Protein fluorescent site-specific labeling method in cell lysate and application thereof

Abstract

This invention discloses a method for site-specific fluorescent labeling of proteins in cell lysates and its applications, belonging to the field of pharmaceutical bioengineering technology. Utilizing genetic code amplification technology, non-natural amino acids containing azide groups are introduced at specific sites of the target protein. A long-lifetime terbium complex modified with dibenzocyclooctylene is directly added to the cell lysate, enabling copper-free click chemistry labeling of the target protein. This method exhibits good biocompatibility and wide applicability, and can construct CoraFluor-DBCO-labeled protein target libraries. Furthermore, a time-resolved fluorescence resonance energy transfer (TR-FRET) screening system is constructed, utilizing the long fluorescence lifetime of the terbium complex to filter out biological background fluorescence and improve the signal-to-noise ratio. Competitive TR-FRET enables homogeneous screening of small molecule ligands targeting the target protein in the lysate. This method avoids protein purification steps, is simple to operate, and has high screening efficiency.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical bioengineering technology, specifically to a method for site-specific labeling of target proteins in cell lysates and its application. Background Technology

[0002] Specific labeling and functional analysis of proteins are core technologies for molecular interaction research and drug screening. Site-specific protein labeling techniques allow for quantitative analysis of protein interactions with other molecules or conformational changes, thereby exploring protein structure and biological function. Currently, traditional labeling methods (such as NHS ester conjugation) have high sample requirements, necessitating target protein purification. The process involves cell disruption, affinity separation, buffer replacement, and other steps, taking ≥48 hours. Furthermore, the purification process can easily lead to protein denaturation and inactivation (membrane proteins) or loss of physiological activity due to the removal of natural interaction partners. Commonly used chemical conjugation methods, such as random chemical conjugation (e.g., lysine ε-amino modification), result in non-site-specific labeling, producing an average of 3-8 random modification sites per protein molecule, potentially masking active sites or interfering with conformation. While fluorescent protein gene fusion expression can achieve fluorescent labeling, it has significant limitations. For example, some fluorescent proteins with dimerization tendencies (such as YFP) may induce abnormal aggregation of target proteins, and the approximately 30 kDa fusion tag may interfere with the spatial accessibility of small molecule binding pockets. Click chemistry is a class of chemical reactions widely used for biomolecular modification, especially azido-alkyne cycloaddition reactions, which enable protein-specific labeling in cellular environments. While traditional copper-catalyzed click reactions (Copper(I)-Catalyzed Alkyne-Azide Cycloaddition, CuAAC) are highly efficient, the Cu(I) and Cu(II) present during the catalytic process can participate in redox reactions, generating reactive oxygen species that can potentially damage protein structure and biological activity, limiting their application in complex biological systems such as lysates and cells. Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC), a copper-free click chemistry method, is suitable for biological samples and can achieve efficient and highly specific labeling in complex biological environments with minimal perturbation to the target protein structure. It is suitable for functional studies requiring the maintenance of native conformation and for metal-sensitive proteins (such as metalloenzymes and transcription factors).

[0003] With the continuous development of molecular biology techniques, genetic code expansion (GCE) technology is increasingly being used for protein-specific site labeling. This technology allows the introduction of non-natural amino acid (nnAAs) tags modified with azide groups at specific protein sites, which can then be used to achieve specific site dye labeling of the target protein through a copper-free click chemistry reaction driven by dibenzocyclooctyne (DBCO).

[0004] Traditional methods for studying protein-ligand interactions include surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence polarization (FP). However, due to the high difficulty in immobilizing protein samples, large consumption, and low detection sensitivity, these methods are insufficient to meet the demands of modern drug screening for high efficiency, high fidelity, and compatibility with complex biological samples.

[0005] Time-resolved fluorescence resonance energy transfer (TR-FRET) uses the long-lived fluorescence dye CoraFluor as the fluorescence donor. Utilizing its millisecond-level fluorescence lifetime, it effectively filters out interference from short-lived background fluorescence in biological samples through time-gated detection technology. At the same time, its homogeneous wash-free detection mode eliminates the need to separate unbound components, completing signal acquisition directly in solution. This simplifies experimental procedures, avoids complex dissociation or signal loss caused by washing steps, and significantly improves the signal-to-noise ratio and data reliability in complex biological environments (such as cell lysates). Summary of the Invention

[0006] This invention provides a method for site-specific labeling of target proteins in cell lysates, and provides specific applications of this method to overcome the shortcomings of existing technologies.

