Recombinant phosphorescent protein containing unnatural amino acid as well as preparation method and application of recombinant phosphorescent protein

Abstract

The invention relates to the technical field of biology, in particular to a recombinant phosphorescent protein containing non-natural amino acid and a preparation method and application thereof, and the recombinant phosphorescent protein is obtained by introducing non-natural amino acid mutation on the basis of LanM protein. The LanM protein is a kind of protein with high affinity to rare earth metal ions; one or more introduction sites of the non-natural amino acid in the LanM protein are provided; the non-natural amino acid is fluorescent non-natural amino acid. The fluorescent non-natural amino acid is introduced near a lanthanide metal binding site of the LanM protein as a sensitizing group, so that the fluorescent non-natural amino acid and the lanthanide metal site are kept at a very close distance in space, and energy of exciting light can be helped to be efficiently transferred to nearby lanthanide metal from the non-natural amino acid; the phosphorescent protein has the sensitivity of fluorescent non-natural amino acid to exciting light, and also has the long luminescence lifetime of lanthanide metal.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant phosphorescent protein containing non-natural amino acids, and a preparation method and application thereof. Background Art

[0002] Fluorescent proteins are widely used in detection and imaging experiments in the biological field due to their high biocompatibility, low cytotoxicity, and ability to be fused with target proteins. They help visualize biological processes and detect molecular regulatory relationships, greatly promoting our understanding of the structure and functional characteristics of biological systems.

[0003] The fluorescence lifetime of traditional fluorescent proteins (such as GFP) is limited to the nanosecond level, making it difficult to eliminate background interference through time-resolved detection; although the protein obtained by chemically labeling long-lived probes has phosphorescence with a microsecond lifetime, this method has defects such as poor labeling selectivity and high background signal.

[0004] While the ff transitions of lanthanide elements can produce narrowband emission and long-lived phosphorescence, their molar absorptivity is low, resulting in very weak luminescence intensity. This requires the "antenna effect" of light-absorbing organic ligands to enhance the luminescence signal. Therefore, while lanthanide metal-binding proteins possess a long phosphorescence lifetime, the lack of photosensitizing groups in the native proteins prevents the lanthanide metal from effectively absorbing the excitation light, resulting in low energy transfer efficiency and an extremely weak phosphorescence signal, hindering further application.

[0005] In addition, the genetic coding system in organisms cannot encode non-natural amino acids containing photosensitive groups into lanthanide metal-binding proteins to enhance the lanthanide metal phosphorescence signal. This is also one of the reasons for the bottleneck in the development of phosphorescent proteins.

[0006] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a recombinant phosphorescent protein containing non-natural amino acids and its preparation method and application, aiming to solve the problems of short luminescence lifetime and low signal-to-noise ratio of existing fluorescent proteins.

[0008] The technical solutions of the present invention are as follows:

[0009] A recombinant phosphorescent protein containing non-natural amino acids, wherein the recombinant phosphorescent protein is obtained by introducing non-natural amino acids into LanM protein; wherein the amino acid sequence of the LanM protein is shown in SEQ ID NO: 1; the introduction sites of the non-natural amino acids in the LanM protein include one or more of T41, F55, D56, K62, G64, T65, L66, A68, K69, L71, P85, N87, T90, L91, K93, K94, Y96, and T114; and the non-natural amino acids are fluorescent non-natural amino acids.

[0010] The recombinant phosphorescent protein containing non-natural amino acids, wherein the recombinant phosphorescent protein includes: a mutant having one or more mutation sites selected from V29A, L66M, and A117T and a non-natural amino acid introduction site selected from T65, the partial amino acid sequence of which is shown in SEQ ID NO: 2; or a mutant having one or more mutation sites selected from F34L, A22E, and A98E and a non-natural amino acid introduction site selected from T90, the partial amino acid sequence of which is shown in SEQ ID NO: 3.

[0011] The recombinant phosphorescent protein containing non-natural amino acids, wherein the non-natural amino acids include one or more of the following structural formulas:

[0012]

[0013]

[0014] A method for preparing a recombinant phosphorescent protein containing unnatural amino acids, comprising the steps of:

[0015] The plasmid containing the LanM protein coding sequence and the plasmid containing the aminoacyl-tRNA synthetase coding sequence are transformed into an expression host, and after adding unnatural amino acids and culturing, a recombinant phosphorescent protein containing unnatural amino acids is obtained;

[0016] The LanM protein coding sequence contains a non-natural amino acid introduction site TAG.

[0017] The method for preparing a recombinant phosphorescent protein containing unnatural amino acids, wherein the amino acid sequence of the aminoacyl-tRNA synthetase is shown in SEQ ID NO: 4; the mutants of the aminoacyl-tRNA synthetase are A302T, N346V, C348A, Y384F, V401L, and W417S.

[0018] An application of the recombinant phosphorescent protein containing unnatural amino acids in detecting lanthanide elements.

[0019] The application, wherein the lanthanide elements include one or more of Pr, Sm, Nd, Eu, Tb, Dy, and Ho.

[0020] A recombinant fusion protein comprises the phosphorescent protein containing unnatural amino acids.

[0021] The recombinant fusion protein, wherein the recombinant phosphorescent protein containing non-natural amino acids is inserted with a protease substrate sequence; and / or the recombinant phosphorescent protein containing non-natural amino acids is fused with a targeting protein.

