amino acid,

(2) Is a truncated variant of (1) having amino acids 76-246, or

(3) Is a sequence having at least 70% sequence identity to the sequence of (1) or (2) and having the mutation of (1) and retaining the ability to bind glyoxylate.

In one or more embodiments, the mutation comprises a mutation selected from the group consisting of E115, N116, I118, G119, optionally, the mutation further comprises a mutation selected from the group consisting of T59, S139, S141.

In one or more embodiments, the mutations include mutations at a site selected from any of (1) I118 and G119, (2) I118, (3) E115, N116, (4) I118, E115, N116, or the mutations comprise mutations ：(5)T59、I118、E115、N116,(6)T59、E115、N116,(7)I118、E115、N116、S139,(8)E115、N116、S139,(9)I118、E115、N116、S141,(10)E115、N116、S141,(11)T133 and I118 at a site selected from any of (12) I118 and S213, (13) I118 and S215.

In one or more embodiments, I118 is mutated to G, Q, T, L, M, V, H, F, S, A, Y or K. In one or more embodiments, G119 is mutated to N, V or S. In one or more embodiments, E115 is mutated to N, S, P, T or Q. In one or more embodiments, N116 is mutated to V, C, K, E, I, T, R or W. In one or more embodiments, T59 is mutated to G or N. In one or more embodiments, S139 is mutated to N or T. In one or more embodiments, S141 is mutated to D, H or G.

In one or more embodiments, the mutation comprises a mutation selected from any one of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K; preferably, the mutation comprises a mutation selected from any one of (6) E115N and N116C, (24) I118F, E N, N V, (28) I118Y, E115S, N116K. Optionally, the mutation further comprises any one or more mutations selected from (a) T59G or T59N, (b) S139N or S139T, (c) S141D or S141G.

In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups ：(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In another aspect the invention provides a glyoxylate optical probe comprising a glyoxylate sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located within the sequence of the glyoxylate sensitive polypeptide or the functional variant thereof. The glyoxylic acid-sensitive polypeptide or a functional variant thereof is separated into a first part and a second part by the optically active polypeptide or a functional variant thereof.

In one or more embodiments, the glyoxylate optical probe comprises a glyoxylate-sensitive polypeptide B and an optically-active polypeptide a, wherein the optically-active polypeptide a is located within the sequence of the glyoxylate-sensitive polypeptide B, dividing the glyoxylate-sensitive polypeptide B into a first part B1 and a second part B2, forming a probe structure of the formula B1-a-B2.

In one or more embodiments, the glyoxylate-sensitive polypeptide comprises a glyoxylate-binding protein or a functional variant thereof. In one or more embodiments, the glyoxylate-sensitive polypeptide is derived from Thermotoga maritima.

In one or more embodiments, the glyoxylate-sensitive polypeptide has:

(1) The sequence shown in SEQ ID No. 1 or a truncated variant thereof having amino acids 76-246 or a sequence which has at least 70% sequence identity thereto and retains binding activity to glyoxylate,

(2) The sequence of the glyoxylate binding protein variant of any one of the embodiments of the first aspect herein, or

(3) Sequences having at least 70% sequence identity to the sequence of (2) and having the mutation of (2) and retaining sensitivity to glyoxylate.

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

In one or more embodiments, the optically active polypeptide is located between residues 17-20,28-31,54-59,67-71,107-110,114-120,128-133,142-149 of the glyoxylate-sensitive polypeptide, numbering corresponding to the full length of the glyoxylate-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more ：17/18,17/19,17/20,17/21,18/19,18/20,18/21,19/20,19/21,20/21,28/29,28/30,28/31,28/32,29/30,29/31,29/32,30/31,30/32,31/32,54/55,54/56,54/57,54/58,54/59,54/60,55/56,55/57,55/58,55/59,55/60,56/57,56/58,56/59,56/60,57/58,57/59,57/60,58/59,58/60,59/60,67/68,67/69,67/70,67/71,67/72,68/69,68/70,68/71,68/72,69/70,69/71,69/72,70/71,70/72,71/72,107/108,107/109,107/110,107/111,108/109,108/110,108/111,109/110,109/111,110/111,114/115,114/116,114/117,114/118,114/119,114/120 114/121,115/115,115/116,115/117,115/118,115/119,115/120,115/121,116/115,116/116,116/117,116/118,116/119,116/120,116/121,117/115,117/116,117/117,117/118,117/119,117/120,117/121,118/115,118/116,118/117,118/118,118/119,118/120,118/121,119/115,119/116,119/117,119/118,119/119,119/120,119/121,120/115,120/116,120/117,120/118,120/119,120/120,120/121,128/129,128/130,128/131,128/132,128/133,128/134,129/130,129/131,129/132,129/133,129/134,130/131,130/132,130/133,130/134,131/132,131/133,131/134,132/133,132/134,133/134,142/143,142/144,142/145,142/146,142/147,142/148,142/149,142/150,143/144,143/145,143/146,143/147,143/148,143/149,143/150,144/145,144/146,144/147,144/148,144/149,144/150,145/146,145/147,145/148 145/149,145/150,146/147,146/148,146/149,146/150,147/148,147/149,147/150,148/149,148/150,149/150. of the following positions of the glyoxylate-sensitive polypeptide, more preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide ：114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115,120/120.

In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the invention may be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide is flanked by linkers of no more than 5 amino acids, e.g., 0, 1,2, 3, 4 amino acids.

In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, the linker Y is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is shown as a first portion B1-Y of the glyoxylate sensitive polypeptide and a second portion B2 of the glyoxylate sensitive polypeptide. In one or more embodiments, the optical probes of the present invention do not comprise a linker.

In one or more embodiments, the optical probes of the invention further comprise a localization sequence for localizing the probe to a specific organelle, e.g., a cell.

In one or more embodiments, the optically active polypeptide is cpYFP, which is located in any one or more ：114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115,120/120, of the following positions selected from the group consisting of glyoxylate-sensitive polypeptides (1) as shown in SEQ ID NO. 1 or a truncated variant thereof having amino acids 76-246, or (2) is a glyoxylate-binding protein variant as described in any one of the embodiments of the first aspect herein.

In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of the following positions 117/120,119/115,119/116,120/115 and 120/117 of the glyoxylate-sensitive polypeptide shown in positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (1) is shown in SEQ ID NO. 1 or is a truncated variant thereof having amino acids 76-246, or (2) is a glyoxylate-binding protein variant according to any one of the embodiments of the first aspect herein.

In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following positions 114/117,114/118,119/119 and 119/120 of the glyoxylate-sensitive polypeptide shown at positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (1) is shown as SEQ ID NO. 1 or is a truncated variant thereof having amino acids 76-246, or (2) is a glyoxylate-binding protein variant as described in any of the embodiments of the first aspect herein.

In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following positions 117/115,118/119,118/120 and 120/121 of the glyoxylate-sensitive polypeptide shown at positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (1) is shown as SEQ ID NO. 1 or is a truncated variant thereof having amino acids 76-246, or (2) is a glyoxylate-binding protein variant as described in any of the embodiments of the first aspect herein.

In one or more embodiments, the optically active polypeptide is cpYFP, which is located in any one or more ：114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115,120/120, of the glyoxylate-sensitive polypeptides (I) as shown in SEQ ID NO. 1 or a truncated variant thereof having amino acids 76-246, or (ii) is a variant of (I) having a mutation selected from any of the group consisting of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K,

(8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of positions 117/120,119/115,119/116,120/115 and 120/117 of the glyoxylate-sensitive polypeptide shown at positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (I) is shown as SEQ ID NO. 1 or is a truncated variant thereof with amino acids 76-246, or (ii) is a variant of (I) having a mutation selected from any of the group consisting of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116C N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,

(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following positions 114/117,114/118,119/119 and 119/120 of the glyoxylate-sensitive polypeptide shown at positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (I) is shown as SEQ ID NO. 1 or which has a truncated variant of amino acids 76-246, or (ii) is a variant of (I) having a mutation selected from any of the group consisting of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116C N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of positions 117/115,118/119,118/120 and 120/121 of the glyoxylate-sensitive polypeptide shown at positions 76-246 of SEQ ID NO. 1, which glyoxylate-sensitive polypeptide (I) is shown as SEQ ID NO. 1 or is a truncated variant thereof with amino acids 76-246, or (ii) is a variant of (I) having a mutation selected from any of the group consisting of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116C N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NOS 10-49 or variants thereof. In one or more embodiments, the optical probes provided herein comprise sequences that are 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identical to any of the amino acid sequences SEQ ID NOs 10-49. Preferably, the optical probes provided by the present invention comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10 to 49.

In another aspect, the invention also provides fusion polypeptides comprising an optical probe as described herein and other polypeptides. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptide is located at the N-terminus and/or the C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, tags for purification, or tags for immunoblotting. The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.

In another aspect, the invention provides a nucleic acid molecule comprising (1) a coding sequence for a polypeptide or probe as described in any of the embodiments herein, or (b) a complement of (a), or (c) a fragment of (a) or (b). The fragments are primers.