[0007] To achieve the above-mentioned objectives, the present invention employs the following technical solution: A method for site-specific labeling of a target protein in cell lysate, the method comprising the following steps: (1) The dibenzocyclooctylene (DBCO) group was coupled with a long fluorescence lifetime terbium complex (CoraFluor) by amide bond condensation to obtain the dibenzocyclooctylene (DBCO) modified long fluorescence lifetime terbium complex CoraFluor-DBCO. (2) Introduce non-natural amino acids containing azide groups into the target protein; (3) The target protein containing non-natural amino acids with azide groups is expressed in large quantities by tool cells. The tool cells are lysed, and CoraFluor-DBCO is added to the cell lysate. The target protein undergoes a copper-free click chemical reaction with the azide groups in the target protein, thereby achieving site-directed labeling of the target protein. (4) Constructing a time-resolved fluorescence resonance energy transfer (TR-FRET) screening system: The target protein is the screening target. The target protein labeled with the CoraFluor-DBCO complex competes with the fluorescent tracer and the compound to be screened in the lysis buffer. The binding is detected by the donor-acceptor energy transfer. (5) The binding signal between the target protein and the substance to be screened in the lysate is detected by TR-FRET signal, and the inhibition curve is fitted to achieve the purpose of high-throughput screening and quantitative analysis of the affinity of the compound to be screened for the target protein.

[0008] The long fluorescence lifetime terbium complex (CoraFluor-DBCO) modified with dibenzocyclooctyne (DBCO) in the above method is a complex in which a dibenzocyclooctyne (DBCO) group is introduced into the long fluorescence lifetime terbium complex through an amino condensation reaction, thereby enabling it to undergo a copper-free click chemical reaction with an azide group.

[0009] Furthermore, in step (2), the codons corresponding to specific amino acids in the target protein coding sequence are replaced with TAG stop codons, and the bioorthogonal tRNA / aminoacyl-tRNA synthetase (aaRS) system is used in... E. coli Expression in the expression system introduces non-natural amino acids containing azide groups into the target protein.

[0010] Taking Halo Tag protein and Galectin-3 protein as examples, step (2) involves replacing the codon corresponding to amino acid F at position 79 of the Halo Tag protein with a TAG stop codon; replacing the codon corresponding to amino acid R at position 129 of the Galectin-3 protein with a TAG stop codon; and then co-transforming the site-directed mutagenesis-mutated Halo Tag protein and Galectin-3 protein into supercompetent cells containing the pEVOL-pAzF plasmid, respectively. This invention can be extended to a variety of proteins, and this method can be extended to rapidly construct protein libraries without protein purification steps.

[0011] Further: In step (2), 4-azido-L-phenylalanine is added during the expression of the target protein.

[0012] Furthermore, in step (3), a mild lysis buffer is used: maintaining near-physiological conditions at pH 7.0-7.4; avoiding buffers containing interfering substances such as trehalose (which may affect subsequent detection); and adding protease inhibitors to prevent protein degradation.

[0013] Furthermore: In step (3), DBCO-alkyne is used as the reactive group: the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction is highly efficient, fast, and selective, and can be efficiently combined with the azide group under physiological conditions.

[0014] Further: In step (3), the CoraFluor-DBCO complex serves as a donor fluorophore for TR-FRET, and the target protein with an azide group is labeled in the lysis buffer.

[0015] Further: In step (3), the copper-free click chemical reaction is carried out in PBS phosphate buffer solution and labeled at 4-37°C for 0.5-24 hours.

[0016] Further: In step (4), the TR-FRET receptor fluorophore Cy5 is modified onto the small molecule ligand Halo Tag ligand of the model target protein Halo Tag, and used as the fluorescent tracer Halo Tag ligand-Cy5 for spectroscopic characterization.

[0017] Further: In step (4), the TR-FRET receptor fluorophore FAM is modified onto the small molecule ligand of the model target protein Galectin-3 as a fluorescent tracer G-FAM for spectroscopic characterization.

[0018] Furthermore, a TR-FRET detection system based on a competitive binding mode was constructed by using fluorescently labeled known ligands (tracers) to compete with the small molecules to be screened for binding to the target protein binding site. This was done by monitoring the changes in TR-FRET signals during experiments where different concentrations of the small molecules to be screened competed with the fluorescent tracers for binding to the target protein.