[0022] A use of the recombinant fusion protein in detecting protease activity and / or immunofluorescence imaging.

[0023] Beneficial effects: The present invention provides a recombinant phosphorescent protein containing non-natural amino acids, and a preparation method and application thereof. The recombinant phosphorescent protein is obtained by introducing non-natural amino acids into LanM protein; wherein the amino acid sequence of the LanM protein is SEQ ID NO: 1; the introduction sites of the non-natural amino acids in the LanM protein include one or more of T41, F55, D56, K62, G64, T65, L66, A68, K69, L71, P85, N87, T90, L91, K93, K94, Y96, and T114. By introducing a fluorescent non-natural amino acid as a sensitizing group near the lanthanide metal binding site of the LanM protein, the fluorescent non-natural amino acid and the lanthanide metal site are kept very close in space. This helps to more efficiently transfer the energy of the excitation light from the non-natural amino acid to the nearby lanthanide metal. This allows the phosphoprotein to have both the sensitivity of the fluorescent non-natural amino acid to the excitation light and the long fluorescence lifetime of the lanthanide metal. Ultimately, the phosphorescence lifetime of the recombinant phosphoprotein reaches over 500 microseconds, and the signal-to-noise ratio is improved by more than 100 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of a recombinant phosphorescent protein containing an unnatural amino acid;

[0025] Figure 2 Schematic diagram of the evolutionary pathway of recombinant phosphorescent proteins containing unnatural amino acids;

[0026] Figure 3 Schematic diagram of matching screening between sensitizing groups and lanthanide metals;

[0027] Figure 4 This is the synthetic route of Acd;

[0028] Figure 5 This is the synthetic route of FAcd;

[0029] Figure 6The synthetic route of SAcd is shown in Figure 2.

[0030] Figure 7 Diagram for the evolution of Acd-RS and validation of site-directed insertion of unnatural amino acids;

[0031] Figure 8 is the mass spectrum of GFP-D190Acd;

[0032] Figure 9 Screening and characterization of UAA introduction sites in LanM protein

[0033] Figure 10 is the distribution of mutation sites of evolved LanM mutants;

[0034] Figure 11 This is a data diagram showing the enhancement effect of the unnatural amino acid SAcd on the phosphorescence signal;

[0035] Figure 12 The diagram shows the photophysical properties of phosphorescent proteins (phosphorescence spectrum, lifetime);

[0036] Figure 13 Schematic diagram of the principle of lanthanide sensor based on LanM-SAcd;

[0037] Figure 14 The data diagram of the detection results of the lanthanide sensor based on LanM-SAcd;

[0038] Figure 15 Based on LanM AMT -Schematic diagram of the principle of the T65SAcd protease activity sensor;

[0039] Figure 16 Based on LanM AMT -T65SAcd test result data chart;

[0040] Figure 17 Based on LanM AMT -Immunoimaging system and effect characterization diagram of T65SAcd. DETAILED DESCRIPTION

[0041] The present invention provides a recombinant phosphorescent protein containing unnatural amino acids, and its preparation method and application. To make the objectives, technical solutions, and effects of the present invention more clear and explicit, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0042] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0043] Even though directed evolution and modification can improve the fluorescence lifetime of existing fluorescent proteins to a certain extent, the lifetime of fluorescent proteins is still limited to the nanosecond level. While chemical labeling can introduce long-lived small molecule probes into proteins, this method also has issues with labeling selectivity and background interference.

[0044] Among the many long-lived fluorescent molecules, lanthanide metals have attracted much attention due to their extremely narrow emission bands and long fluorescence lifetimes. The fluorescence lifetime of lanthanide metals can reach 500-1000μs, which is a long lifespan, but lanthanide metals usually have very weak fluorescence signals due to their low absorbance. However, studies have found that strongly absorbing organic ligands, when coordinated with lanthanide metals, can sensitize lanthanide metals through intramolecular energy transfer. This sensitization strategy significantly enhances the emission intensity of lanthanide complexes while maintaining their long luminescence lifetime, providing the possibility for the development of a variety of long-lived lanthanide probes.

[0045] Based on this, the present invention provides a recombinant phosphorescent protein containing non-natural amino acids, wherein the recombinant phosphorescent protein is obtained by introducing non-natural amino acids into LanM protein; wherein the amino acid sequence of the LanM protein is SEQ ID NO: 1; the introduction sites of the non-natural amino acids in the LanM protein include one or more of T41, F55, D56, K62, G64, T65, L66, A68, K69, L71, P85, N87, T90, L91, K93, K94, Y96, and T114; and the non-natural amino acids are fluorescent non-natural amino acids.

[0046] Wherein, the amino acid sequence of SEQ ID NO: 1 is MAFRLSSAVLLAALVAAPAYAAPTTTTKVDIAAFDPDKDGTIDLKEALAA GSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLA AVEAQFKAANPDNDGTIDARELASPAGSALVNLIR.

[0047] In this embodiment, the schematic diagram of the recombinant phosphorescent protein is as follows Figure 1As shown, by introducing a fluorescent non-natural amino acid as a sensitizing group near the lanthanide metal binding site of the LanM protein, the fluorescent non-natural amino acid and the lanthanide metal site are kept very close in space, which helps to more efficiently transfer the energy of the excitation light from the non-natural amino acid to the nearby lanthanide metal. As a result, the recombinant phosphorescent protein has both the sensitivity of the fluorescent non-natural amino acid to the excitation light and the long fluorescence lifetime of the lanthanide metal. Ultimately, the phosphorescence lifetime of the recombinant phosphorescent protein reaches more than 500 microseconds, and the signal-to-noise ratio is improved by more than 100 times.