In one or more embodiments, the nucleic acid sequence comprises an amino acid sequence encoding any one of SEQ ID NOs 23 to 49. Preferably, the nucleic acid sequence comprises any one of the nucleotide sequences SEQ ID NO. 23 or a variant thereof. More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any of the nucleotide sequences SEQ ID NO. 23, or comprises a nucleotide sequence substantially similar to or identical to any of the nucleotide sequences SEQ ID NO. 23.

The invention also relates to the complementary sequences of the above-mentioned nucleic acid sequences or variants thereof, which may comprise nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the optical probes or fusion proteins of the invention or the complementary sequences thereof.

The invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes an optical probe or fusion polypeptide of the invention.

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

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

In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

The invention also provides a host cell comprising (1) an optical probe or fusion polypeptide according to any of the embodiments of the invention, (2) a nucleic acid molecule according to any of the embodiments of the invention, or (3) a nucleic acid construct according to any of the embodiments of the invention. The host cell is preferably E.coli.

In a further aspect the invention provides a glyoxylic acid detection kit comprising an optical probe as described herein or a fusion polypeptide or polynucleotide or an optical probe prepared by a method as described herein.

In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of buffers, media, glyoxylate standards.

The present invention provides methods of making an optical probe described herein comprising providing a host cell expressing an optical probe or fusion polypeptide described herein, culturing the host cell under conditions in which the cell is expressed, and isolating the optical probe or fusion polypeptide.

In one or more embodiments, a method of making a glyoxylate optical probe or fusion polypeptide described herein comprises the steps of 1) transferring an expression vector encoding a glyoxylate optical probe described herein into a host cell, 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the glyoxylate optical probe.

The invention also provides a method of detecting glyoxylic acid in a sample comprising contacting an optical probe or fusion polypeptide described herein or prepared as described herein with the sample and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular or in situ. Such as blood.

Also provided herein are methods of quantifying glyoxylate in a sample comprising contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, detecting a change in an optically active polypeptide, and quantifying glyoxylate in the sample based on the change in the optically active polypeptide.

The invention also provides a method of screening for a compound (e.g., a drug) comprising contacting an optical probe or fusion polypeptide as described herein or an optical probe or fusion polypeptide prepared as described herein with a candidate compound in a glyoxylic acid-containing system, detecting a change in an optically active polypeptide, and screening the compound for a change in the optically active polypeptide. The method can screen compounds with high throughput.

In one or more embodiments, the host cell described herein is contacted with a candidate compound in a glyoxylate-containing system, and an optical change in the optically active polypeptide is indicative of whether the candidate compound modulates cellular uptake of glyoxylate.

In a further aspect the invention provides a method for intracellular and/or extracellular localization of glyoxylate comprising contacting a glyoxylate-containing system with the optical probe or the host cell and detecting an optical change in an optically active polypeptide.

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

In a further aspect the invention provides the use of a glyoxylate optical probe or fusion polypeptide or host cell as described herein for detecting glyoxylate, screening compounds or intracellular/extracellular localization of glyoxylate in a sample. In one or more embodiments, the positioning is real-time positioning.

The glyoxylate optical probe provided by the application has the beneficial effects that the glyoxylate optical probe is easy to mature, has large dynamic change of fluorescence and good specificity, can be expressed in cells by a gene operation method, can be used for positioning inside and outside the cells in real time, has high flux and quantitatively detects glyoxylate, and omits the time-consuming step of processing samples. The experimental effect shows that the highest response of the glyoxylate optical probe provided by the application to glyoxylate reaches more than 5 times of that of a control, and the glyoxylate optical probe can be used for positioning, qualitatively and quantitatively detecting cells in subcellular structures such as cytoplasm, mitochondria, cell nucleus, endoplasmic reticulum, lysosomes, golgi apparatus and the like, and can be used for high-throughput compound screening and glyoxylate quantitative detection in blood.

Drawings

The invention is further described below with reference to the drawings and examples.

FIG. 1 is a SDS-PAGE diagram of an exemplary glyoxylate optical probe as described in example 1;

FIG. 2 is a graph of titration of response of an exemplary glyoxylate optical probe to different concentrations of glyoxylate as described in example 7;

FIG. 3 is a bar graph of specific detection of 4 glyoxylate analogs by an exemplary glyoxylate optical probe as described in example 7;

FIG. 4 is a photograph of subcellular organelle localization of an exemplary glyoxylate optical probe of example 8 in a mammalian cell;

FIG. 5 is a schematic representation of dynamic monitoring of glyoxylate concentrations in a cytosol in a mammalian cell using an exemplary glyoxylate optical probe of example 8;

FIG. 6 is a plot of high throughput compound screening at the living cell level for an exemplary glyoxylate optical probe as described in example 9;

FIG. 7 is a bar graph of the quantification of glyoxylate in mouse and human blood using an exemplary glyoxylate optical probe described in example 10;

Detailed Description

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

The terms "comprising," including, "and equivalents thereof as used herein include the meaning of" containing "and" consisting of, for example, a composition "comprising" X may consist of X alone or may contain other materials, such as X+Y.

The term "glyoxylate-sensitive polypeptide" or "glyoxylate-responsive polypeptide" as used herein refers to a polypeptide that responds to glyoxylate, including any response of a chemical, biological, electrical or physiological parameter of the polypeptide that is associated with the interaction of the sensitive polypeptide. Responses include small changes, e.g., changes in the orientation of amino acids or peptide fragments of a polypeptide, e.g., changes in the primary, secondary, or tertiary structure of a polypeptide, including, e.g., changes in protonation, electrochemical potential, and/or conformation. A "conformation" is a three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule comprising pendant groups in the molecule, which changes when the three-dimensional structure of the molecule changes. Examples of conformational changes include a transition from an alpha-helix to a beta-sheet or from a beta-sheet to an alpha-helix. It will be appreciated that the detectable change need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered. The glyoxylate-sensitive polypeptides described herein can also include functional variants thereof. Functional variants of a glyoxylate-sensitive polypeptide include, but are not limited to, variants that can interact with glyoxylate to effect the same or similar changes as the parent glyoxylate-sensitive polypeptide.

Glyoxylate-sensitive polypeptides of the invention include, but are not limited to glyoxylate-binding proteins IcIR or variants having greater than 90% homology thereto. The exemplary glyoxylate binding proteins IcIR of the present invention are derived from Thermotoga maritima. IclR is a member of a large family of co-folding transcription regulators that includes a DNA binding domain that interacts with an operator sequence and a C-terminal domain that binds small molecules (C-IclR). The aceBAK operon is an enzyme encoding glyoxylate shunt, which is necessary for the growth of these enzymes on acetate. IclR inhibits expression of the aceBAK operon and its own genes by binding to specific operational sequences of the promoter region. The term "optical probe" as used herein refers to a glyoxylate-sensitive polypeptide fused to an optically active polypeptide. The inventors have found that conformational changes resulting from binding of a glyoxylate-sensitive polypeptide, such as a glyoxylate binding protein, specifically to a physiological concentration of glyoxylate, result in a conformational change of an optically active polypeptide (e.g. a fluorescent protein) which in turn results in a change of the optical properties of the optically active polypeptide. The presence and/or level of glyoxylate can be detected and analyzed by plotting a standard curve from the fluorescence of the fluorescent protein measured at different glyoxylate concentrations. Exemplary IcIR proteins are shown in SEQ ID NO. 1, and exemplary IcIR truncated variants are fragments of SEQ ID NO. 1 comprising amino acids 76-246. When describing the optical probes, glyoxylate-sensitive polypeptides or glyoxylate binding proteins of the invention (e.g. when describing insertion sites or mutation sites), reference is made to the amino acid residue numbers all referring to SEQ ID NO:1.

The "optical probe" of the present invention refers to a glyoxylate-sensitive polypeptide fused to an optically-active polypeptide (e.g., a fluorescent protein) operably inserted into the glyoxylate-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used as a means to detect the responsiveness of an optical probe of the invention. As used herein, the term "fluorescent properties" refers to molar extinction coefficient, fluorescence quantum efficiency, shape of excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of excitation at two different wavelengths, emission amplitude ratio at two different wavelengths, excited state lifetime or fluorescence anisotropy at an appropriate excitation wavelength. The measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrates of the invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a particular wavelength or the integration of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are readily distinguishable in the unactivated and activated conformational state. Optically active polypeptides described herein can also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can undergo a change in the same or similar fluorescent properties as the parent optically active polypeptide.

"Linker" or "junction region" refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the invention. In the present invention, the number of amino acids at the amino terminus of the linking region between the glyoxylate-sensitive polypeptide and the optically-active polypeptide is exemplified by 0 to 3, and the number of amino acids at the carboxy terminus is exemplified by 0 to 2, and when the recombinant optical probe is linked to the functional protein as a basic unit, the amino acids or the carboxy terminus of the recombinant optical probe may be fused. The linker sequence may be a short peptide chain consisting of one or more flexible amino acids, such as Y.