[0019] Synthesis of fluorescent tracers: High-affinity ligands corresponding to the target protein can be selected, and multiple fluorophores can be covalently coupled, not limited to those mentioned in this patent. Taking Halo Tag protein and Galectin-3 protein as examples, the fluorescent tracer Halo Tag ligand-Cy5 and the small molecule to be detected are incubated with the fluorescently labeled Halo Tag protein lysate at room temperature for 1.5 h, and then loaded into a 384-well microplate; or the fluorescent tracer G-FAM and the small molecule to be detected are incubated with the fluorescently labeled Galectin-3 protein lysate at room temperature for 1.5 h, and then loaded into a 384-well microplate.

[0020] Further: In step (5), the spectral data analysis is performed using time-resolved fluorescence scanning. The fluorescence intensity of the donor and acceptor in the TR-FRET system is measured by statistically analyzing the total number of photons of the donor and acceptor within a certain time window. The FRET ratio is calculated by the ratio of acceptor to donor fluorescence intensity. The dissociation constant Kd and inhibition constant Ki are calculated using a unit point-total binding fitting model to analyze the interaction mode between the small molecule to be screened and the target protein in the lysis buffer.

[0021] The method of site-specific labeling of target proteins in cell lysates has wide applications: In the lysate, candidate drug molecules, fluorescent tracers, and labeled target proteins compete for binding, achieving the effect of directly screening the interaction between candidate drug molecules and target proteins; the lysate is closer to the physiological environment, retaining many intracellular cofactors and chaperone proteins, and better reflects the activity under physiological conditions than purified enzymes. Therefore, enzyme activators or inhibitors can be screened in the lysate to study the effect of drugs on enzyme reaction kinetics; utilizing the lysate environment, other factors required to maintain protein-protein interactions (PPIs) may be present, and small molecules or peptides that disrupt or stabilize specific PPIs can also be screened.

[0022] The method of site-specific labeling of target proteins in cell lysates is used in screening substances that bind to target proteins.

[0023] The application of the method for site-specific labeling of target proteins in cell lysates in drug screening.

[0024] This invention utilizes genetic code amplification technology to introduce non-natural amino acids modified with azide groups at specific sites of proteins, and then achieves long-lifetime dye labeling of specific sites of the target protein through a copper-free click chemistry reaction driven by dibenzocyclooctylene. With the help of small molecule ligand fluorescent tracers, high-throughput screening of homogeneous drugs based on TR-FRET can be directly achieved in cell lysate.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects: This invention proposes a site-specific modification technique for recombinant proteins, utilizing strain-promoted azido-acetylene cycloaddition (SPAAC) to achieve copper-free click labeling. This eliminates the need for recombinant protein purification, allowing direct manipulation in cell lysis buffer, significantly simplifying the experimental procedure, shortening time, and reducing costs. It also avoids protein inactivation or conformational changes that may occur during traditional purification processes, thus maximizing the preservation of the protein's native functional state. Highly efficient fluorescent site-specific labeling of target proteins is achieved by directly adding a long-lived terbium complex (CoraFluor-DBCO) modified with dibenzocyclooctylene to the lysis buffer. This method exhibits good biocompatibility and broad protein applicability, suitable for various recombinant proteins, and can construct drug protein target libraries labeled with long-lived terbium complexes at specific sites. Based on this, a time-resolved fluorescence resonance energy transfer (TR-FRET) screening system is established, utilizing the high stability, narrow emission spectrum, and large Stokes shift of CoraFluor dye, as well as the low background interference due to its long fluorescence lifetime, to achieve efficient and highly specific homogeneous screening in complex biological environments. This method can accurately assess the binding affinity between small molecules and target proteins by detecting changes in TR-FRET signals caused by the competitive binding of small molecules and fluorescent tracers to the target protein in the lysis buffer, achieving high-throughput screening with low background and high sensitivity. This method can be flexibly extended to drug screening for different targets, providing a novel, efficient, low-cost, and widely applicable technology platform for early high-throughput drug screening and lead compound discovery, significantly improving screening efficiency and reducing R&D costs. Attached Figure Description

[0026] Figure 1 This is a flowchart of the technology of the present invention.

[0027] Figure 2 This diagram illustrates the expression of the target protein and its fluorescent labeling in the lysis buffer, where A and B represent the target protein HaloTag. F79pAzF / Galectin-3 R129pAzF Co-transformation verification with a biologically orthogonal tRNA / aminoacyl-tRNA synthetase (aaRS) plasmid; C, D: dye CoraFluor-DBCO in lysis buffer with target protein Halo Tag F79pAzF / Galectin-3 R129pAzF Click reaction coupling and fluorescence imaging.