[0048] Specifically, the recombinant phosphoprotein binds to a lanthanide-binding protein (LanM) via a photosensitive non-natural amino acid. Genetic codon expansion technology is used to achieve site-specific insertion of the non-natural amino acid, which then binds to the lanthanide chelated by the LanM protein to form an energy transfer pair, generating a phosphorylation signal. Directed evolution and chemical optimization significantly enhance the lanthanide phosphorescence signal. The recombinant phosphoprotein has a lifetime of up to 500 microseconds, with signal intensity hundreds of times higher than that of wild-type LanM. Furthermore, the recombinant phosphoprotein is suitable for use in high-sensitivity lanthanide metal sensors, protease activity detection platforms, and immunofluorescence imaging. It exhibits an ultra-high signal-to-noise ratio in time-resolved detection, with a detection limit below 10 nanomolar, providing a novel tool for bioimaging and molecular sensing.

[0049] In some embodiments, the LanM protein can be replaced by other proteins belonging to lanthanide binding proteins; in this example, the LanM protein derived from Methylorubrum extorquens AM1, PDB: 6mi5, is used as a lanthanide binding protein, which has a binding site for lanthanide metals and obtains the fluorescent properties of lanthanide metals after coordinating lanthanide metals.

[0050] In some embodiments, the evolutionary pathway of recombinant phosphorescent proteins is shown in FIG. Figure 2 As shown, the phosphorescent protein includes: a mutant (LanM AMT -T65TAG), the partial amino acid sequence of which is shown in SEQ ID NO: 2; or a mutant in which the mutation sites are one or more of F34L, A22E and A98E and the non-natural amino acid introduction site is T90 (LanM ELE-T90TAG), the partial amino acid sequence of which is shown in SEQ ID NO: 3. Leveraging the high-affinity binding properties of the LanM protein to lanthanide metals, a LanM variant with a 50-fold increased phosphorescence signal was generated through directed evolution. Further chemical evolution was used to modify the structure of the sensitizing group, further increasing the phosphorescence signal by 10-fold. Key mutations included F34L, A22E, and A98E for the T90 mutant, and V29A, L66M, and A117T for the T65 mutant.

[0051] Specifically, the sequence of SEQ ID NO: 2 is:

[0052] APTTTTKADIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGXM DAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDG TIDTRELASPAGSALVNLIR*;

[0053] The sequence of SEQ ID NO:3 is:

[0054] EPTTTTKVDIAALDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLD AKELKGRVSEADLKKLDPDNDGXLDKKEYLEAVEAQFKAANPDNDGTI DARELASPAGSALVNLIR*; wherein X is a non-natural amino acid, which is TAG in the nucleotide sequence.

[0055] In some embodiments, the unnatural amino acid comprises one or more of the following structural formulas:

[0056]

[0057]

[0058] The energy of the triplet excited state of the above-mentioned non-natural amino acid matches that of the lanthanide metal, and the introduction of LanM protein can greatly improve the intensity of the phosphorescence signal of the lanthanide metal; preferably, the non-natural amino acid is FAcd. Based on the inductive effect, the absorption spectrum and emission spectrum of FAcd are both red-shifted relative to Acd, thereby increasing the overlap effect of its emission spectrum with the absorption spectrum of the lanthanide metal ion.

[0059] Specifically, the use of these unnatural amino acids as photosensitive groups, whose absorption spectra match the excited-state energy levels of lanthanides, facilitates efficient energy transfer from the unnatural amino acids to the lanthanide metals. This allows phosphorescent proteins to combine the sensitivity of the unnatural amino acids to excitation light with the long luminescence lifetime of the lanthanides. Furthermore, optimizing the unnatural amino acids can improve energy transfer efficiency. For example, sulfur substitution (SAcd) can extend the triplet state lifetime and enhance the intensity of the phosphorescent signal.

[0060] In addition, the present invention also provides a method for preparing a recombinant phosphorescent protein containing unnatural amino acids, comprising the steps of:

[0061] The plasmid containing the LanM protein coding sequence and the plasmid containing the aminoacyl-tRNA synthetase coding sequence are transformed into an expression host, and after adding unnatural amino acids and culturing, a recombinant phosphorescent protein containing unnatural amino acids is obtained;

[0062] The LanM protein coding sequence contains a non-natural amino acid introduction site TAG.

[0063] In this embodiment, the recombinant phosphoprotein utilizes genetic codon expansion technology to achieve site-specific insertion of photosensitive non-natural amino acids. This protein binds to the lanthanide chelated by the LanM protein to form an energy transfer pair, generating a phosphorylation signal. Directed evolution and chemical optimization significantly enhance the lanthanide phosphorescence signal. This recombinant phosphoprotein exhibits a lifetime of up to 500 microseconds, with signal intensity hundreds of times higher than that of wild-type LanM. Furthermore, this recombinant phosphoprotein is suitable for use in high-sensitivity lanthanide metal sensors, protease activity detection platforms, and immunofluorescence imaging. It exhibits an ultra-high signal-to-noise ratio in time-resolved detection, with a detection limit below 10 nM, providing a novel tool for bioimaging and molecular sensing.