As used herein, the terms "chromophore", "fluorophore" and "fluorescent protein" are synonymous and refer to proteins that fluoresce upon irradiation with excitation light. Fluorescent protein is used as basic detection means in the field of bioscience, such as green fluorescent protein GFP, cyclic rearranged blue fluorescent protein (cpBFP) derived from mutation of the protein, cyclic rearranged green fluorescent protein (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP) and the like, as well as red fluorescent protein RFP, which is commonly used in the field of technology, and cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate and the like. Exemplary fluorescent proteins have the sequence shown in any one of SEQ ID NOS.2-9.

The glyoxylate optical probes of the invention comprise glyoxylate-sensitive polypeptides B, e.g., glyoxylate-binding proteins or variants thereof, and optically-active polypeptides A, e.g., fluorescent proteins. The optical activity polypeptide A is inserted into the glyoxylate sensitive polypeptide B, the B is divided into two parts of B1 and B2 to form a probe structure of a B1-A-B2 formula, and the interaction of the glyoxylate sensitive polypeptide B and the glyoxylate leads to the optical signal of the optical activity polypeptide A to be strong.

In the optical probes of the invention, the optically active polypeptide may be located at any position of the glyoxylate-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located in the N-C direction anywhere along the N-C direction of the glyoxylate sensitive polypeptide. In particular, the optically active polypeptide is located in a flexible region of the glyoxylate-sensitive polypeptide, which refers to a specific structure such as a cyclic domain that exists in the higher structure of a protein, which has higher mobility and flexibility than other higher structures of the protein, and which can undergo a dynamic change in spatial structure conformation upon binding of the protein to a ligand. The flexible region in the present invention mainly refers to the region where the insertion site in glyoxylate binding protein is located, such as amino acid residues 17-20,28-31,54-59,67-71,107-110,114-120,128-133, and 142-149. Illustratively, the optically active polypeptide is located at 17/18,17/19,17/20,17/21,18/19,18/20,18/21,19/20,19/21,20/21,28/29,28/30,28/31,28/32,29/30,29/31,29/32,30/31,30/32,31/32,54/55,54/56,54/57,54/58,54/59,54/60,55/56,55/57,55/58,55/59,55/60,56/57,56/58,56/59,56/60,57/58,57/59,57/60,58/59,58/60,59/60,67/68,67/69,67/70,67/71,67/72,68/69,68/70,68/71,68/72,69/70,69/71,69/72,70/71,70/72,71/72,107/108,107/109,107/110,107/111,108/109,108/110,108/111,109/110,109/111,110/111,114/115,114/116,114/117,114/118,114/119,114/120,114/121,115/115,115/116,115/117,115/118,115/119,115/120,115/121,116/115,116/116,116/117,116/118,116/119,116/120,116/121,117/115,117/116,117/117,117/118,117/119,117/120,117/121,118/115,118/116,118/117,118/118,118/119,118/120,118/121,119/115,119/116,119/117,119/118,119/119,119/120,119/121,120/115,120/116,120/117,120/118,120/119,120/120,120/121,128/129,128/130,128/131,128/132,128/133,128/134,129/130,129/131,129/132,129/133,129/134,130/131,130/132,130/133,130/134,131/132,131/133,131/134,132/133,132/134,133/134,142/143,142/144,142/145,142/146,142/147,142/148,142/149,142/150,143/144,143/145,143/146,143/147,143/148,143/149,143/150,144/145,144/146,144/147,144/148,144/149,144/150,145/146,145/147,145/148 145/149,145/150,146/147,146/148,146/149,146/150,147/148,147/149,147/150,148/149,148/150,149/150 of the amino acid sequence of the glyoxylate binding protein.

In the present context, in the position indicated in the "X/Y" form, the optically active polypeptide has a portion of the glyoxylate-sensitive polypeptide at each of its two ends, wherein the optically active polypeptide has an N-terminal starting amino acid (e.g., any of amino acids 1 to 76) to an X-terminal amino acid of the glyoxylate-sensitive polypeptide sequence and a C-terminal amino acid (e.g., any of amino acids Y to 246) of the glyoxylate-sensitive polypeptide sequence at its C-terminal amino acid. Wherein, if two numbers in the positions indicated in the form of "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids indicated in the number, for example, insertion position 147/148 means that the optically active polypeptide is located between amino acids 147 and 148 of the glyoxylate-sensitive polypeptide, if two numbers in the form of "X/Y" are not consecutive integers and X is smaller than Y, it means that the optically active polypeptide replaces amino acids between the amino acids indicated in the number, for example, insertion position 174/185 means that the optically active polypeptide replaces amino acids 175-184 of the glyoxylate-sensitive polypeptide, if X in the position indicated in the form of "X/Y" is greater than or equal to Y, it means that the portion of the glyoxylate-sensitive polypeptide located at the N-terminal of the optically active polypeptide terminates at amino acid position from amino acid position 147 to 148 of the glyoxylate-sensitive polypeptide sequence, for example, insertion position 119/115 means that the N-terminal portion of the optically active polypeptide is fused with the glyoxylate-sensitive polypeptide sequence from amino acid position 1 to amino acid position 246 at the N-terminal end (for example, any one of amino acids from amino acid position 1 to position 119 to the end of the optically active polypeptide) and fused amino acid sequence at the N-terminal end of the glyoxylate-sensitive polypeptide is fused to position 115, exemplary structures are (glyoxylate-sensitive polypeptide sequences from any one of amino acids (1-76) to amino acid 119) - (optically active polypeptide) - (glyoxylate-sensitive polypeptide sequences from amino acid 115 to amino acid 246).

In one or more embodiments, the optical probe comprises, in order from the N-terminus to the C-terminus, residues Z-X of SEQ ID NO.1, SEQ ID NO:2-9 and the optically active polypeptide as set forth in any one of SEQ ID NOs: residues Y-246 of 1, wherein Z is any integer from 1 to 76 (preferably 1 or 76), and X and Y are selected from any of the following groups: (1) X is 17, Y is 18, (2) X is 17, Y is 19, (3) X is 17, Y is 20, (4) X is 29, Y is 21, (5) X is 18, Y is 19, (6) X is 18, Y is 20, (7) X is 18, Y is 21, (8) X is 19, Y is 20, (9) X is 19, Y is 21, (10) X is 20, Y is 21, (11) X is 28, Y is 29, (12) X is 28, Y is 30, (13) X is 28, Y is 31, (14) X is 28, Y is 32, (15) X is 29, Y is 30, (16) X is 29, Y is 31, (17) X is 29, Y is 32, (18) X is 30, Y is 31, (19) X is 30, Y is 32, (20) X is 31, Y is 32, (21) X is 54, Y is 55, (22) X is 54, Y is 56, (23) X is 54, Y is 57, (24) X is 54, Y is 24, Y is 58, (14) X is 28, Y is 32, (15) X is 29, Y is 30, (16) X is 29, Y is 31, (16) X is 29, Y is 31, (17) X is 29, Y is 31, Y is 30, Y is 32, (20) X is 30, and (20) X is 32 is 54, and (24) X is 54, and is 55, and is 25, and is 28, and is 30, and is 31, and, (35) X is 56, Y is 60, (36) X is 57, Y is 58, (37) X is 57, Y is 59, (38) X is 57, Y is 60, (39) X is 58, Y is 59, (40) X is 58, Y is 60, (41) X is 59, Y is 60, (42) X is 67, Y is 68, (43) X is 67, Y is 69, (44) X is 67, Y is 70, (45) X is 67, Y is 71, (46) X is 67, Y is 72, (47) X is 68, Y is 69, (48) X is 68, Y is 70, (49) X is 68, Y is 71, (50) X is 68, Y is 72, (51) X is 69, Y is 70, (52) X is 69, Y is 71, (53) X is 69, Y is 72, (54) X is 70, Y is 71, (55) X is 70, Y is 72, (56) X is 71, Y is 72 (57) X is 107, Y is 108, (58) X is 107, Y is 109, (59) X is 107, Y is 110, (60) X is 107, Y is 111, (61) X is 108, Y is 109, (62) X is 108, Y is 110, (63) X is 108, Y is 111, (64) X is 109, Y is 110, (65) X is 109, Y is 111, (66) X is 110, Y is 111, (67) X is 114, Y is 115, (68) X is 114, Y is 116, (69) X is 114, Y is 117, (70) X is 114, Y is 118, (71) X is 114, Y is 119, (72) X is 114, Y is 120, (73) X is 114, Y is 121, (74) X is 115, Y is 115, (75) X is 115, Y is 116, (76) X is 115, Y is 117, (77) X is 115, Y is 118, (78) X is 115, Y is 119, (79) X is 115, Y is 120, (80) X is 115, Y is 121, (81) X is 116, Y is 115, (82) X is 116, Y is 116, (83) X is 116, Y is 117, (84) X is 116, Y is 118, (85) X is 116, Y is 119, (86) X is 116, Y is 120, (87) X is 116, Y is 121, (88) X is 117, Y is 115, (89) X is 117, Y is 116, (90) X is 117, Y is 117, (91) X is 117, Y is 118, (92) X is 117, Y is 119, (93) X is 117, Y is 120, (94) X is 117, Y is 121, (95) X is 118, Y is 115, (96) X is 118, Y is 116, (97) X is 118, Y is 117,