[0028] Figure 3 In the middle: A is the fluorescent tracer Halo Tag. Fluorescence characterization spectrum of ligand-Cy5; B represents the fluorescence tracer Halo Tag ligand-Cy5 and the model target protein Halo Tag in the lysis buffer in Example 3. F79pAzFProtein binding analysis; C represents the fluorescent tracer from Example 4 and the small molecule to be screened, Halo Tag ligand-Cl, along with the model target protein Halo Tag in the lysis buffer. F79pAzF Protein competitive binding analysis.

[0029] Figure 4 In the image: A is the fluorescence characterization spectrum of the fluorescent tracer G-FAM; B is the fluorescence characterization spectrum of the fluorescent tracer G-FAM and the model target protein Galectin-3 in the lysis buffer in Example 3. R129pAzF Protein binding analysis; C represents the fluorescent tracer G-FAM and the small molecule TDP-139 to be screened in Example 4, and the model target protein Galectin-3 in the lysis buffer. R129pAzF Protein competitive binding analysis. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description, in conjunction with specific embodiments and accompanying drawings, further illustrates the invention. Obviously, the described embodiments are only a portion, not all, of the embodiments disclosed in this invention. All other embodiments obtained by those skilled in the art based on the embodiments disclosed in this invention without inventive effort are within the scope of protection of this invention.

[0031] TR-FRET eliminates the need for coating and washing, enabling direct detection in complex systems such as lysis buffers, thus improving screening efficiency. Utilizing a long-lived terbium complex (CoraFluor) as a donor reduces background fluorescence interference in complex biological environments, enhancing the signal-to-noise ratio. Homogeneous, wash-free detection simplifies experimental procedures, reduces operational complexity, and saves costs. This technology supports high-throughput screening of compounds, significantly accelerating the discovery and validation of promising compounds.

[0032] like Figure 1 As shown, this invention introduces azide groups at specific sites on target proteins using genetic code amplification technology, and achieves precise covalent labeling using long-lifetime terbium complexes modified with dibenzocyclooctylene (DBCO). Finally, the affinity between the compound and the target protein is detected via TR-FRET signaling. This method uses terbium complexes with long fluorescence lifetimes, which can serve as TR-FRET donors, and small-molecule fluorescent dyes as TR-FRET acceptors. Time-gated detection effectively eliminates background fluorescence interference inherent in biological samples.

[0033] Example 1: Expression of target protein containing azide groups 1. Design target protein mutation sites Based on the three-dimensional conformation of proteins in the PDB, non-natural mutation sites were designed. The mutation sites of Halo Tag protein and Galectin-3 protein are shown in Table 1.

[0034] Table 1

[0035] 2. Preparation of supercompetent cells from pEVOL-pAzF plasmid Take 50 μL of glycerol bacteria containing the pEVOL-pAzF plasmid and inoculate it into 5 mL of fresh, sterile LB medium. Incubate overnight at 37°C until the bacterial concentration reaches an absorbance (OD600) of 0.6–0.8 at 600 nm. Inoculate the seed culture at a ratio of 1% into a 500 mL Erlenmeyer flask containing 100 mL of fresh LB medium and incubate at 37°C for 2.5–4 h until the bacterial concentration reaches an absorbance (OD600) of 0.5–0.6 at 600 nm. Place the bacterial culture on ice for 30 min (all subsequent steps should be performed on ice), and collect the bacterial cells by centrifugation at 3000 rpm for 10 min at 4°C. Remove the supernatant and gently resuspend the cells in 4 mL of sterile Inoue buffer. Repeat the above solution replacement procedure twice, then resuspend the cells in 8 mL of Inoue buffer, slowly add 8 mL of DMSO and mix well. Incubate on ice for 10 min. Aliquot 100 μL of competent cells into pre-chilled centrifuge tubes and store at -80°C.