[0064] Specifically, fluorescent non-natural amino acids were used as lanthanide metal sensitizing groups and site-specifically inserted into the LanM mutant protein using genetic codon expansion technology. This enabled energy transfer to the lanthanide metal ions, resulting in the successful construction of a phosphorescent protein with phosphorescence signal intensity several dozen times higher than that of the wild-type LanM protein. This was achieved primarily by exploiting the overlap between the emission spectrum of the non-natural amino acids and the absorption spectrum of the lanthanide metal ions. Furthermore, after introduction into the LanM protein, the non-natural amino acids were brought into close proximity with the lanthanide metal ions coordinated to the LanM protein, enabling efficient energy transfer.

[0065] In some embodiments, the amino acid sequence of the aminoacyl-tRNA synthetase (Acd-RS) is as shown in SEQ ID NO: 4; the mutation sites of the aminoacyl-tRNA synthetase include A302T, N346V, C348A, Y384F, V401L and W417S.

[0066] The present invention also provides a use of a recombinant phosphoprotein containing a non-natural amino acid for detecting lanthanides. The recombinant phosphoprotein containing a non-natural amino acid is used as a sensor for detecting lanthanides. When the phosphoprotein containing a non-natural amino acid binds to a lanthanide metal, it emits long-lived fluorescence upon irradiation with excitation light, thereby detecting lanthanides. The sensor exhibits metal selectivity exceeding 1000-fold, a signal-to-noise ratio exceeding 100-fold in time-resolved detection, and a detection limit below 10 nM.

[0067] Specifically, LanM-based phosphorescent proteins have both high affinity for lanthanide metals and the ability to efficiently sensitize lanthanide metals (sensitization effect of non-natural amino acids), thus achieving a lanthanide metal detection limit of less than 10nM and a selectivity of more than 1000 times.

[0068] In some embodiments, the lanthanide elements include, but are not limited to, one or more of Pr, Sm, Nd, Eu, Tb, Dy, and Ho.

[0069] In some embodiments, the sensor is sensitive to Eu 3+ The concentration range of the assay was from 10 nM to 10 μM.

[0070] In addition, the present invention also provides a recombinant fusion protein comprising the recombinant phosphorescent protein containing non-natural amino acids.

[0071] In this embodiment, by utilizing the recombinant phosphorescent protein containing non-natural amino acids to recombinantly obtain a recombinant fusion protein, the application of the recombinant phosphorescent protein in multiple fields can be realized; preferably, the recombinant fusion protein is formed by fusing the C-terminus or N-terminus of the recombinant phosphorescent protein containing non-natural amino acids to other protein or polypeptide sequences.

[0072] In some embodiments, the recombinant phosphorescent protein containing an unnatural amino acid is inserted into a protease substrate sequence; and / or the recombinant phosphorescent protein containing an unnatural amino acid is fused with a targeting protein.

[0073] Specifically, when using the phosphoprotein with a protease substrate sequence inserted into it to detect protease activity, the protease recognizes and cleaves the phosphoprotein containing the sequence, and the protease activity is detected by a decrease in the phosphorescence signal. The phosphoprotein is fused to the target protein as a secondary antibody marker, and time-resolved imaging is used to achieve multi-channel detection with background interference eliminated.

[0074] In some embodiments, the protease substrate sequence includes but is not limited to ENLYFQS, DEVD or LEVLFQGP; the protease cleavage sequence is inserted into the flexible region of the phosphoprotein, and the protease activity is detected by the change of the phosphorescence signal, and the detection effect is more sensitive and stable.

[0075] In some embodiments, the targeting protein includes but is not limited to Affibody; LanM fused with Affibody is used as a phosphorescent secondary antibody that specifically recognizes the primary antibody and is applied to a multi-channel time-resolved imaging system, which has the advantages of low background and high signal intensity.

[0076] At the same time, the present invention also provides an application of the recombinant fusion protein in detecting protease activity and / or immunofluorescence imaging.

[0077] Specifically, the recombinant fusion protein obtained by recombining phosphoproteins supports wash-free multi-channel imaging and is suitable for complex biological environments. The main reason is that the free lanthanide ions in the solution show weak phosphorescence due to the lack of sensitizer binding, and the sensitizer alone cannot chelate the lanthanide metal. The fluorescence lifetime of the free photosensitive non-natural amino acid (sensitizing group) is very short, and its signal can be excluded by time window detection. Therefore, long-lifetime phosphorescence will only come from phosphoproteins that contain both UAAs and chelated lanthanide metals. Even if the free lanthanide metals are not washed away during imaging, it will not affect the imaging effect. In addition, since there are both fluorescent non-natural amino acids and lanthanide metals sensitized by non-natural amino acids on the phosphoprotein, there are luminescence signals from two channels at the same time, namely the fluorescence signal of the amino acid itself and the phosphorescence signal of the lanthanide metal.

[0078] The present invention will be described in detail with reference to the following examples. It should also be understood that the following examples are only intended to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above disclosure of the present invention fall within the scope of protection of the present invention.

[0079] Example 1

[0080] This example screens pairs of photosensitizing groups (non-natural amino acids) and rare earth metal ions, including the following steps:

[0081] Seven unnatural amino acids, 7MW, 7CNW, AzAla, pBpa, Acd, FAcd, and SAcd, were mixed with seven lanthanide metals at equal concentrations. The enhancement of the characteristic lanthanide metal emission intensity after a 100 microsecond delay was measured at the excitation wavelength corresponding to the maximum absorption of each amino acid. Figure 3 As shown, Acd exhibits the highest energy transfer efficiency to Eu(III), and the phosphorescence intensity is enhanced more than tenfold after a 100 μs delay.