(98) X is 118, Y is 118, (99) X is 118, Y is 119, (100) X is 118, Y is 120,

(101) X is 118, Y is 121, (102) X is 119, Y is 115, (103) X is 119, Y is 116,

(104) X is 119, Y is 117, (105) X is 119, Y is 118, (106) X is 119, Y is 119,

(107) X is 119, Y is 120, (108) X is 119, Y is 121, (109) X is 120, Y is 115,

(110) X is 120, Y is 116, (111) X is 120, Y is 117, (112) X is 120, Y is 118,

(113) X is 120, Y is 119, (114) X is 120, Y is 120, (115) X is 120, Y is 121,

(116) X is 128, Y is 129, (117) X is 128, Y is 130, (118) X is 128, Y is 131,

(119) X is 128, Y is 132, (120) X is 128, Y is 133, (121) X is 128, Y is 134,

(122) X is 129, Y is 130, (123) X is 129, Y is 131, (124) X is 129, Y is 132,

(125) X is 129, Y is 133, (126) X is 129, Y is 134, (127) X is 130, Y is 131,

(128) X is 130, Y is 132, (129) X is 130, Y is 133, (130) X is 130, Y is 134,

(131) X is 131, Y is 132, (132) X is 131, Y is 133, (133) X is 131, Y is 134,

(134) X is 132, Y is 133, (135) X is 132, Y is 134, (136) X is 133, Y is 134,

(137) X is 142, Y is 143, (138) X is 142, Y is 144, (139) X is 142, Y is 145,

(140) X is 142, Y is 146, (141) X is 142, Y is 147, (142) X is 142, Y is 148,

(143) X is 142, Y is 149, (144) X is 142, Y is 150, (145) X is 143, Y is 144,

(146) X is 143, Y is 145, (147) X is 143, Y is 146, (148) X is 143, Y is 147,

(149) X is 143, Y is 148, (150) X is 143, Y is 149, (151) X is 143, Y is 150,

(152) X is 144, Y is 145, (153) X is 144, Y is 146, (154) X is 144, Y is 147,

(155) X is 144, Y is 148, (156) X is 144, Y is 149, (157) X is 144, Y is 150,

(158) X is 145, Y is 146, (159) X is 145, Y is 147, (160) X is 145, Y is 148,

(161) X is 145, Y is 149, (162) X is 145, Y is 150, (163) X is 146, Y is 147,

(164) X is 146, Y is 148, (165) X is 146, Y is 149, (166) X is 146, Y is 150,

(167) X is 147, Y is 148, (168) X is 147, Y is 149, (169) X is 147, Y is 150,

(170) X is 148, Y is 149, (171) X is 148, Y is 150, (172) X is 149, and Y is 150.

Preferably cpYFP as an optically active polypeptide is located at 114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115,120/120 of the amino acid sequence of the glyoxylate binding protein as shown in SEQ ID NO 10-22.

The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function but different sequences of the polypeptide or protein. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. Such variants include, but are not limited to, sequences obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxy-terminal and/or amino-terminal end thereof. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitution with amino acids having similar or similar properties typically does not alter the function of the polypeptide or protein. Amino acids of similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, succinic acid), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, succinic acid, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the polypeptide or protein. Exemplary truncated variants of IcIR proteins are fragments of SEQ ID NO. 1 comprising amino acids 76-246 which retain the binding function of IcIR protein to glyoxylate and do not affect the change in optical properties of the inserted optically active polypeptide in response to glyoxylate binding.

The optical probes of the present invention may comprise a glyoxylate-sensitive polypeptide having a mutation and an optically-active polypeptide having a mutation. Glyoxylate binding protein variants having a mutation at a site selected from the group consisting of E115, N116, I118, G119, T59, S139, S141 of SEQ ID NO. 1 or a truncated variant thereof exhibit a binding activity different from glyoxylate. The amino acid mutation includes modification, substitution or deletion of amino acids.

Wherein, as an example in the examples, in SEQ ID NO. 1 or a truncated variant thereof, I118 is mutated to G, Q, T, L, M, V, H, F, S, A, Y or K. In one or more embodiments, G119 is mutated to N, V or S. In one or more embodiments, E115 is mutated to N, S, P, T or Q. In one or more embodiments, N116 is mutated to V, C, K, E, I, T, R or W. In one or more embodiments, T59 is mutated to G or N. In one or more embodiments, S139 is mutated to N or T. In one or more embodiments, S141 is mutated to D, H or G.

In preferred embodiments, the mutation of the glyoxylate binding protein variant comprises a mutation selected from any one of the following groups:

(1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116K N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,

The present invention provides glyoxylate binding protein variants having these mutations and optical probes described herein comprising such glyoxylate binding protein variants as glyoxylate sensitive polypeptides. Thus, included herein are optical probes wherein the optically active polypeptide is located at 17/18,17/19,17/20,17/21,18/19,18/20,18/21,19/20,19/21,20/21,28/29,28/30,28/31,28/32,29/30,29/31,29/32,30/31,30/32,31/32,54/55,54/56,54/57,54/58,54/59,54/60,55/56,55/57,55/58,55/59,55/60,56/57,56/58,56/59,56/60,57/58,57/59,57/60,58/59,58/60,59/60,67/68,67/69,67/70,67/71,67/72,68/69,68/70,68/71,68/72,69/70,69/71,69/72,70/71,70/72,71/72,107/108,107/109,107/110,107/111,108/109,108/110,108/111,109/110,109/111,110/111,114/115,114/116,114/117,114/118,114/119,114/120 114/121,115/115,115/116,115/117,115/118,115/119,115/120,115/121,116/115,116/116,116/117,116/118,116/119,116/120,116/121,117/115,117/116,117/117,117/118,117/119,117/120,117/121,118/115,118/116,118/117,118/118,118/119,118/120,118/121,119/115,119/116,119/117,119/118,119/119,119/120,119/121,120/115,120/116,120/117,120/118,120/119,120/120,120/121,128/129,128/130,128/131,128/132,128/133,128/134,129/130,129/131,129/132,129/133,129/134,130/131,130/132,130/133,130/134,131/132,131/133,131/134,132/133,132/134,133/134,142/143,142/144,142/145,142/146,142/147,142/148,142/149,142/150,143/144,143/145,143/146,143/147,143/148,143/149,143/150,144/145,144/146,144/147,144/148,144/149,144/150,145/146,145/147,145/148 145/149,145/150,146/147,146/148,146/149,146/150,147/148,147/149,147/150,148/149,148/150,149/150, preferably 119/115, of the amino acid sequence of a glyoxylate binding protein variant which is shown as SEQ ID NO. 1 or a truncated variant thereof comprising amino acids 76-246 and which comprises a mutation selected from any of the group consisting of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G.

In some embodiments, the glyoxylate-sensitive polypeptide in the optical probe is shown in amino acids 76-246 of SEQ ID NO:1, the optically-active polypeptide is shown in SEQ ID NO:2, the optically-active polypeptide is located at position 119/115 of the glyoxylate-sensitive polypeptide, and the glyoxylate-sensitive polypeptide has a mutation shown in any one of (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, and (10) E115T and N116V,(11)E115N、N116E,(12)E115N、N116I,(13)E115N、N116T,(14)E115N、N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G. the optical probe has the structure:

(amino acids 76 to 119 of the glyoxylate-sensitive polypeptide sequence) - (optically-active polypeptide) - (amino acids 115 to 246 of the glyoxylate-sensitive polypeptide sequence). The amino acid sequence of the optical probe shown in item (1) is shown as SEQ ID NO. 23, the amino acid sequence of the optical probe shown in item (2) is shown as SEQ ID NO. 24, the amino acid sequence of the optical probe shown in item (5) is shown as SEQ ID NO. 25, the amino acid sequences of the optical probes shown in items (18) to (29) are shown as SEQ ID NO. 26-37, respectively, and the amino acid sequences of the optical probes shown in items (30), (32), (34), (36), (38), (40), (42), (44), (46), (48), (50), (52) are shown as SEQ ID NO. 38-49, respectively.

In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NOs 10-49 or a variant thereof. In one or more embodiments, the optical probes provided herein comprise sequences that are 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identical to any of the amino acid sequences SEQ ID NOs 10-49. In a preferred embodiment, the optical probes provided herein comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10 to 49.

In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) as compared and aligned for maximum correspondence over a comparison window or designated region, using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25:3389 and Altschul et al (1990) J.mol.biol.215:403, respectively.

It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example.

The terms "functional variant," "derivative," and "analog" as used herein refer to a protein that retains substantially the same biological function or activity as the original polypeptide or protein (e.g., icIR protein or fluorescent protein). The functional variant, derivative or analogue of a polypeptide or protein of the invention (e.g. IcIR protein or fluorescent protein) may be (i) a protein having one or more, preferably conservative or non-conservative amino acid residues substituted, which may or may not be encoded by the genetic code, or (ii) a protein having a substituent in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g. a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to the protein sequence (e.g. a secretion sequence or a sequence used to purify the protein or a pro-protein sequence, or fusion protein with the formation of an antigen IgG fragment). Such functional variants, derivatives and analogs are within the scope of those skilled in the art, as determined by the teachings herein.