[0036] 3. Halo Tag F79pAzF / Galectin-3 R129pAzF Co-transformation with pEVOL-pAzF plasmid Thaw the supercompetent cells in an ice bath and take 2 μL of Halo Tag. F79pAzF / Galectin-3 R129pAzF Plasmids were added to thawed supercompetent cells, mixed and incubated on ice for 30 min, then heat-shocked at 42 ℃ for 90 s. The cells were then revived in antibiotic-free LB medium at 37 ℃ for 1 h. The bacterial culture was then evenly spread onto solid medium and incubated upside down at 37 ℃ overnight. Single colonies were picked as templates, and after expansion culture, nucleic acids were extracted and verified by gel electrophoresis. Cells with correct band sizes were then further amplified. Figure 2 As shown in Figure A, lane 1 is the Halo Tag. F79pAzF The plasmids appeared in a supercoiled state; lane 2 contained the pEVOL-pAzF plasmid, and lane 3 contained the Halo Tag plasmid. F79pAzF The co-transformation of the plasmid with the pEVOL-pAzF plasmid resulted in the appearance of HaloTag. F79pAzFThe plasmid and the pEVOL-pAzF plasmid showed bands, both exhibiting a supercoiled state. For example... Figure 2 As shown in Figure B, lane 1 is Galectin-3. R129pAzF The plasmids appeared in a supercoiled state; lane 2 contained pEVOL-pAzF plasmids, and lane 3 contained Galectin-3. R129pAzF The co-transformation of the plasmid with pEVOL-pAzF plasmid also resulted in the appearance of Galectin-3. R129pAzF The plasmid and the pEVOL-pAzF plasmid showed bands, but due to their similar molecular weights, the gel separation effect was not significant, and bright and thick plasmid bands appeared after co-transformation.

[0037] 4. Halo Tag F79pAzF / Galectin-3 R129pAzF Protein-induced amplification Take 50 μL of Halo Tag F79pAzF / Galectin-3 R129pAzF +pEVOL-pAzF E. coli BL21(DE3) culture was incubated in 50 mL of LB resistant medium (Amp: 100 μg / mL; CAM: 25 μg / mL) at 37 ℃ and 220 rpm for 6–7 h. Seed culture was inoculated at a ratio of 1% into 500 mL Erlenmeyer flasks containing 100 mL of fresh LB medium and cultured at 37 ℃ for 2.5–4 h until the cell concentration reached an absorbance (OD 600) of 0.6–0.8 at 600 nm. 4-Azide-L-phenylalanine was added to a final concentration of 200 mg / L in a clean bench, followed by induction of expression with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The culture was then incubated at 12 ℃ and 180 rpm for 22 h in a shaker.

[0038] 5. Halo Tag F79pAzF / Galectin-3 R129pAzF Collection of cell lysate of protein 500 mL of *E. coli* bacterial culture induced for 22 h was centrifuged at 8000 g for 10 min on a refrigerated centrifuge at 4 °C. The supernatant was discarded, and the cells were resuspended twice in phosphate buffer and placed in an ice box. Then, bacterial lysis buffer was used for 20 min of lysis, followed by 10 min of lysis using an ultrasonic cell disruptor at 150 W. After cell disruption, the cells were centrifuged at 8000 g for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm pore size filter to obtain the supernatant containing Halo Tag. F79pAzF / Galectin-3 R129pAzF Protein lysis buffer.

[0039] Example 2: Protein site-specific labeling 1. Halo Tag F79pAzF / Galectin-3 R129pAzF Labeling of protein in cell lysate The expression level of the target protein in the lysis buffer was calculated using Western blotting. The protein was diluted with phosphate buffer to a final concentration of 4-6 μM, with a dye molar ratio of (protein:CoraFluor-DBCO = 1:0 / 5 / 10 / 20 / 40). The HaloTag was then used. F79pAzF / Galectin-3 R129pAzF Protein lysis buffer was mixed with CoraFluor-DBCO at 4-37 °C and reacted for 0.5-24 h by rotation. Halo Tag in the lysis buffer was detected by SDS-PAGE protein electrophoresis. F79pAzF / Galectin-3 R129pAzF To assess protein labeling, the SDS-PAGE gel was placed on a UV-Vis light cutting stage after gel electrophoresis for gel imaging. Labeling efficiency was verified based on the color of the fluorescent band at the 35 kDa molecular weight. Figure 2 As shown in C, Halo Tag F79pAzF The protein has a molecular weight of approximately 35 kDa, and as the labeling ratio increases, the fluorescent band at the 35 kDa molecular weight gradually increases in color from left to right in the lane, demonstrating that CoraFluor-DBCO successfully binds to the Halo Tag in the lysis buffer. F79pAzF Protein binding was observed, but the presence of lysis buffer background and other fluorescent bands in the lanes indicated partial non-specific binding. Figure 2 As shown in D, due to Galectin-3 R129pAzF The protein fusion expressing the SUMO tag has a molecular weight of approximately 35 kDa, and a fluorescent band appears at this molecular weight. Furthermore, as the labeling ratio increases, the color of the fluorescent band at the 35 kDa molecular weight gradually increases from left to right in the lane, demonstrating that CoraFluor-DBCO successfully binds to Galectin-3 in the lysis buffer.R129pAzF Protein binding was observed, but due to the lysis buffer background and the presence of other fluorescent bands in the lanes, it was determined that some non-specific binding occurred.

