Methods for detection and / or screening of target protein expressing cell lines based on fluorescence indication

Abstract

The present application relates to a method for detecting and / or screening a target protein expression cell line based on fluorescence indication, aiming to realize rapid detection of specific protein conformation in a chassis cell, and at the same time, to efficiently screen chassis cells with different functional states, activities and target protein expression levels. The essence of the present technology is different from the conventional protein fusion fluorescent tag used to indicate the protein. The present method first reflects the conformational change of the protein through a cross-linking agent, and reflects the functional state corresponding to the conformation of the protein based on the change of the fluorescence color of the cross-linking agent skeleton under different protein conformations. This technology provides a convenient, efficient and non-invasive method for the field of biological research, which can be widely used in the fields of cell activity research, drug screening and protein function research.

Description

Technical Field

[0001] This invention relates to a method for detecting and / or screening cell lines expressing target proteins based on fluorescence indicators. Background Technology

[0002] Protein expression and structure determination are core issues in biological science research. As major functional macromolecules in life processes, proteins play various important roles within cells, including signal transduction, metabolic regulation, and structural support. Their expression levels and structural states directly affect the physiological processes and functional performance of organisms (Nat. Rev. Mol. CellBiol., 2020, 21, 327−340). Therefore, accurately characterizing protein expression levels and structural states is crucial for understanding cellular biological processes, disease mechanisms, and new drug development.

[0003] Currently, in protein expression detection, except for a few proteins with self-luminescent properties, most proteins typically require the use of fluorescently fused expression tags. However, for endogenous proteins that do not possess self-luminescent properties, it is difficult to accurately indicate their expression status and functional state in situ. Although existing methods such as mass spectrometry can detect protein expression and its conformational state (Proc. Natl. Acad. Sci. USA 2021, 118,e2023360118; J. Biol. Chem. 2017, 292, 1187−1196), these methods usually require cell lysis, thereby disrupting the protein's expression and functional state in situ. Such methods are difficult to directly detect the expression and functional state of proteins without fluorescent tags within cells, thus limiting their application in the screening and culture of target proteins.

[0004] Therefore, a new technology is needed to overcome the current limitations of protein expression detection. This technology should be able to directly indicate whether a target protein is expressed and characterize its functional state in situ, without the need for labeling or disrupting the cellular environment. Such an innovative technology will provide a more efficient and accurate means for cell screening and protein function research, and will drive the development of the field of cell biology, providing reliable technical support for protein research, biological research, disease treatment, and drug development. Summary of the Invention

[0005] This invention provides a method for detecting and / or screening target protein expression cell lines based on fluorescence indicators. This technology enables rapid detection of specific protein expression levels and / or structural states within cells and efficiently screens chassis cells in different functional states. By targeting a cross-linking agent with targeting and fluorescence properties to the target protein in chassis cells for a chemical cross-linking reaction, the differences in the target protein's own conformation and / or chemical microenvironment cause changes in the cross-linking agent's skeletal structure, resulting in the cross-linking agent emitting fluorescence of different colors. This allows for accurate and real-time characterization of protein structures within chassis cells.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] 1) Design and construction of target protein chassis cells: Transfect the target protein plasmid into one of the chassis cells such as HEK 293T, HeLa, E. coli, yeast, or Bacillus subtilis with a lentivirus, culture the cells or bacteria, and continuously screen the lentivirus-transfected chassis cells for 1 day to 1 month using one or more of puromycin, G418, kanamycin, and tetracycline to obtain chassis cells that can stably express the target protein, thereby achieving overexpression of the target protein in chassis cells.

[0008] 2) Design of targeted fluorescent indicator crosslinking agents:

[0009] (a) The reactive groups of the crosslinking agent are selected from one or more of the following: benzenesulfonyl fluoride group, phenoxysulfonyl fluoride group, N-hydroxysuccinimide group, o-phthalaldehyde group, and bisacrylidine group, so as to achieve a reaction rate of 1 s-1 h and a reaction efficiency of 80%-100%;

[0010] (b) The length of the spacer arm of the crosslinking agent is 1-200 Å;

[0011] (c) The water solubility of the crosslinking agent is achieved by introducing one or more of the following groups into the crosslinking agent backbone: hydroxyl group, amide group, sulfonic acid group, amino group, carboxyl group, and polyethylene glycol group;

[0012] (d) By introducing one or more of amino groups, positively charged groups, amide groups, and polyethylene glycol groups into the crosslinking agent backbone, the membrane permeability of the crosslinking agent is achieved.

[0013] (e) The cross-linking agent backbone achieves targeting by introducing one or more of the following into the cross-linking agent backbone: an inhibitor of the target protein, an affinity group of the target protein, an antibody of the target protein, and a charge opposite to that of the target protein.