The difference between the analog and the original polypeptide or protein may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, as well as by site-directed mutagenesis or other known molecular biological techniques.

The analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It will be appreciated that the glyoxylate-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives and analogs listed above. Modified (typically without altering the primary structure) forms include chemically derivatized forms of the protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

Fusion polypeptides of the invention include optical probes and other polypeptides described herein. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. Other polypeptides may be located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, tags for purification, or tags for immunoblotting. The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.

Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, golgi apparatus, lysosomes, peroxisomes, and the like. In some embodiments, the tag for purification or for immunoblotting comprises 6 histidine (6 xhis), glutathione-s-transferase (GST), flag.

The invention also provides a preparation method of the glyoxylate optical probe, which comprises the following steps of 1) incorporating a nucleic acid sequence encoding the glyoxylate optical probe into an expression vector, 2) transferring the expression vector into a host cell, 2) culturing the host cell under the condition suitable for the expression of the expression vector, and 3) separating the glyoxylate optical probe.

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The term "variant" as used herein when referring to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution pattern of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded protein. The nucleic acids of the invention may comprise a nucleotide sequence 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 to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR).

The full-length sequence of the optical probe or fusion protein of the present invention or a fragment thereof can be generally obtained by PCR amplification, artificial synthesis or recombinant methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the nucleotide sequence is larger than 2500bp, the PCR amplification is preferably carried out for 2-6 times, and then the amplified fragments are spliced together according to the correct sequence. The PCR amplification procedure and system are not particularly limited, and conventional PCR amplification procedures and systems in the art can be adopted. The sequences of interest can also be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating and purifying the relevant polypeptide or protein from the proliferated host cells by conventional methods. Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In the present invention, when the nucleotide sequence of the optical probe is less than 2500bp, the optical probe can be synthesized by adopting an artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in the field, and has no other special requirements. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or functional variants, derivatives or analogues thereof) entirely by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Mutations can be introduced into the protein sequences of the present invention by mutation PCR or chemical synthesis, etc.

After the nucleotide sequence for coding the optical probe is obtained, the nucleotide sequence for coding the optical probe is incorporated into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein to refer to a prokaryotic or eukaryotic vector well known in the art, such as a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors, which are capable of replication and stable expression in a host, an important feature of such recombinant vectors being that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element operably linked to a gene of interest that regulates the transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translational control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc., the choice of expression control sequence being dependent upon the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operably linked" refers to the attachment of a nucleotide sequence of interest to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Methods for constructing expression vectors comprising the fusion protein coding sequences of the invention and appropriate transcriptional/translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E.coli, the lambda phage PL promoter, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses and some other known promoters which control gene expression in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one or more embodiments, the expression vector may be a commercially available pCDF vector, without other special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with HindIII and XhoI, respectively, and then the digested products of the two are ligated to obtain a recombinant expression vector. The specific steps and parameters of the digestion and the connection are not particularly limited, and the steps and parameters conventional in the art are adopted.

After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide comprising the fusion protein. Such transfer may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention is a cell capable of receiving and accommodating recombinant DNA molecules, is a site for amplifying recombinant genes, and ideal recipient cells should satisfy both conditions of easy acquisition and proliferation. "host cells" according to the invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples are bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HEK293 cells, or Bowes melanoma cells, among others, including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being well known and commonly used in the art. An exemplary host cell for use in embodiments of the invention is the E.coli BL21-DE3 strain. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Methods of transferring to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably a CaCl ₂ method or MgCl ₂ method treatment, using procedures well known in the art. When the host cell is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

After the expression vector is transferred into a host cell, the host cell transferred into the expression vector is amplified, expressed and cultured, and the glyoxylate optical probe is obtained by separation. The host cell amplification expression culture can be carried out by adopting a conventional method. The medium used in the culture may be various conventional media depending on the kind of host cell used. The culture is carried out under conditions suitable for the growth of the host cell.

In the present invention, the optical probe is expressed in a cell, on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated or purified by various isolation methods using their physical, chemical and other properties. The method for separating the glyoxylate fluorescent protein is not particularly limited, and a fusion protein separation method conventional in the art can be adopted. Such methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatment, salting-out methods, centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one or more embodiments, the separation of the optical probe is performed using His-tag affinity chromatography.

The invention also provides application of the glyoxylate optical probe in real-time positioning and quantitative detection of glyoxylate and high-flux compound screening. In one aspect, the glyoxylate optical probe is preferably connected with signal peptides at different parts of the cell, is transferred into the cell, performs real-time localization of glyoxylate by detecting the intensity of fluorescent signals in the cell, and performs quantitative detection of corresponding glyoxylate by using a glyoxylate standard dripping curve. The standard dripping curve of glyoxylate is drawn according to fluorescent signals of glyoxylate optical probes under the condition of different concentrations of glyoxylate. The glyoxylate optical probe is directly transferred into cells, and a time-consuming sample treatment process is not needed in the process of real-time positioning and quantitative detection of glyoxylate, so that the glyoxylate optical probe is more accurate. When the glyoxylate optical probe is used for high-flux compound screening, different compounds are added into a cell culture solution, and the change of the glyoxylate content is measured, so that the compounds with influence on the change of the glyoxylate content are screened. The application of the glyoxylate optical probe in the invention in the real-time positioning and quantitative detection of glyoxylate and the screening of high-flux compounds is non-diagnosis and treatment purposes, and does not relate to the diagnosis and treatment of diseases.

Concentrations, amounts, percentages, and other numerical values may be expressed herein in terms of ranges. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, as well as to include all the individual numerical values or sub-ranges encompassed within that range.

Examples

The glyoxylate optical probes provided in the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.

I. experimental materials and reagents

In the examples, the conventional cloning method, cell culture and imaging methods of the genetically engineered molecular biology are mainly used, and these methods are well known to those skilled in the art, for example, jianluo Skems et al, J. Sambrook, D.W. Lassel, huang Peitang et al, ind. Molecular cloning laboratory Manual (third edition, 8 months 2002, published by Sci.11, beijing), fei Leixie ni et al, ind. Basic technical Specification (fifth edition), zhang Jingbo, xu Cunshuan et al, J.S. Bonefferson, M.dar et al, ind. Cell biological laboratory Manual, zhang Jingbo et al. Modifications and variations as would be apparent to one skilled in the art are within the scope of the following claims, as may be made in the practice of the application.

The pCDF-cpYFP-based, pCDF-glyoxylate binding protein plasmid used in the examples was constructed from the university of Dongpo protein laboratory, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry as correct by Shanghai large gene bioengineering technologies, inc. The expression plasmids constructed in the examples were all subjected to sequence determination, which was performed by Huada gene company and Jie Li Cexu company. Taq DNA polymerase used in each example was purchased from Dongsheng, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Co., ltd, PRIMESTAR DNA polymerase was purchased from TaKaRa Co., ltd, and the corresponding polymerase buffer and dNTPs were added when all three polymerases were purchased. BamHI, bglII, hindIII, ndeI, xhoI, ecoRI, speI, etc., a T4 ligase, a T4 phosphorylase (T4 PNK) are purchased from Fermentas, inc., and corresponding buffers are added thereto. Transfection reagent Lip2000 Kit was purchased from Invitrogen company. Glyoxylic acid and like compounds are all available from Aladin corporation. Unless otherwise specified, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich corporation. HEPES salts, ampicillin and streptomycin were purchased from Ameresco. 96-well assay blackboard, 384 Kong Yingguang assay blackboard, purchased from Grenier company.

The DNA purification kit used in the examples was purchased from BBI (Canada), and the ordinary plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Co. Clone strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column packing were all from GE HEALTHCARE company.

The main instruments used in the examples include Biotek Synergy 2 multifunctional enzyme-labeled instrument (Bio-Tek Co., ltd., U.S.A.), X-15R high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), microfuge22R bench-type high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), PCR amplification instrument (Biometa, germany), ultrasonoscope (Ningbo Zhi Co., ltd.), nucleic acid electrophoresis instrument (Shencan Bo Co., fluorescent spectrophotometer (Varian Co., ltd.), CO ₂ constant temperature cell incubator (SANYO), inverted fluorescent microscope (Nikon Co., ltd.).

II molecular biology method and cell experiment method

II.1 Polymerase Chain Reaction (PCR):

1. Amplification of the fragment of interest PCR:

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

TABLE 1PCR amplification reaction System

TABLE 2 PCR amplification procedure

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

The long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, a technique for obtaining site-directed mutagenesis in the following examples. Reverse PCR primers were designed at the mutation sites, wherein the 5' end of one primer contained the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.

