Fluorescent fixed-point labeling method for protein in cell lysis buffer and application of fluorescent fixed-point labeling method

Abstract

The invention discloses a fluorescent fixed-point labeling method for protein in a cell lysis solution and application, and belongs to the technical field of medical bioengineering. The method comprises the following steps: introducing non-natural amino acid containing an azide group into a specific site of a target protein by using a genetic code expansion technology, directly adding a dibenzocyclooctyne modified terbium complex with long fluorescence lifetime into a cell lysis solution, and carrying out fixed-point labeling on the target protein through copper-free click chemistry. The method disclosed by the invention is good in biocompatibility and wide in applicability, and can be used for constructing a protein target library marked by the CoFluor-DBCO. A time-resolved fluorescence resonance energy transfer screening system is further constructed, biological background fluorescence is filtered out by utilizing the long fluorescence lifetime of the terbium complex, and the signal-to-noise ratio is increased. By adopting the competitive TR-FRET, the homogeneous screening of the small molecule ligand of the targeted target protein in the lysis solution can be realized, the protein purification step is avoided, the operation is simple and convenient, and the screening efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical biological engineering technology, and in particular to a method for site-specific labeling of target proteins in a cell lysate and application thereof. BACKGROUND

[0002] Specific labeling and functional analysis of proteins are core technologies for molecular interaction research and drug screening. With the help of site-specific protein labeling technology, the interaction of proteins with other molecules or the conformational changes of proteins can be quantitatively analyzed, and the structure and biological function of proteins can be explored. At present, traditional labeling methods (such as NHS ester coupling) require high sample requirements, and the target protein needs to be purified. The process involves cell disruption, affinity separation, buffer replacement and other steps, which takes ≥48 hours, and the purification process easily leads to protein denaturation and inactivation (membrane proteins), or loses physiological activity due to the removal of natural interaction partners. Common chemical coupling methods, such as random chemical coupling (such as lysine epsilon-amino modification), lead to non-site-specific labeling, with an average of 3-8 random modification sites per protein molecule, which may mask the active sites of the protein or interfere with the conformation. Although fluorescent protein gene fusion expression can achieve fluorescent labeling, it has significant limitations, such as the dimerization tendency of some fluorescent proteins (such as YFP) may induce abnormal aggregation of target proteins, and the fusion tag of about 30 kDa may interfere with the spatial accessibility of small molecule binding pockets. Click chemistry is a class of chemical reactions widely used for modification of biological molecules, especially azide-alkyne cycloaddition, which can achieve specific labeling of proteins in a cell environment. Although the traditional copper-catalyzed click reaction (Copper(I)-Catalyzed Alkyne-Azide Cycloaddition, CuAAC) has high efficiency, Cu(I) and Cu(II) involved in the catalytic process can participate in redox reactions, producing reactive oxygen species, which may potentially damage the structure and biological activity of proteins, limiting its application in complex biological systems such as lysates and cells. Strain-promoted azide-alkyne cycloaddition (Strain-Promoted Azide-Alkyne Cycloaddition, SPAAC) is a copper-free click chemistry method suitable for biological samples, which can achieve efficient and highly specific labeling in complex biological environments with minimal disturbance to the structure of the target protein, making it suitable for functional studies that require maintenance of the native conformation and for proteins sensitive to metals (such as metalloenzymes and transcription factors).

[0003] With the continuous development of molecular biology technology, genetic code expansion technology (GCE) is more and more applied to protein specific site labeling. Through this technology, a non-natural amino acid (nnAA) tag modified with an azido group can be introduced at a specific site of a protein, and then specific site dye labeling of the target protein can be realized through a copper-free click chemistry reaction driven by dibenzocyclooctyne (DBCO).

[0004] Traditional protein-ligand interaction research methods include surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), fluorescence polarization (FP), etc. However, due to the high difficulty of protein sample immobilization, large consumption, low detection sensitivity and other defects, it is difficult to meet the needs of modern drug screening for high efficiency, high fidelity and complex biological sample compatibility.

[0005] Time-resolved fluorescence resonance energy transfer (TR-FRET) uses a long fluorescence lifetime dye CoraFluor as a fluorescent donor. By using its millisecond fluorescence lifetime characteristics, the time-gated detection technology effectively filters out the interference of short-lived background fluorescence in biological samples. At the same time, its homogeneous wash-free detection mode does not need to separate the unbound components, and directly completes the signal acquisition in the solution, simplifies the experimental operation process, avoids the complex dissociation or signal loss caused by the washing step, and greatly improves the signal-to-noise ratio and data reliability in complex biological environments (such as cell lysates). SUMMARY

[0006] The present application provides a method for site-specific labeling of target proteins in cell lysates, and provides specific applications of the method to overcome the shortcomings of the prior art.

[0007] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: A method for site-specific labeling of target proteins in cell lysates, the method comprising the following steps: (1) coupling dibenzocyclooctyne (DBCO) group with long fluorescence lifetime terbium complex (CoraFluor) by amide bond condensation to obtain dibenzocyclooctyne (DBCO) modified long fluorescence lifetime terbium complex CoraFluor-DBCO; (2) introducing non-natural amino acids containing azido groups into target proteins; (3) A large number of target proteins containing unnatural amino acids with azido groups are expressed by tool cells, the tool cells are lysed, CoraFluor-DBCO is added to the cell lysate, and a copper-free click chemistry reaction occurs with the azido group in the target protein, thereby achieving site-specific labeling of the target protein; (4) A time-resolved fluorescence resonance energy transfer (TR-FRET) screening system is constructed: the target protein is the screening target, and the target protein labeled by the CoraFluor-DBCO complex competes with the fluorescent tracer and the screened compound for binding in the lysate, and the energy transfer of the donor-acceptor is detected; (5) The binding signal of the target protein and the screened substance in the lysate is detected by TR-FRET signal, and the inhibition curve is fitted, so as to achieve the purpose of high-throughput screening and quantitative analysis of the affinity of the screened compound to the target protein.