[0014] (f) The fluorescent properties of the crosslinking agent backbone are achieved by introducing one or more of the fluorescent molecules rhodamine, fluorescein, and Alexa Fluor series molecules and their derivatives into the crosslinking agent backbone;

[0015] 3) Harvesting of chassis cells: Cells on the culture dish are obtained by scraping or digesting with trypsin, centrifuging and washing to obtain chassis cell samples. The chassis cells are washed with one or more of the following buffer solutions with pH 7.0-10.0: ammonium bicarbonate buffer solution, phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution to remove impurities such as culture medium during chassis cell culture. The washed chassis cell samples are then collected by centrifugation.

[0016] 4) Preparation of crosslinking solution: Prepare a 0.1mM-1M solution of the crosslinking agent described in 2) using one or more organic solvents selected from organic alcohols, aprotic polar solvents, organic bases, and organic acids. Add the solution to one or more cell buffer solutions selected from ammonium bicarbonate buffer solution, phosphate buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, or tris(hydroxymethyl)aminomethane buffer solution with a pH of 7.1-10, so that the final concentration of the crosslinking agent is 0.1mM-100mM.

[0017] 5) Crosslinking reaction: Add the crosslinking solution obtained in 4) to the chassis cell sample collected in 3). The volume ratio of crosslinking agent solution to chassis cells is 100:1-1:1. Use one or more of the following: pipette, dropper, or mixer to evenly disperse the chassis cells in the crosslinking solution. Carry out the crosslinking reaction in one or more of the following: constant temperature mixer, shaker, shaker, or test tube rack. Set the temperature to 20-50℃ and the crosslinking reaction time to 1 s-1 h.

[0018] 6) Harvest the chassis cells that have completed the cross-linking reaction: Collect the cross-linked chassis cell samples by centrifugation, and wash the chassis cells with one or more of the following buffer solutions with pH 7.0-10.0: ammonium bicarbonate buffer solution, phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution to wash away the unreacted cross-linking agent.

[0019] 7) Protein conformation detection in chassis cells: One or more of the following techniques are used to monitor the fluorescence signals emitted by the cross-linking agent backbone in different conformational states, in order to obtain information on the structural state of the target protein in chassis cells: fluorescence resonance energy transfer, fluorescence activated cell sorting, fluorescence microscopy sorting, and fluorescence live cell imaging sorting.

[0020] 8) Screening and separation of chassis cells based on conformational analysis results: By detecting changes in the fluorescence signal emitted by the cross-linking agent backbone using one or more of the following techniques—fluorescence resonance energy transfer (FRET), fluorescence-activated cell sorting (FIRS), fluorescence microscopy sorting, and fluorescence live-cell imaging sorting—the expression levels and structural states of proteins in different chassis cells can be determined. Based on this information, chassis cells in specific conformational states can be selected to achieve efficient sorting and screening of chassis cells in different functional states.

[0021] The method described in this invention has the following advantages:

[0022] 1) Rapid detection of specific protein expression levels: This technology can rapidly detect the expression levels of specific proteins in chassis cells, greatly shortening the research process and time;

[0023] 2) Efficient screening of chassis cells in different functional states: By analyzing the different color fluorescence signals emitted by the cross-linking agent, chassis cells in different functional states can be screened efficiently, thereby distinguishing the state of intracellular protein structure.

[0024] 3) Convenient and non-invasive: This technology uses a non-invasive method that does not cause additional impact on the chassis cells, maintains the original state of the cells, and ensures the reliability and reproducibility of the research results;

[0025] 4) Providing accurate intracellular information: The application of quantitative analysis of fluorescence signals and high-resolution fluorescence microscopy enables this technology to provide accurate structural information of target proteins within cells. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of a method for detecting and / or screening cell lines expressing target proteins based on fluorescence indicators.

[0027] Figure 2 This is a confocal image of the target protein in the chassis cell cross-linked by the cross-linking agent; only the cross-linked target protein exhibits fluorescence. Detailed Implementation

[0028] The present invention provides the following embodiments to illustrate the present invention, but does not limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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.

[0029] Synthesis of targeted fluorescent crosslinking agents:

[0030]

[0031] Synthesis of compound 3:

[0032] Compound 1 (2.93 g, 8 mmol, Rhodamine 110) was dissolved in 80 mL of DMSO, and compound 2 (4.2 g, 8 mmol, reference: Hang Gao, et al., Anal. Chem. 2022, 94, 21, 7551–7558) was dissolved in 20 mL of DMSO and added dropwise to the solution. The mixture was stirred at room temperature for 5 min and purified using semi-preparative reversed-phase high-performance liquid chromatography (SPC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 20%-50%, 30 min, flow rate 50 mL / min, monitored at 200 nm UV wavelength). The target product was freeze-dried at 30℃ for 24 h to obtain 1.6 g of a pale yellow viscous oily substance with a yield of 38.1% and LCMS[M+H]+=740.23.