TABLE 3 Long fragment (> 2500 bp) amplification PCR reaction System

TABLE 4 Long fragment (> 2500 bp) amplification PCR amplification procedure

TABLE 5 Long fragment (> 2500 bp) amplification PCR amplification procedure

II.2 endonuclease cleavage reaction:

the system for double digestion of plasmid vectors is shown in Table 6, where n represents the amount of sterilized ultra-pure water μL added to make the system total volume.

TABLE 6 plasmid vector double enzyme digestion system

II.3 5' -terminal phosphorylation of DNA fragments

The plasmid or genome extracted from the microorganism contains phosphate groups at the terminal, but the PCR product does not, so that the 5' -terminal base of the PCR product needs to be subjected to phosphate group addition reaction, and only DNA molecules containing phosphate groups at the terminal can undergo ligation reaction. The phosphorylation reaction system is shown in Table 7, wherein T4 PNK is a shorthand for T4 polynucleotide kinase, and is used for addition reaction of 5' -terminal phosphate group of DNA molecule.

TABLE 7 phosphorylation reaction System

II.4 ligation of the fragment of interest and the vector

The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present invention

1. Blunt end ligation of blunt end short fragments and linearized vectors

The principle of the method is that a blunt end product obtained by PCR carries out phosphorylation reaction on the 5' end of a DNA fragment under the action of T4 PNK, and then is connected with a linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

TABLE 8 blunt end fragment ligation reaction System

2. Ligation of DNA fragments containing cohesive ends and vector fragments containing cohesive ends

DNA fragments cleaved by restriction enzymes will typically produce protruding cohesive ends and thus can be ligated to cohesive end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is shown in Table 9, wherein the mass ratio of PCR product fragments to carrier double cleavage products is approximately between 2:1 and 6:1.

TABLE 9 viscous terminal ligation reaction System

3. Ligation of the product of 5' -phosphorylated DNA fragments by self-cyclization after introduction of site-directed mutagenesis by inverse PCR

The DNA fragment with phosphorylated 5' end is connected with the 3' end and the 5' end of the linearization vector through self cyclization connection reaction to obtain the recombinant plasmid. The self-cyclized ligation reaction system is shown in Table 10.

TABLE 10 self-cyclizing ligation reaction System

II.5 preparation and transformation of competent cells

Preparation of competent cells:

1. single colonies (e.g., mach 1) were picked and inoculated into 5mL LB medium, and shaken overnight at 37 ℃.

2. 0.5-1ML of the overnight cultured bacterial liquid is transferred to 50mL of LB culture medium, and cultured for 3 to 5 hours at 37 ℃ and 220rpm until the OD600 reaches 0.5.

3. The cells were pre-chilled in an ice bath for 2 hours.

Centrifuge at 4000rpm for 10 min at 4.4 ℃.

5. The supernatant was discarded, and the cells were resuspended in 5mL of pre-chilled buffer, and after homogenization, the resuspension buffer was added to a final volume of 50mL.

6. Ice bath for 45 minutes.

The bacteria were resuspended by centrifugation at 4000rpm at 7.4℃for 10 minutes with 5mL of ice-chilled storage buffer.

8. Mu.L of bacterial liquid was placed in each EP tube and frozen at-80℃or with liquid nitrogen.

Resuspension buffer CaCl ₂(100mM)、MgCl₂ (70 mM), naAc (40 mM)

Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 XCaCl ₂(1M)、1mL 10×MgCl₂(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH₂ O

Transformation of competent cells:

1. 100. Mu.L of competent cells were thawed on an ice bath.

2. Add the appropriate volume of ligation product, gently blow mix, ice bath for 30 minutes. The ligation product is typically added in a volume of less than 1/10 of the competent cell volume.

3. The bacterial liquid is placed in a 42 ℃ water bath for heat shock for 90 seconds, and is quickly transferred to an ice bath for 5 minutes.

4. Mu.L of LB was added and incubated at 200rpm on a 37℃constant temperature shaker for 1 hour.

5. The bacterial liquid was centrifuged at 4000rpm for 3 minutes, 200. Mu.L of supernatant was left to blow the bacterial cells evenly, and the cells were spread evenly on the surface of an agar plate containing the appropriate antibiotics, and the plate was inverted overnight in a 37℃incubator.

II.6 expression, purification and fluorescence detection of proteins

1. Expression vectors (e.g., pCDF-based glyoxylate optical probe expression vectors) were transformed into BL21 (DE 3) cells, cultured upside down overnight, cloned into 250ml Erlenmeyer flasks were picked from plates, placed in a 37℃shaker, cultured at 220rpm to OD=0.4-0.8, 1/1000 (v/v) IPTG (1M) was added, and expression was induced at 18℃for 24-36 hours.

2. After the induction expression was completed, the cells were collected by centrifugation at 4000rpm for 30 minutes, and the cell pellet was resuspended in 50mM phosphate buffer and sonicated until the cells were clarified. Centrifugation was performed at 9600rpm at 4℃for 20 minutes.

3. The supernatant was purified by self-contained nickel column affinity chromatography to obtain protein, and the protein after nickel column affinity chromatography was further passed through self-contained desalting column to obtain protein dissolved in 100mM HEPES buffer (pH 7.4).

4. After SDS-PAGE identification of the purified proteins, the probes were diluted with assay buffer (100mM HEPES,100mM NaCl,pH 7.4) to a final concentration of 0.2-5. Mu.M protein solution. Glyoxylate was prepared as a stock solution with a final concentration of 50mM using assay buffer (100mM HEPES,100mM NaCl,pH 7.4).

5. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10min, glyoxylic acid titration was added, and the fluorescence intensities of 528nm emission after 420nm excitation and 528nm emission after 485nm excitation were measured. The fluorescence excitation and emission measurement of the sample are completed by a multifunctional fluorescence enzyme-labeling instrument.

6. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10min, glyoxylic acid was added, and the absorption spectrum and fluorescence spectrum of the protein were determined. The measurement of the absorption spectrum and fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.

II.7 transfection and fluorescence detection of mammalian cells

1. Plvx-based glyoxylate optical probe plasmids were transfected into HEK293 by the transfection reagent Lipofectamine2000 (Invitrogen) and incubated in a 37 ℃ cell incubator with 5% CO ₂. And (4) performing fluorescence detection after the exogenous gene is fully expressed for 24-36 hours.

2. After the induction of expression is completed, the adherent HEK293 cells are washed three times by PBS and placed in HBSS solution for detection by a fluorescence microscope and an enzyme-labeled instrument respectively.

EXAMPLE 1 glyoxylate binding protein plasmids

The IcIR (76-246) gene in Thermotoga maritima genes is amplified by PCR, and after the gel electrophoresis of the PCR products is recovered, bamHI and XhoI are used for digestion, and the pCDF vector is subjected to corresponding double digestion. After ligation with T4 DNA ligase, DH 5. Alpha. Was transformed with the product, and the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 100 ug/mL) and incubated overnight at 37 ℃. The growing DH5 alpha transformants were subjected to plasmid extraction and PCR identification. The positive plasmid is sequenced correctly and then the subsequent plasmid construction is carried out.

Example 2 expression and detection of cpYFP optical probes at different insertion sites

In this example, the following site insert cpYFP was selected based on pCDF-IcIR to give the corresponding pCDF-IcIR-cpYFP plasmid ：17/18,17/19,17/20,17/21,18/19,18/20,18/21,19/20,19/21,20/21,28/29,28/30,28/31,28/32,29/30,29/31,29/32,30/31,30/32,31/32,54/55,54/56,54/57,54/58,54/59,54/60,55/56,55/57,55/58,55/59,55/60,56/57,56/58,56/59,56/60,57/58,57/59,57/60,58/59,58/60,59/60,67/68,67/69,67/70,67/71,67/72,68/69,68/70,68/71,68/72,69/70,69/71,69/72,70/71,70/72,71/72,107/108,107/109,107/110,107/111,108/109,108/110,108/111,109/110,109/111,110/111,114/115,114/116,114/117,114/118,114/119,114/120 114/121,115/115,115/116,115/117,115/118,115/119,115/120,115/121,116/115,116/116,116/117,116/118,116/119,116/120,116/121,117/115,117/116,117/117,117/118,117/119,117/120,117/121,118/115,118/116,118/117,118/118,118/119,118/120,118/121,119/115,119/116,119/117,119/118,119/119,119/120,119/121,120/115,120/116,120/117,120/118,120/119,120/120,120/121,128/129,128/130,128/131,128/132,128/133,128/134,129/130,129/131,129/132,129/133,129/134,130/131,130/132,130/133,130/134,131/132,131/133,131/134,132/133,132/134,133/134,142/143,142/144,142/145,142/146,142/147,142/148,142/149,142/150,143/144,143/145,143/146,143/147,143/148,143/149,143/150,144/145,144/146,144/147,144/148,144/149,144/150,145/146,145/147,145/148 145/149,145/150,146/147,146/148,146/149,146/150,147/148,147/149,147/150,148/149,148/150,149/150.