[0008] The dibenzo cyclooctyne (DBCO) modified long fluorescence lifetime terbium complex (CoraFluor-DBCO) in the above method is a complex in which a dibenzo cyclooctyne (DBCO) group is introduced on the long fluorescence lifetime terbium complex through amino condensation reaction, so that a copper-free click chemistry reaction can occur with the azido group.

[0009] Further, in the step (2), the codon corresponding to the specific amino acid in the target protein coding sequence is replaced with a TAG stop codon, and a biological orthogonal tRNA / aminoacyl tRNA synthetase (aaRS) system is used to express in E. coli an expression system, and introduce unnatural amino acids containing azido groups into the target protein.

[0010] Taking Halo Tag protein and Galectin-3 protein as examples, in step (1), the codon corresponding to the 79th F amino acid of the Halo Tag protein is replaced with a TAG stop codon; the codon corresponding to the 129th R amino acid of the Galectin-3 protein is replaced with a TAG stop codon; and the plasmid of the site-mutated Halo Tag protein and the plasmid of the Galectin-3 protein are co-transfected into super-competent cells containing a pEVOL-pAzF plasmid. The present application can be expanded to a variety of proteins, and the method can be expanded for rapid construction of a protein library without a protein purification step.

[0011] Further, in the 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: maintain near-physiological conditions at pH 7.0-7.4; avoid buffers containing interfering substances such as Tris and trehalose (which may affect subsequent detection); and add 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 image: A is the fluorescence characterization spectrum of the fluorescent tracer Halo Tag ligand-Cy5; B is the fluorescence characterization spectrum of the fluorescent 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 and the small molecule to be screened, Halo Tag ligand-Cl, in Example 4, and the model target protein Halotag 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 the target protein mutation site: 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: Table 1 .

[0034] 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.

[0035] 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 A, lane 1 is a Halo tag. F79pAzF The plasmids appeared in a supercoiled state; lane 2 contained the pEVOL-pAzF plasmid, and lane 3 contained the Halo tag. F79pAzF The co-transformation of the plasmid with 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.

[0036] 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 solution was incubated in 50 mL LB resistant medium (Amp: 100 μg / mL; CAM: 25 μg / mL) at 37 ℃ and 220 rpm for 6-7 h. The seed culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of fresh LB at a ratio of 1%. The flask was incubated at 37 °C for 2.5–4 h until the cell concentration at 600 nm absorbance (OD 600) reached between 0.6 and 0.8. 4-Azide-L-phenylalanine was added to a final concentration of 200 mg / L in a clean bench. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression. The flask was then incubated at 12 °C and 180 rpm / min for 22 h in a shaker.

[0037] 5. Halo tag F79pAzF / Galectin-3 R129pAzF Collection of cell lysate of proteins: 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 Halo tag-containing supernatant. F79pAzF / Galectin-3 R129pAzF Protein lysis buffer.

[0038] 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 by Western blotting. The protein was diluted with phosphate buffer to a final concentration of 4-6 μM, and the dye molar ratio was (protein:CoraFluor-DBCO=1:0 / 5 / 10 / 20 / 40). Halotag was then used. F79pAzF / Galectin-3 R129pAzF Protein lysis buffer was mixed with CoraFluor-DBCO at 4-37 °C and reacted by rotation for 0.5-24 h. Halo tags in the lysis buffer were 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 3 As shown in C, 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.

[0039] 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 are characterized as follows: Figure 4 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.

[0040] The spectroscopic properties of G-FAM and CoraFluor-DBCO are characterized as follows: Figure 3 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.

[0041] 2. TR-FRET verification of Halo tag ligand-Cy5 and Halo tag in lysis buffer F79pAzF Protein binding: Halo tag in lysis buffer with 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 when only the FRET donor is present (FRET ratio (0)) (Equation b).

[0042] (Formula a); (Formula b).

[0043] Depend on Figure 4 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 in the lysis buffer... F79pAzF The protein can bind to the ligand Halo tag ligand-Cy5 and undergo FRET.

[0044] 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 3Results 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.

[0045] 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 Halo tag in lysis buffer F79pAzF Protein competitive binding Halo tag in lysis buffer with 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 4 The results show that with the increase of Halotag Ligand-Cl concentration (2.5 nM-320 nM), the FRET ratio and relative ratio gradually decrease. An inhibition plateau occurs when the Halotag Ligand-Cl concentration reaches 20 nM. This indicates that with the increase of Halotag Ligand-Cl concentration, the concentration of Halotag 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 half-inhibition rate constant Ki was calculated to be 4.64 nM, indicating that the Halo tag... F79pAzFThe protein exhibits a strong binding affinity to the small molecule Halo Tag ligand-Cl to be screened.

[0046] 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). ​ 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 As the protein binds, FRET gradually decreases. Using a single-binding-site competitive binding curve fitting model, its half-inhibition rate 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.

[0047] 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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