[0033] Synthesis of compound 5:

[0034] Compound 3 (1.6 g, 2.16 mmol) was dissolved in 50 mL of DMSO, and compound 4 (865 mg, 4 mmol, Bis-PEG2-NHS ester) was dissolved in 10 mL of DMSO and added dropwise to the solution. The mixture was stirred at room temperature for 5 min and purified using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 30%-60%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). After lyophilization at 30 °C for 24 h, 720 mg of the target product was obtained as a pale yellow viscous oil, yield: 45%, LCMS [M+H]+ = 1025.32.

[0035] Synthesis of compound 7:

[0036] Compound 5 (720 mg, 0.7 mmol) was dissolved in 10 mL of DMSO, and compound 6 (143 mg, 0.7 mmol) was dissolved in 1.5 mL of DMSO. The solutions were then added dropwise. The mixture was stirred at room temperature for 5 min and purified using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 30%-70%, 30 min, flow rate 50 mL / min, monitored at 200 nm UV wavelength). The product was lyophilized at 30 °C for 24 h to obtain 216 mg of the target product as a pale yellow viscous oil, yield: 30%, LCMS [M+H]+ = 1113.33.

[0037] Synthesis of compound 9:

[0038] Compound 7 (216 mg, 0.19 mmol) was dissolved in 5 mL of DMSO, and compound 6 (39 mg, 0.19 mmol) was dissolved in 1 mL of DMSO and added dropwise to the solution. The mixture was stirred at room temperature for 5 min and purified using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 30%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). After lyophilization at 30 °C for 24 h, 110.2 mg of the target product was obtained as a pale yellow viscous oil, yield: 51%, LCMS [M+H]+ = 1201.35. The compound structure was confirmed to be consistent with the expected structure by nuclear magnetic resonance, mass spectrometry, and liquid chromatography.

[0039] Synthesis of compound 10:

[0040] Compound 7 (216 mg, 0.19 mmol) was dissolved in 5 mL of DMSO, and compound 8 (42 mg, 0.19 mmol) was dissolved in 1 mL of DMSO and added dropwise to the solution. The mixture was stirred at room temperature for 5 min and purified using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 30%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). After lyophilization at 30 °C for 24 h, 105.8 mg of the target product was obtained as a pale yellow viscous oil, yield: 49%, LCMS [M+H]+ = 1217.32. The compound structure was confirmed to be consistent with the expected structure by NMR, mass spectrometry, and liquid chromatography.

[0041] Synthesis of crosslinking agent 1:

[0042] Compound 9 (110.2 mg, 0.09 mmol) was dissolved in 3 mL PBS, and ubquitin antibody (0.2 mmol) was added to the solution. The reaction was carried out at room temperature for 90 min, followed by purification using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 50%-70%, 30 min, flow rate 50 mL / min, monitored at 200 nm UV wavelength). The target product was obtained by lyophilization at 30 °C for 24 h.

[0043] Synthesis of crosslinking agent 2:

[0044] Compound 10 (105.8 mg, 0.09 mmol) was dissolved in 3 mL of PBS, and SIRT2 antibody (0.2 mmol) was added to the solution. The reaction was carried out at room temperature for 90 min, followed by purification using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 50%-70%, 30 min, flow rate 50 mL / min, monitored at 200 nm UV wavelength). The target product was obtained by lyophilization at 30 °C for 24 h.

[0045] Synthesis of crosslinking agent 3:

[0046] Compound 10 (105.8 mg, 0.09 mmol) was dissolved in 3 mL PBS, and FGL1 antibody (0.2 mmol) was added to the solution. The reaction was carried out at room temperature for 90 min, followed by purification using semi-preparative reversed-phase high-performance liquid chromatography (SHPLC) (Hanbang Technology, model: NS4000, A: 0.1% TFA / H2O, B: ACN, linear gradient method set as: B: 50%-70%, 30 min, flow rate 50 mL / min, monitored at 200 nm UV wavelength). The target product was obtained by lyophilization at 30 °C for 24 h.

[0047] The ubquitin sequence is:

[0048] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

[0049] The SIRT2 sequence is:

[0050] MEGVPADLRAKQSVVCTGSFYEEYDTLETMRNLVVLDAGSEAVWRALELNLVDCKYKSHPHPEFNLVVPRGRSLGKGLDFIYKMKGDSPFRGKFAVYAGVADTQPLEKGIDPNSKGQKPFVTDYRRKHTLQDNIVFNLPNVSFYETAK DLTKWVDIQHCFKGHAEFSFSYRRGEVNFEYGDYHVVGTAEVSLANAKNDTWTGERKVLTTHFNVIDFLENKLQTDKRDEAPYQGVPFIFELIDGSSVHRNIYIWDTAGGSSVAEAQGESSSDPSMKGPDASDEDSQQESGKQETSLD