[0040] Example 3: TR-FRET verification experiment on the binding of fluorescent tracer to target protein in lysis buffer 1. Spectroscopic characterization of fluorescent tracers Halo Tag ligand-Cy5 and G-FAM The spectroscopic properties of Halo Tag ligand-Cy5 and CoraFluor-DBCO were characterized as follows: Figure 3 Figure A shows the excitation and emission spectra of HaloTag ligand-Cy5 (red dashed line) and Halo Tag ligand-Cy5 (green solid line); and the emission spectrum of CoraFluor-DBCO (blue solid line). Because CoraFluor has a narrow and sharp fluorescence emission peak, there is less spectral crosstalk between the donor and acceptor. The fluorescence signal at 552 nm was used as the donor CoraFluor-DBCO signal, and the fluorescence signal at 650 nm was used as the acceptor Halo Tag ligand-Cy5 signal.

[0041] The spectroscopic properties of G-FAM and CoraFluor-DBCO are characterized as follows: Figure 4 As shown in Figure A, the excitation spectrum and emission spectrum of G-FAM (red dashed line) and G-FAM (green solid line) are displayed; the emission spectrum of CoraFluor-DBCO (blue solid line) is also shown. The fluorescence signal at 552 nm is used as the donor CoraFluor-DBCO signal, and the fluorescence signal at 530 nm is used as the acceptor G-FAM signal.

[0042] The structural formula of the dibenzocyclooctylene (DBCO) modified long fluorescence lifetime terbium complex (CoraFluor-DBCO) is shown below:

[0043] CoraFluor™ 1,amine reactive reagent (12 mg, 0.01 mmol) was dissolved in anhydrous N,N-dimethylformamide (1.0 mL), and N,N-diisopropylethylamine (2.0 mg, 0.015 mmol) and azadibenzocyclooctylenylamine (CAS: 1255942-06-3, 5.5 mg, 0.02 mmol) were added. The mixture was stirred at room temperature in the dark for 2 h. CoraFluor™ 1,amine reactive reagent was a commercially available reagent purchased from Tocris (Bio-Techne, Cat. No. 7920). After the reaction, the crude product was purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Separation was performed using gradient elution with 0.1 M triethylammonium bicarbonate buffer as mobile phase A and acetonitrile as mobile phase B. The fractions corresponding to the target peaks were collected, combined, and lyophilized to obtain CoraFluor-DBCO solid. The obtained product was identified by positive ion mode electrospray ionization mass spectrometry (ESI-MS). Identification results: HRMS (ESI) m / z for C 76 H 88 N 14 O 14 Tb [M] + calcd 1577.5696, found1577.5751.

[0044] The structural formulas of the fluorescent tracer Halo Tag ligand-Cy5 and its competing ligand Halo Tag ligand-Cl are shown below: ; Where A is Halo Tag ligand-Cy5; B is Halo Tag ligand-Cl.

[0045] The structural formulas of the fluorescent tracer G-FAM and the competing ligand TDP-139 are shown below: ; Where A stands for G-FAM and B stands for TDP-139.

[0046] 2. TR-FRET verification of Halo Tag ligand-Cy5 and Halo Tag in lysis buffer F79pAzF protein binding Halo Tag in the lysis buffer to a final concentration of 30 nM F79pAzFThe protein and serially diluted Halo Tagligand-Cy5 (0-1280 nM) were incubated together in detection buffer (20 mM Tris, 50 mM NaCl, 0.1% Tween, pH 8.0) at room temperature for 1 h before TR-FRET measurement. The FRET ratio and relative FRET ratio were calculated by statistically analyzing the total number of photons from the CoraFluor donor (I_CoraFluor) and Cy5 acceptor (I_Cy5) within a certain time window. The FRET ratio was calculated by dividing the fluorescence intensity of the Cy5-acceptor by the fluorescence intensity of the CoraFluor donor (Equation a). The relative FRET ratio represents the relative change of the FRET ratio compared to the FRET ratio in the presence of only the FRET donor (FRET ratio(0)) (Equation b). .