[0082] Example 2

[0083] This example designs and synthesizes unnatural amino acid homologues

[0084] 1) Synthesis and characterization of Acd, its synthetic route is as follows Figure 4 As shown, the specific steps include:

[0085] S1 (68 g, 230 mmol) was dissolved in dichloromethane, pyridine (29 g, 360 mmol) was added, and the temperature was then cooled to -78°C. After stirring at -78°C for 10 minutes, trifluoromethanesulfonic anhydride (68 g, 240 mmol) was slowly added, and the mixture was stirred at -78°C for 30 minutes, then warmed to room temperature and allowed to react overnight. After the reaction was completed, the solids in the reaction system were filtered to remove the solids. The resulting filtrate was first cooled to -78°C and then quenched with 10% aqueous citric acid solution. The quenched organic phase was washed three times with water, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the yellow solid product S2 (81 g, 82% yield).

[0086] In a glove box, S2 (1 g, 2.34 mmol), S3-1 (424 mg, 2.81 mmol), palladium acetate (52 mg, 0.23 mmol), rac-BINAP (293 mg, 0.47 mmol), and cesium carbonate (1.08 g, 3.34 mmol) were mixed in a flask. Toluene (6 mL, 0.4 M) was added and the flask was sealed with a rubber stopper. The flask was then removed from the glove box and heated at 120°C for 48 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solids in the reaction system were removed by filtration. The filtrate was then dried and separated by column chromatography to obtain S4-1 (910 mg, 91% yield).

[0087] Dissolve S4-1 (3 g, 7.0 mmol) in tetrahydrofuran and add aqueous lithium hydroxide (840 mg, 35 mmol, 20 mL of water). Heat the reaction mixture to reflux for 4 hours. After the reaction is complete, cool the reaction mixture to room temperature and extract with ethyl acetate. The resulting organic phase is then dried to yield crude product S5-1. S5-1 is used directly in the next step without further purification.

[0088] Add polyphosphoric acid (45 mL) to the flask, slowly add 12 mL of water and mix well, then add the above S5-1. Heat the reaction system to 135 ° C and react for 3 hours. After the reaction is completed, cool to room temperature, adjust the pH to 5.5 with 10M sodium hydroxide aqueous solution, and react at room temperature overnight. After the reaction is completed, collect the solid by centrifugation, resuspend it with water, adjust the pH to 9 with 5M sodium hydroxide aqueous solution, remove the solid by centrifugation, adjust the pH of the upper solution to 5 with 1M hydrochloric acid, then place it in a refrigerator at 4 ° C overnight, let it stand for crystallization, and filter to obtain a light yellow solid product Acd (809 mg, yield 41%).

[0089] Acd H NMR spectrum data1 H NMR (600 MHz, D2O) δ8.07 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 3.49 (t, J = 6.5 Hz, 1H), 2.98 (dd, J = 13.5, 5.7 Hz, 1H), 2.85 (dd, J = 13.7, 7.3 Hz, 1H). 13 C NMR (151 MHz, D2O) δ 182.18, 178.08, 141.79, 140.79, 134.90, 133.37, 131.33, 125.02, 124.76, 121.47, 119.40, 119.24, 118.90, 118.69, 57.36, 40.60. High-resolution mass spectrometry theoretical value C 16 H 13 N2O3 - [MH] - :281.0932, experimental value 281.0933. (Note: When preparing NMR samples, solid sodium hydroxide was added to promote dissolution).

[0090] 2) Synthesis and characterization of FAcd, its synthetic route is as follows Figure 5 As shown,

[0091] A tyrosine analogue is used as a starting material, reacted with Tf2O to generate a coupling reaction precursor, and a carbon-nitrogen bond coupling product is obtained through a Buchwald-Hartwig reaction. The carboxylic acid compound obtained by hydrolysis is deprotected under polyphosphoric acid conditions, and then subjected to Friedel-Crafts acylation and cyclization to obtain the final product FAcd. The method specifically includes the following steps:

[0092] S1 (68 g, 230 mmol) was dissolved in dichloromethane, pyridine (29 g, 360 mmol) was added, and the temperature was then cooled to -78°C. After stirring at -78°C for 10 minutes, trifluoromethanesulfonic anhydride (68 g, 240 mmol) was slowly added, and the mixture was stirred at -78°C for 30 minutes before warming to room temperature and reacting overnight. After the reaction was completed, the solids in the reaction system were filtered to remove the solids. The resulting filtrate was first cooled to -78°C and then quenched with 10% aqueous citric acid. The quenched organic phase was washed three times with water, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the yellow solid product S2 (81 g, 82% yield).

[0093] In a glove box, S2 (1 g, 2.34 mmol), S3-2 (476 mg, 2.81 mmol), palladium acetate (52 mg, 0.23 mmol), rac-BINAP (293 mg, 0.47 mmol), and cesium carbonate (1.08 g, 3.34 mmol) were mixed in a flask. Toluene (6 mL, 0.4 M) was added and the flask was sealed with a rubber stopper. The flask was then removed from the glove box and heated at 120°C for 48 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solids in the reaction system were removed by filtration. The filtrate was then dried and separated by column chromatography to obtain S4-2 (887 mg, 85% yield).