And amplifying by using a PCR technology to obtain a cpYFP DNA fragment and a pCDF-IcIR linearization vector, wherein the 5 'and 3' extreme ends of the DNA fragment respectively have completely identical sequences (15 bp-20 bp) corresponding to the two extreme ends of cpYFP. The linearized pCDF-IcIR and cpYFP fragments were subjected to homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH 5. Alpha. And the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 100 ug/mL) and incubated overnight at 37 ℃. Positive clones identified by PCR were sequenced after drawing the plasmid. Sequencing was accomplished by Jie Li Cexu.

After sequencing correctly, the recombinant plasmid was transformed into BL21 (DE 3) to induce expression, and the protein was purified and sized around 47.2kDa by SDS-PAGE electrophoresis. The size of the recombinant DNA is consistent with the size of the IcIR-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-IcIR-cpYFP. The results are shown in FIG. 1.

Glyoxylate response screening was performed with the disrupted supernatant of E.coli expressing IcIR-cpYFP fusion protein, and the detection signal of the fusion fluorescent protein containing 10mM glyoxylate was divided by the detection signal of the fusion fluorescent protein of glyoxylate. The results are shown in Table 1, and the detection results show that 13 optical probes with the fold change of response to glyoxylate lower than 0.8 or higher than 1.2 are provided, and the sequences of the probes are shown in SEQ ID NOS 10-22 at 114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115,120/120.

TABLE 1

Example 3 expression and detection of cpGFP optical probes at different insertion sites

The glyoxylic acid green fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpGFP as in example 2. As shown in Table 3, the detection results showed that the optical probes having a fold change of less than 0.8 or more than 1.2 in response to glyoxylate had optical probes performing insertion at the 117/120,119/115,119/116,120/115 and 120/117 positions or the corresponding amino acid positions of the family proteins.

TABLE 2

Example 4 expression and detection of cpBFP optical probes at different insertion sites

The glyoxylic acid blue fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpBFP as in example 2. As shown in Table 3, the detection results showed that the optical probes having a fold change of less than 0.8 or more than 1.2 in response to glyoxylate had optical probes performing insertion at the 114/117,114/118,119/119 and 119/120 positions or the corresponding amino acid positions of the family proteins.

TABLE 3 Table 3

Example 5 expression and detection of cpmApple optical probes at different insertion sites

The glyoxylic acid red fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpmApple as in example 2. As shown in Table 4, the detection results showed that the optical probes having a fold change of less than 0.8 or more than 1.2 in response to glyoxylate had optical probes performing insertion at the 117/115,118/119,118/120 and 120/121 positions or the corresponding amino acid positions of the family proteins thereof

TABLE 4 Table 4

EXAMPLE 6 expression and detection of mutated cpYFP optical probes

For the optical probes with the fold change of the response to glyoxylate lower than 0.8 or higher than 1.2 obtained in example 2, namely, 13 optical probes inserted at 114/115,114/119,114/120,117/115,117/116,117/118,117/121,118/118,118/119,119/115,119/116,120/115 and 120/120 sites were linearized by inverse PCR, the sequences of mutation sites were introduced into the primers, homologous recombination was performed on the obtained PCR products under the action of Hieff Clone Enzyme, and a mutation library was established. The recombinant plasmid of the mutant library was transformed into BL21 (DE 3) to induce expression, and the broken supernatant of E.coli expressing the probe protein was used to perform glyoxylate response screening by dividing the detection signal of the fusion fluorescent protein containing 10mM glyoxylate by the detection signal of the fusion fluorescent protein without glyoxylate. The results are shown in Table 5, and the optical probes having a response to glyoxylate of 3-fold or more are shown below.

TABLE 5

Sequence(s)	Insertion site	Mutation	R_420/485
				SEQ ID NO:23	119/115	I118G/G119N	3.03
SEQ ID NO:24	119/115	I118Q	3.02
					119/115	I118T	3.07
	119/115	I118L	3.11
				SEQ ID NO:25	119/115	E115N/N116V	3.27
	119/115	E115N/N116C	3.24
					119/115	E115S/N116K	3.46
	119/115	E115S/N116C	3.04
					119/115	E115P/N116C	3.18
	119/115	E115T/N116V	3.12
					119/115	E115N/N116E	3.78
	119/115	E115N/N116I	3.11
					119/115	E115N/N116T	3.02
	119/115	E115N/N116R	3.17
					119/115	E115Q/N116K	3.21
	119/115	E115N/N116W	3.06
					119/115	E115P/N116V	3.12
SEQ ID NO:26	119/115	I118M/E115N/N116V	5.74
				SEQ ID NO:27	119/115	I118T/E115N/N116V	5.70
SEQ ID NO:28	119/115	I118V/E115N/N116V	5.80
				SEQ ID NO:29	119/115	I118L/E115N/N116V	5.26
SEQ ID NO:30	119/115	I118H/E115N/N116V	5.01
				SEQ ID NO:31	119/115	I118V/E115N/N116V	4.98
SEQ ID NO:32	119/115	I118F/E115N/N116V	5.00
				SEQ ID NO:33	119/115	I118S/E115N/N116C	5.24
SEQ ID NO:34	119/115	I118L/G119V/E115N/N116C	4.97
				SEQ ID NO:35	119/115	I118A/G119S/E115N/N116C	4.98
SEQ ID NO:36	119/115	I118Y/E115S/N116K	5.34
				SEQ ID NO:37	119/115	I118K/E115S/N116K	8.6

Mutation is carried out on amino acids 59, 139 and 141 of IcIR protein on the basis of 118F/115N/116V, 118Y/115S/116K, I K/E115S/N116K so as to improve the specificity of a glyoxylate optical probe, and then the probe is linearized by inverse PCR, and the sequence of a mutation site is introduced into a primer. Site-directed mutagenesis plasmids were transformed into BL21 (DE 3) for induction of expression, and the lysates of E.coli expressing the probe proteins were used for glyoxylate response screening by dividing the detection signal of fusion fluorescent protein containing 10mM glyoxylate and 10mM pyruvate by the detection signal of fusion fluorescent protein without glyoxylate and pyruvate. The results are shown in Table 6, and the optical probes whose specificity for glyoxylate was significantly improved are shown below.

TABLE 6

Sequence(s)	Mutation site	Mutation	R_420/485
				SEQ ID NO:38	59	IcIR-T59G/I118F/E115N/N116V	3.75
	59	IcIR-T59N/I118F/E115N/N116V	3.77
				SEQ ID NO:39	59	IcIR-T59G/E115N/N116V	4.19
	59	IcIR-T59N/E115N/N116V	3.36
				SEQ ID NO:40	59	IcIR-T59G/I118Y/E115S/N116K	3.82
	59	IcIR-T59N/I118Y/E115S/N116K	3.64
				SEQ ID NO:41	59	IcIR-T59G/I118K/E115S/N116K	8.2
	59	IcIR-T59N/I118K/E115S/N116K	7.6
				SEQ ID NO:42	139	IcIR-I118F/E115N/N116V/S139N	3.69
	139	IcIR-I118F/E115N/N116V/S139T	3.77
				SEQ ID NO:43	139	IcIR-E115N/N116V/S139N	3.36
	139	IcIR-E115N/N116V/S139T	2.05
				SEQ ID NO:44	139	IcIR-I118Y/E115S/N116K/S139N	4.36
	139	IcIR-I118Y/E115S/N116K/S139T	2.64
				SEQ ID NO:45	139	IcIR-I118K/E115S/N116K/S139N	9.6
	139	IcIR-I118K/E115S/N116K/S139T	6
				SEQ ID NO:46	141	IcIR-I118F/E115N/N116V/S141D	2.97
	141	IcIR-I118F/E115N/N116V/S141G	2.01
				SEQ ID NO:47	141	IcIR-E115N/N116V/S141D	1.5
	141	IcIR-E115N/N116V/S141G	2.87
				SEQ ID NO:48	141	IcIR-I118Y/E115S/N116K/S141D	1.93
	141	IcIR-I118Y/E115S/N116K/S141G	2.52
				SEQ ID NO:49	141	IcIR-I118K/E115S/N116K/S141D	4.2
	141	IcIR-I118K/E115S/N116K/S141G	4.42

Example 7 Properties of optical Probe mutants

Glyoxylate was detected at a concentration gradient (0-10 mM) for a portion of glyoxylate optical probes in Table 5 described in example 6 and for all glyoxylate optical probes in Table 6, respectively. After 10 minutes of probe treatment, the change in the ratio of the fluorescence intensity at 528nm emission from 420nm excitation to the fluorescence intensity at 528nm emission from 485nm excitation was detected. The results of the probe titration are shown in fig. 2, and the results show that the different mutants have different affinities for glyoxylate.

The glyoxylate probes of tables 5 and 6 were specifically tested for reactivity with glyoxylate structural analogs, pyruvic acid, malic acid, oxaloacetic acid, alpha ketoglutaric acid, fumaric acid, succinic acid, citric acid and isocitric acid, respectively, and the results showed good specificity as shown in FIG. 3.

Example 8 subcellular organelle localization of optical probes and the Performance of optical probes within subcellular organelles

In this example, different localization signal peptides were used to fuse with the optical probe IcIR-T59G/E115N/N116C to localize the optical probe to different organelles. HEK293 cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and fluorescence detected under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 4. The glyoxylic acid optical probe can be localized to the plasma, mitochondria, nucleus, nuclear exclusion, outer membrane, endoplasmic reticulum by fusion with different specific localization signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.