[0047] Depend on Figure 3 Results B show that with the increase of Halo Tag ligand-Cy5 concentration (5 nM-1280 nM), the FRET ratio and relative ratio also gradually increased, and gradually showed a binding plateau trend. This indicates that the Halo Tag concentration in the lysis buffer... F79pAzF The protein can bind to the ligand Halo Tag ligand-Cy5 and undergo FRET.

[0048] 3. TR-FRET verification of G-FAM and Galectin-3 in the lysis buffer R129pAzF protein binding Galectin-3 in the lysis buffer to a final concentration of 5 nM R129pAzF The protein and serially diluted G-FAM (0-1280 nM) were incubated together in detection buffer (20 mM Tris, 50 mM NaCl, 0.1% Tween, pH 8.0) at room temperature for 1 h before TR-FRET measurement. The FRET ratio and relative FRET ratio were calculated by statistically analyzing the total number of photons from the CoraFluor donor (I_CoraFluor) and FAM acceptor (I_FAM) within a certain time window. The FRET ratio was calculated by dividing the fluorescence intensity of the FAM-acceptor by the fluorescence intensity of the CoraFluor donor (Equation a). The relative FRET ratio represents the relative change in the FRET ratio compared to the FRET ratio in the presence of only the FRET donor (FRET ratio(0)) (Equation b). Figure 4Results B show that with the increase of G-FAM concentration (5 nM-1280 nM), the FRET ratio and relative ratio also gradually increase, and gradually show a binding plateau trend. This indicates that Galectin-3 in the lysis buffer... R129pAzF The protein can bind to the ligand G-FAM and undergo FRET.

[0049] Example 4: TR-FRET validation experiment of competitive binding between fluorescent tracer / small molecule to be screened and target protein in lysis buffer 1. TR-FRET verification of Halo Tag ligand-Cy5 / Halo Tag Ligand-Cl and HaloTag in lysis buffer F79pAzF Protein competitive binding Halo Tag in the lysis buffer to a final concentration of 10 nM F79pAzF The protein was incubated with serially diluted Halo Tag Ligand-Cl (2.5-320 nM) and 10 nM fluorescent tracer Halo Tag ligand-Cy5 in detection buffer (20 mM Tris, 50 mM NaCl, 0.1% Tween, pH 8.0) at room temperature for 1.5 h before TR-FRET measurement. The FRET ratio and relative FRET ratio were calculated by statistically analyzing the total number of photons from the CoraFluor donor (I_CoraFluor) and Cy5 acceptor (I_Cy5) within a certain time window. The FRET ratio was calculated by dividing the fluorescence intensity of the Cy5-acceptor by the fluorescence intensity of the CoraFluor donor (Equation a). The relative FRET ratio represents the relative change in the FRET ratio compared to the FRET ratio when only the FRET donor is present (FRET ratio(0)) (Equation b). Figure 3 The results show that with the increase of Halo Tag Ligand-Cl concentration (2.5 nM-320 nM), the FRET ratio and relative ratio gradually decrease. A plateau is observed when the Halo Tag Ligand-Cl concentration reaches 20 nM. This indicates that with increasing Halo Tag Ligand-Cl concentration, the concentration of Halo Tag in the lysis buffer decreases. F79pAzF The active site of the protein is gradually occupied by Halo Tag Ligand-Cl, inhibiting the interaction of the fluorescent tracer Halo Tag ligand-Cy5 with Halo Tag. F79pAzF Protein binding leads to a gradual decrease in FRET. Using a single-binding-site competitive binding curve fitting model, the inhibition constant Ki was calculated to be 4.64 nM, indicating that Halo Tag... F79pAzFThe protein exhibits a strong binding affinity to the small molecule Halo Tag ligand-Cl to be screened.

[0050] 2. TR-FRET verification of G-FAM / TPD-139 and Galectin-3 in the lysis buffer R129pAzF Protein competitive binding Galectin-3 protein in a final concentration of 5 nM lysis buffer was incubated with serially diluted TDP-139 (5-1280 nM) and 5 nM fluorescent tracer G-FAM in a detection buffer (20 mM Tris, 50 mM NaCl, 0.1% Tween, pH 8.0) at room temperature for 1.5 h before TR-FRET measurement. The FRET ratio and relative FRET ratio were calculated by statistically analyzing the total number of photons from the CoraFluor donor (I_CoraFluor) and FAM acceptor (I_FAM) within a certain time window. The FRET ratio was calculated by dividing the fluorescence intensity of the FAM-acceptor by the fluorescence intensity of the CoraFluor donor (Equation a). The relative FRET ratio represents the relative change in the FRET ratio compared to the FRET ratio when only the FRET donor is present (FRET ratio(0)) (Equation b). Figure 4 Results C show that as the TDP-139 concentration (5 nM-1280 nM) increases, the FRET ratio and relative ratio gradually decrease. A plateau is observed when the TDP-139 concentration reaches 640 nM. This indicates that as the TDP-139 concentration increases, the concentration of Galectin-3 in the lysis buffer decreases. R129pAzF The active site of the protein was gradually occupied by TDP-139, inhibiting the fluorescent tracers G-FAM and Galectin-3. R129pAzF Protein binding leads to a gradual decrease in FRET. Using a single-binding-site competitive binding curve fitting model, the inhibition constant Ki was calculated to be 7.24 nM, indicating that Galectin-3... R129pAzF The protein exhibits a strong binding affinity to the small molecule TDP-139 to be screened.