[0094] Dissolve S4-2 (3.1 g, 7.0 mmol) in tetrahydrofuran and add aqueous lithium hydroxide (840 mg, 35 mmol, 20 mL of water). Heat the reaction mixture to reflux for 4 hours. After the reaction is complete, cool the reaction mixture to room temperature and extract with ethyl acetate. The resulting organic phase is then dried to yield crude S5-2. S5-2 is used directly in the next step without further purification.

[0095] Add polyphosphoric acid (45 mL) to the flask, slowly add 12 mL of water and mix well, then add the above S5-1. Heat the reaction system to 135 ° C and react for 3 hours. After the reaction is completed, cool to room temperature, adjust the pH to 5.5 with 10M sodium hydroxide aqueous solution, and react at room temperature overnight. After the reaction is completed, collect the solid by centrifugation and resuspend it with water, adjust the pH to 9 with 5M sodium hydroxide aqueous solution, remove the solid by centrifugation, adjust the pH of the upper solution to 5 with 1M hydrochloric acid, then place it in a refrigerator at 4 ° C overnight, let it stand for crystallization, and filter to obtain a light brown solid product FAcd (777 mg, yield 31%).

[0096] FAcd: H NMR data 1 H NMR (600 MHz, D2O) δ7.39 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.11-7.05 (m, 1H), 7.02 (td, J = 8.5, 2.9 Hz, 1H), 6.75 (dd, J = 9.1, 4.5 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 3.43 (t, J = 7.2 Hz, 1H), 2.83 (dd, J = 13.9, 5.8 Hz, 1H), 2.67 (dd, J = 14.0, 7.6 Hz, 1H). 13CNMR (151MHz, D2O) δ181.97, 176.95, 157.68, 156.09, 137.49 (d, J = 165.9Hz) 134.95, 131.57, 124.30, 122.34 (d, J = 12.4Hz), 119.39 (d, J = 3.77Hz), 119.01 (d, J = 3.77Hz), 117.83, 117.45, 108.23 (d, J = 11.3Hz), 57.34, 40.57. 19 F NMR (377MHz, D2O) δ-120.44. High resolution mass spectrometry theoretical value C 16 H 12 FN2O3 - [MH] - :299.0837, experimental value 299.0839. (Note: When preparing NMR samples, solid sodium hydroxide was added to promote dissolution)

[0097] 3) Synthesis and characterization of SAcd, its synthetic route is as follows Figure 6 As shown, the specific steps include:

[0098] Disperse zinc powder (2.15 g, 32.7 mmol) in N,N-dimethylformamide (11 mL, 1 M) and add iodine pellets (139 mg, 0.55 mmol) at room temperature. Stir for ten minutes, then add S6 (3.6 g, 10.9 mmol). Stir at room temperature for ten minutes, and then add iodine pellets (139 mg, 0.55 mmol) again. Allow to react at room temperature for four hours to convert S6 to S7, which can be used directly in the next step without purification.

[0099] In a glove box, S8 (2.44 g, 8.38 mmol) and tetrakistriphenylphosphine palladium (1.94 g, 1.68 mmol) were mixed in a flask, sealed, and N,N-dimethylformamide (10 mL) was added. The reaction system was heated to 60°C, and then the above-mentioned S7 was slowly added to the flask. The reaction was allowed to react at 60°C overnight. After the reaction was completed, the reaction was cooled to room temperature and quenched by adding saturated ammonium chloride aqueous solution. The product was diluted with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the light yellow solid product S9 (3.46 g, 83% yield).

[0100] Dissolve S9 (3.9 g, 9.4 mmol) in tetrahydrofuran / water (30 / 10 mL) and add lithium hydroxide (1.1 g, 47 mmol). Allow to react at room temperature for 2 hours. After the reaction is complete, dilute with water, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain crude product S10. S10 can be used directly in the next step without purification.

[0101] The crude product S10 was added to a flask, and a solution of hydrogen chloride in 1,4-dioxane (20 mL, 4 mol / L) was added dropwise at room temperature. The reaction was allowed to proceed for two hours. After the reaction, the solvent was evaporated to dryness, and the resulting crude oil was washed with petroleum ether (20 mL, once) and dichloromethane (20 mL, once). After washing, the crude product was dissolved in methanol (5 mL), heated to 40°C to dissolve completely, and then diethyl ether (25 mL) was slowly added dropwise to allow the product to crystallize. The crystals were filtered to obtain the product SAcd (1.4 g, 45% yield) as a light yellow solid. SAcd precipitated as the hydrochloride salt.

[0102] SAcd: H NMR data 1 H NMR 1 H NMR (400 MHz, D2O) δ8.07 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.52-7.43 (m, 1H), 7.38-7.23 (m, 3H), 7.22 (d, J = 8.3 Hz, 1H), 3.44 (t, J = 5.6 Hz, 1H), 2.91 (dd, J = 13.6, 5.6 Hz, 1H), 2.74 (dd, J = 13.6, 7.5 Hz, 1H). 13 C NMR (151 MHz, D2O) δ 181.58, 180.53, 137.31, 136.50, 135.58, 133.72, 132.41, 128.56, 128.37, 127.03, 127.00, 126.09, 125.89, 125.59, 57.22, 40.81. High-resolution mass spectrometry theoretical value C 16 H 12 NO3S - [MH] - :298.0543, experimental value 298.0545. (Note: When preparing NMR samples, solid sodium hydroxide was added to promote dissolution)

[0103] Example 3

[0104] This embodiment performs the screening and verification of Acd-RS, including the following:

[0105] By screening the mutation library in the laboratory, an Acd-RS mutant (A302T / N346V / C348A / Y384F / V401L / W417S) that can efficiently recognize Acd, FAcd, SAcd, and SeAcd was obtained. Figure 7GFP-D190TAG was used as a reporter protein and expressed under 1mM Acd conditions. The molecular weight was consistent with the theoretical value by mass spectrometry, further confirming that Acd, FAcd, and Acd were successfully introduced into the 190 site of GFP, as shown in Figure 2. Figure 8 shown.