HEK293 cells were transfected with cytoplasmic expression optical probe plasmid for 36 hours, rinsed with PBS, placed in HBSS solution, and the change in the ratio of fluorescence intensity at 420nm excitation 528nm emission to fluorescence intensity at 485nm excitation 528nm emission was detected over a 30min period. The results are shown in FIG. 5. 10mM glyoxylic acid was added and detection continued for 30 minutes. The 485/420 of the glyoxylic acid added sample was gradually increased up to 8 times the initial value, while the 485/420 of the control group without glyoxylic acid added remained essentially unchanged.

Example 9 high throughput Compound screening in living cells based on optical probes

In this example, we used HEK293 cells expressing IcIR-T59G/E115N/N116C for high throughput compound screening.

Transfected HEK293 cells were rinsed with PBS, placed in HBSS solution (no glyoxylic acid) for 1 hour and then treated with 10 μm compound for 1 hour. Glyoxylic acid was added dropwise to each sample. The change in the ratio of the fluorescence intensity at the 528nm emission of 420nm excitation to the fluorescence intensity at the 528nm emission of 485nm excitation was recorded using a microplate reader. Samples not treated with any compound were normalized as controls. The results are shown in FIG. 6. Of the 2000 compounds used, the vast majority of compounds had minimal effect on glyoxylate entry into the cell. 8 compounds can improve the uptake capacity of cells for glyoxylic acid, and 7 compounds can obviously reduce the uptake capacity of cells for glyoxylic acid.

Example 10 quantitative detection of glyoxylate in blood with an optical probe

In this example, purified IcIR-T59G/E115N/N116C was used to analyze glyoxylate in mouse and human blood supernatants.

After IcIR-T59G/E115N/N116C was mixed with the diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at 528nm emission at 420nm excitation to the fluorescence intensity at 528nm emission at 485nm excitation was detected using a microplate reader. As a result, as shown in FIG. 7, the glyoxylate content in the blood of the mice was about 30. Mu.M, and the glyoxylate content in the blood of the human was about 25. Mu.M.

The embodiment shows that the glyoxylate optical probe provided by the invention has the advantages of relatively small molecular weight, easiness in maturation, large dynamic change of fluorescence, good specificity, capability of expressing in cells by a gene operation method, capability of detecting glyoxylate in real time and quantitatively inside and outside the cells, and capability of carrying out high-flux compound screening.

Other embodiments

This specification describes a number of embodiments. It will be appreciated that various modifications may be made by those skilled in the art from a reading of this specification without departing from the spirit and scope of the invention, and are intended to be included within the scope of the appended claims.

Claims

1. A glyoxylate binding protein variant, which:

(1) having a sequence as shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five, six or seven positions selected from the group consisting of: E115, N116, I118, G119, T59, S139, S141, wherein the mutation comprises a modification, substitution or deletion of an amino acid,

(2) is a truncated variant of (1) having amino acids 76 to 246, or

(3) a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to glyoxylate,

Preferably, the mutation site is selected from 1, 2, 3 or 4 of any one of the following groups: E115, N116, I118, G119. Optionally, the mutation further comprises a mutation at any one of the following sites: T59, S139, S141,

More preferably, the mutation includes a mutation at a site selected from any one of the following groups: (1) I118 and G119, (2) I118, (3) E115, N116, (4) I118, E115, N116; (5) T59, I118, E115, N116, (6) T59, E115, N116, (7) I118, E115, N116, S139, (8) E115, N116, S139, (9) I118, E115, N116, S141, (10) E115, N116, S141, (11) T133 and I118, (12) I118 and S213, (13) I118 and S215,

Further preferably, I118 mutates to G, Q, T, L, M, V, H, F, S, A, Y or K; G119 mutates to N, V or S; E115 mutates to N, S, P, T or Q; N116 mutates to V, C, K, E, I, T, R or W; T59 mutates to G or N; S139 mutates to N or T; S141 mutates to D, H or G.

2. An optical probe comprising a glyoxylic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the glyoxylic acid-sensitive polypeptide, the glyoxylic acid-sensitive polypeptide (1) having the sequence shown in SEQ ID NO:1 or a truncate thereof having amino acids 76-246, or a sequence having at least 70% sequence identity therewith and retaining sensitivity to glyoxylic acid, or (2) being a glyoxylic acid-binding protein variant as described in claim 1, wherein the optically active polypeptide is a fluorescent protein or a functional variant thereof.

3. The optical probe of claim 2, wherein the optically active polypeptide is located between residues 17-20, 28-31, 54-59, 67-71, 107-110, 114-120, 128-133, 142-149 of the glyoxylate-sensitive polypeptide.

Preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide: 17/18, 17/19, 17/20, 17/21, 18/19, 18/20, 18/21, 19/20, 19/21, 20/21, 28/29, 28/30, 28/31, 28/32, 29/30, 29/31, 29/32, 30/31, 30/32, 31/32, 54/55, 54/56, 54/57, 54/58, 54/59, 54/60, 55/56, 55/57, 55/58, 55/59, 55/60, 56/57, 56/58, 56/59, 56/60, 57/5 8,57/59,57/60,58/59,58/60,59/60,67/68,67/69,67/70,67/71,67/72, 68/69, 68/70, 68/71, 68/72, 69/70, 69/71, 69/72, 70/71, 70/72, 71/72, 10 7/108,107/109,107/110,107/111,108/109,108/110,108/111,109/110, 109/111,110/111,114/115,114/116,114/117,114/118,114/119,114/120 114/121,115/115,115/116,115/117,115/118,115/119,115/120,115/121,116/115,116/116,116/117,116/118,116 /119,116/120,116/121,117/115,117/116,117/117,117/118,117/119,117/120,117/121,118/115,118/116,118/117 ,118/118,118/119,118/120,118/121,119/115,119/116,119/117,119/118,119/119,119/120,119/121,120/115,120 /116,120/117,120/118,120/119,120/120,120/121,128/129,128/130,128/131,128/132,128/133,128/134,129/130 ,129/131,129/132,129/133,129/134,130/131,130/132,130/133,130/134,131/132,131/133,131/134,132/133,132 /134,133/134,142/143,142/144,142/145,142/146,142/147,142/148,142/149,142/150,143/144,143/145,143/146 ,143/147,143/148,143/149,143/150,144/145,144/146,144/147,144/148,144/149,144/150,145/146,145/147,145 /148145/149,145/150,146/147,146/148,146/149,146/150,147/148,147/149,147/150,148/149,148/150,149/150,

More preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide selected from: 114/115, 114/119, 114/120, 117/115, 117/116, 117/118, 117/121, 118/118, 118/119, 119/115, 119/116, 120/115, 120/120.

4. The optical probe according to claim 2, wherein the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein,

Preferably, the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9.

5. A nucleic acid molecule comprising:

(a) a coding sequence of the glyoxylate binding protein variant according to claim 1 or the optical probe according to any one of claims 2 to 4,

(b) The complementary sequence of (a).

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5,

Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

7. A host cell, wherein:

(1) expressing the glyoxylate binding protein variant of claim 1 or the optical probe of any one of claims 2 to 4;

(2) comprising the nucleic acid molecule of claim 5; or

(3) comprising the nucleic acid construct according to claim 6.

8. A detection kit comprising:

(1) The optical probe according to any one of claims 2 to 4,

(2) The nucleic acid sequence according to claim 5,

(3) The nucleic acid construct according to claim 6, or

(4) The host cell according to claim 7,

The detection kit optionally further comprises other reagents required for detecting glyoxylic acid using an optical probe.

Preferably, the detection kit further comprises one or more reagents selected from the following: buffer, culture medium, glyoxylic acid standard.

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

10. Use of the optical probe according to any one of claims 2 to 4, the nucleic acid sequence according to claim 5, the nucleic acid construct according to claim 6 or the host cell according to claim 7 in detecting glyoxylic acid in a sample, screening compounds, and localizing glyoxylic acid inside or outside a cell.

Preferably,

Detecting glyoxylic acid in a sample comprises the steps of contacting the optical probe or the host cell with the sample, detecting an optical change of the optically active polypeptide, and detecting glyoxylic acid in the sample according to the optical change of the optically active polypeptide.

The screening compound comprises the steps of: contacting the optical probe or host cell with a candidate compound in a system containing glyoxylic acid, detecting an optical change of an optically active polypeptide, and screening the compound according to the optical change of the optically active polypeptide; preferably, the screening compound comprises the steps of: contacting the host cell with a candidate compound in a system containing glyoxylic acid, and the optical change of the optically active polypeptide indicates whether the candidate compound regulates the uptake of glyoxylic acid by the cell,

The intracellular/extracellular localization of glyoxylic acid comprises the steps of: contacting a system containing glyoxylic acid with the optical probe or the host cell, and detecting an optical change of an optically active polypeptide.

More preferably, the system is a solution system, a cell system or a subcellular system.