[0051] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for site-specific labeling of a target protein in cell lysate, characterized in that, The method includes the following steps: (1) The dibenzocyclooctylene DBCO group was coupled with the long fluorescence lifetime terbium complex CoraFluor via amide bond condensation to prepare the dibenzocyclooctylene DBCO-modified long fluorescence lifetime terbium complex CoraFluor-DBCO. (2) Introduce non-natural amino acids containing azide groups into the target protein; (3) The target protein containing non-natural amino acids with azide groups is expressed in large quantities by tool cells. The tool cells are lysed, and CoraFluor-DBCO is added to the cell lysate. The target protein undergoes a copper-free click chemical reaction with the azide groups in the target protein, thereby achieving site-directed labeling of the target protein. (4) Constructing a time-resolved fluorescence resonance energy transfer (TR-FRET) screening system: The target protein is the screening target. The target protein labeled with the CoraFluor-DBCO complex competes with the fluorescent tracer and the compound to be screened in the lysis buffer. The binding is detected by the donor-acceptor energy transfer. (5) The binding signal between the target protein and the substance to be screened in the lysate is detected by TR-FRET signal, and the inhibition curve is fitted to achieve the purpose of high-throughput screening and quantitative analysis of the affinity of the compound to be screened for the target protein.

2. A long-fluorescence-lifetime terbium complex, CoraFluor-DBCO, modified with dibenzocyclooctylene (DBCO), characterized in that... The CoraFluor-DBCO complex is formed by introducing a dibenzocyclooctylene DBCO group into a long fluorescence lifetime terbium complex via an amino condensation reaction, thereby enabling a copper-free click chemistry reaction with the azide group.

3. The method for site-specific labeling of target proteins in cell lysate as described in claim 1, characterized in that, In step (2), the codons corresponding to specific amino acids in the target protein coding sequence are replaced with TAG stop codons, and a bioorthogonal tRNA / aminoacyl-tRNA synthetase system is used in... E. coli Expression in the expression system introduces non-natural amino acids containing azide groups into the target protein; 4-Azide-L-phenylalanine was added during the expression of the target protein.

4. The method for site-specific labeling of target proteins in cell lysate as described in claim 1, characterized in that, In step (3), a mild lysis buffer with pH 7.0-7.4 is used, and a protease inhibitor is added to prevent protein degradation. The copper-free click chemical reaction is carried out in PBS phosphate buffer solution and labeled at 4-37°C for 0.5-24 hours.

5. The method for constructing a TR-FRET screening system as described in claim 1, characterized in that, In step (4), a known ligand tracer labeled with fluorescence is used to compete with the small molecule to be screened for binding to the target protein binding site. By monitoring the changes in TR-FRET signal in experiments where different concentrations of the small molecule to be screened compete with the fluorescent tracer for binding to the target protein, a TR-FRET detection system based on the competitive binding mode is constructed.

6. As described in claim 1, characterized in that, In step (5), the spectral data is analyzed by time-resolved fluorescence scanning. The fluorescence intensity of the donor and acceptor in the TR-FRET system is measured by statistically analyzing the total number of photons of the donor and acceptor within a certain time window. The TR-FRET ratio is calculated by the ratio of acceptor to donor fluorescence intensity. The dissociation constant Kd and inhibition constant Ki are calculated using a unit point-total binding fitting model to analyze the affinity of the small molecule to be screened for the target protein in the lysis buffer.

7. The application of the method for site-specific labeling of target proteins in cell lysates as described in claim 1 in screening substances that bind to target proteins.

8. The application of the method for site-specific labeling of target proteins in cell lysate as described in claim 1 in drug screening.

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