[0106] Example 4

[0107] This example screens for non-natural amino acid insertion sites, including the following:

[0108] Based on the structure of LanM and its lanthanide binding sites, 42 sites in LanM were mutated into TAG codons, and then transformed into E. coli together with a plasmid containing Acd-RS. After induced expression, the phosphorescence signal intensity was measured to screen out sites where Acd specific incorporation can achieve efficient energy transfer. After screening, the position of Acd significantly affected the phosphorescence signal of LanM, among which LanM proteins with Acd located at sites T65, T90 and T114 showed significantly higher phosphorescence signals, such as Figure 9 shown.

[0109] The specific preparation method of phosphorescent protein is as follows: Taking the LanM-T65Acd mutant (one of the 42 TAG mutants) as an example, the plasmid containing LanM-T65TAG and the plasmid containing Acd-RS are simultaneously transformed into Escherichia coli, cultured at 37°C overnight, and 40 μL of the bacterial liquid is inoculated into 2 mL of fresh LB medium (2% inoculation ratio). After further culture at 37°C for 2 hours, the unnatural amino acid Acd and inducer are added to the culture medium. After inducing the LanM-T65Acd mutant at 30°C overnight, the bacteria are lysed. The lysate contains the LanM-T65Acd mutant protein, which can be directly added with metal Eu. 3+ The intensity of the phosphorescent signal is measured. Furthermore, because the LanM-T65Acd mutant carries a 6×his tag, the protein can be purified via nickel affinity chromatography for use in biosensors or bioimaging experiments. This method is simple to use and the resulting phosphorescent protein has very high biocompatibility and purity.

[0110] Example 5

[0111] This example performs mutation and directed evolution of phosphorescent proteins, including the following:

[0112] Through directed protein evolution, two mutants LanM with significantly improved phosphorescence signals were obtained. AMT -T65TAG (V29A / L66M / A117T) and LanM ELE-T90TAG (A22E / F34L / A98E), the corresponding sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The phosphorescence signal intensity is increased by about 50 times relative to the starting LanM-WT. Figure 10 shown.

[0113] Example 6

[0114] This example performs optimization screening of photosensitizing groups to verify the improvement of phosphorescent protein signal intensity, including the following:

[0115] The three unnatural amino acids prepared in Example 2 were introduced into LanM using Acd-RS. AMT -T65TAG and LanM ELE After -T90TAG, it was found that the introduction of FAcd only slightly increased the intensity of the phosphorescence signal, while SAcd extended the triplet lifetime due to the heavy atom effect, and its phosphorescence signal was significantly improved compared with Acd after its insertion into LanM.

[0116] It can be seen that after chemical structure optimization, the energy transfer efficiency of the sensitizer to rare earth metals is significantly improved, which is hundreds of times higher than that of the starting LanM-WT. Figure 11 shown.

[0117] Example 7

[0118] This example measures and characterizes the photophysical properties of phosphoproteins, including the following:

[0119] Combine Acd-RS with LanM AMT -T65TAG、LanM ELE -T90TAG were co-transfected into E. coli DH10B competent cells, 1mM SAcd was added to the culture system to induce the expression of mutant protein, and LanM was purified. AMT -T65SAcd and LanM ELE-T90SAcd two mutant proteins were mixed with the rare earth metal Eu(III) and their fluorescence spectra under excitation at 390nm (the optimal excitation wavelength of SAcd) were measured as the delay time increased. The results showed that when the delay time was 0μs, both mutants could detect the fluorescence signal of SAcd (450nm) and the fluorescence signal of Eu(III) with comparable signal intensity (615nm); when the delay time increased, the fluorescence signal intensity of SAcd decreased rapidly, while the fluorescence signal of Eu(III) decreased very slowly, indicating that SAcd on both mutants can efficiently transfer energy to the rare earth metal Eu(III), and the emission light lifetime of Eu(III) is much longer than that of SAcd, further proving that phosphorescent protein mutants with long excitation wavelength (390nm), long fluorescence lifetime (500μs), and high phosphorescence intensity were successfully obtained.

[0120] LanM AMT -T65TAG was used as an example, and the phosphorescence spectrum and phosphorescence lifetime were characterized. AMT -T65Acd and LanM AMT -T65SAcd has a lifetime of up to 500 μs, which can be significantly distinguished from the signals of unnatural amino acids with short fluorescence lifetimes, such as Figure 12 shown.

[0121] Example 8

[0122] This embodiment designs and applies lanthanide metal sensors based on phosphorescent proteins, including the following:

[0123] Schematic diagram of the principle of lanthanide metal sensor based on phosphorescent protein Figure 13 As shown, it was detected that 50nM LanM-SAcd (including LanM AMT -T65SAcd and LanM ELE -T90SAcd) to Eu 3+ The detection limit is 8nM, the signal-to-noise ratio is >100, and other metal ions (such as Ca 2+ Mg 2+ ) No cross-reaction, such as Figure 14 shown.

[0124] Example 9

[0125] This example designs and applies a phosphoprotein-based protease activity sensor, including the following:

[0126] Schematic diagram of the principle of the protease activity sensor based on phosphorylated protein Figure 15 As shown, it is tested and it is known that in LanM AMT-T65SAcd was inserted into the P2 position of the TEV protease cleavage sequence (ENLYFQS), and the phosphorescence signal decreased by more than 90% after enzymatic cleavage. Similar strategies were applied to Caspase-3 and HRV 3C protease, and the orthogonality between the three proteases was very good. These phosphorescent protein substrates showed significant phosphorescence changes when cleaved by specific proteases, showing excellent orthogonality, indicating its potential as a screening platform for sequence-specific proteases, such as Figure 16 shown.

[0127] Example 10

[0128] This example develops a phosphoprotein-based immunophosphorescence imaging reagent, including the following:

[0129] Affibody-fused LanM AMT -T65SAcd is used to label PD-L1 in HeLa cells. Under 405nm excitation, the fluorescence signal of the 615nm channel co-localizes with the 450nm fluorescence channel, and the background interference is significantly reduced. Figure 17 shown.

[0130] In summary, the present invention provides a recombinant phosphorescent protein containing non-natural amino acids, and a preparation method and application thereof. The recombinant phosphorescent protein is obtained by introducing non-natural amino acids into LanM protein; wherein the amino acid sequence of the LanM protein is SEQ ID NO: 1; and the introduction sites of the non-natural amino acids in the LanM protein include one or more of T41, F55, D56, K62, G64, T65, L66, A68, K69, L71, P85, N87, T90, L91, K93, K94, Y96, and T114. The present invention introduces a fluorescent non-natural amino acid as a sensitizing group near the lanthanide metal binding site of the LanM protein, so that the fluorescent non-natural amino acid and the lanthanide metal site maintain a very close spatial distance. This can help the energy of the excitation light to be transferred from the non-natural amino acid to the nearby lanthanide metal more efficiently. As a result, the phosphorescent protein has both the sensitivity of the fluorescent non-natural amino acid to the excitation light and the long fluorescence lifetime of the lanthanide metal. Ultimately, the phosphorescence lifetime of the recombinant phosphorescent protein reaches more than 500 microseconds, and the signal-to-noise ratio is improved by more than 100 times.

[0131] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A recombinant phosphorescent protein containing unnatural amino acids, characterized in that: The recombinant phosphorescent protein is obtained by introducing non-natural amino acids into the LanM protein; wherein the amino acid sequence of the LanM protein is shown in SEQ ID NO: 1; the introduction sites of the non-natural amino acids in the LanM protein include one or more of T41, F55, D56, K62, G64, T65, L66, A68, K69, L71, P85, N87, T90, L91, K93, K94, Y96, and T114; and the non-natural amino acids are fluorescent non-natural amino acids.

2. The recombinant phosphorescent protein containing unnatural amino acids according to claim 1, wherein The recombinant phosphorescent protein includes: a mutant in which the mutation site is one or more of V29A, L66M, and A117T and the non-natural amino acid introduction site is T65, and the partial amino acid sequence of the protein is shown in SEQ ID NO: 2; or a mutant in which the mutation site is one or more of F34L, A22E, and A98E and the non-natural amino acid introduction site is T90, and the partial amino acid sequence of the protein is shown in SEQ ID NO:

3.

3. The phosphorescent protein containing an unnatural amino acid according to claim 1, wherein The non-natural amino acids include one or more of the following structural formulas:

4. A method for preparing a recombinant phosphorescent protein containing an unnatural amino acid according to any one of claims 1 to 3, characterized in that: Including steps: The plasmid containing the LanM protein coding sequence and the plasmid containing the aminoacyl-tRNA synthetase coding sequence are transformed into an expression host, and after adding unnatural amino acids and culturing, a recombinant phosphorescent protein containing unnatural amino acids is obtained; Wherein, the LanM protein coding sequence contains a non-natural amino acid introduction site TAG.

5. The method for preparing a recombinant phosphorescent protein containing unnatural amino acids according to claim 4, wherein: The amino acid sequence of the aminoacyl-tRNA synthetase is shown in SEQ ID NO: 4; the mutants of the aminoacyl-tRNA synthetase are A302T, N346V, C348A, Y384F, V401L and W417S.

6. Use of the recombinant phosphorescent protein containing an unnatural amino acid according to any one of claims 1 to 3 in detecting lanthanides.

7. The use according to claim 6, characterized in that The lanthanide elements include one or more of Pr, Sm, Nd, Eu, Tb, Dy, and Ho.

8. A recombinant fusion protein, characterized in that The invention relates to a recombinant phosphorescent protein comprising a non-natural amino acid as claimed in any one of claims 1 to 3.

9. The recombinant fusion protein according to claim 8, characterized in that The recombinant phosphorescent protein containing non-natural amino acids is inserted with a protease substrate sequence; and / or the recombinant phosphorescent protein containing non-natural amino acids is fused with a targeting protein.

10. Use of the recombinant fusion protein according to any one of claims 8 to 9 in detecting protease activity and / or immunofluorescence imaging.

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