[0051] The FGL1 sequence is:

[0052] MVPPLLLLSGLALWLQLCSLFSLHSTLPDSQVCQDLQTRKKRLHVCADARFYHLPLSAQQAEGKTIYFPDLHATPKDITQIGLQNGIRVWVHKGAHVVPNRVCEKFIHHSTNYLQKVAEYFQSGKGQSESTEHLLKASSLNDPLPKCKRLVVPDDIFGVEGFHRGEYLFSVPGPNELEFPRNPMSLGAADWPKFLSHEHH

[0053] Example 1

[0054] 1) Construction of chassis cells expressing the target protein: The ubquitin gene was transfected into HEK 293T cells via the NcmDH5-α vector (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the cells were cultured. The chassis cells transfected with lentivirus were continuously screened using puromycin (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the chassis cells stably expressing the target protein were cultured to a density of 80%-90%.

[0055] 2) Design of a targeted fluorescent indicator crosslinking agent 1: The reactive group of the crosslinking agent was selected as benzenesulfonyl fluoride group, the reaction time was 1 min, and the reaction efficiency was 50%; the length of the spacer arm of the crosslinking agent was 150 Å; water solubility of the crosslinking agent was achieved by introducing polyethylene glycol groups into the crosslinking agent backbone, and membrane permeability of the crosslinking agent was achieved by introducing amide groups; targeting of the crosslinking agent backbone was achieved by introducing an antibody of the target protein into the crosslinking agent backbone, and fluorescence properties of the crosslinking agent backbone were achieved by introducing the fluorescent molecule rhodamine; the preparation process and structure of crosslinking agent 1 are described in the section "Synthesis of a Targetable Fluorescent Crosslinking Agent";

[0056] 3) Harvesting chassis cells: Gently place the scraper tip on the cell culture dish and scrape along the surface to collect chassis cell samples. Centrifuge to collect chassis cell samples. Wash chassis cells with 10mM phosphate buffer solution at pH 7.4 to remove impurities such as culture medium during chassis cell culture. Collect washed chassis cell samples by centrifugation.

[0057] 4) Preparation of crosslinking solution: Prepare a 1M solution of the crosslinking agent described in 2) using the aprotic polar solvent dimethyl sulfoxide, and add it to a 10mM phosphate buffer solution with a pH of 7.4 to make the final concentration of the crosslinking agent 100mM.

[0058] 5) Crosslinking reaction: Add the crosslinking solution obtained in 4) to the chassis cell sample collected in 3). The volume ratio of crosslinking agent solution to chassis cells is 10:1. Use a pipette to blow and disperse the chassis cells evenly in the crosslinking solution. Carry out the crosslinking reaction in a constant temperature mixer at 37℃ for 1 min.

[0059] 6) Harvest the chassis cells that have completed the cross-linking reaction: Collect the cross-linked chassis cell samples by centrifugation, and wash the chassis cells with a 10mM phosphate buffer solution at pH 7.4 to remove unreacted cross-linking agents.

[0060] 7) Protein conformation detection in chassis cells: Fluorescence live-cell imaging sorting technology was used to monitor the fluorescence signals emitted by the cross-linking agent backbone in different conformational states (Reference: Daniel Schraivogel et al., High-speed fluorescence image–enabled cell sorting. Science 2022, 375, 315-320.), thereby obtaining information on the structural state of the target protein in chassis cells. Red fluorescence signals indicate that the target protein is in a compact structural state (the protein structure is similar to a sphere), while green fluorescence signals indicate that the target protein is in an extended structural state (the protein structure is similar to a line). No fluorescence signal indicates that the cross-linking agent has not reacted with the target protein. (See attached diagram) Figure 2 As shown.

[0061] 8) Screening and separation of chassis cells based on conformational analysis results: Fluorescence signal emitted by the cross-linking agent backbone is detected using fluorescence live-cell imaging sorting technology to determine the protein expression level and structural state in different chassis cells. Stronger fluorescence signals indicate higher target protein expression levels. The experimental procedure is detailed in the appendix. Figure 1 Using fluorescence live-cell imaging sorting technology, chassis cells emitting red fluorescence signals were sorted into a collection pool, while those emitting green fluorescence signals were discarded, thus separating chassis cells with a compact structure of the target protein. Next, using fluorescence live-cell imaging sorting technology, chassis cells emitting strong red fluorescence signals (reaching 10-1) were sorted into a collection pool. 2 (Above), cells with weak red fluorescence signals are discarded, thereby isolating chassis cells with high expression of the target protein and a compact structural state. The number of cells obtained in this step is 10% of the initial total number of cells.

[0062] 9) Application: Collect and select chassis cells emitting red fluorescent signals. The target protein in these chassis cells is in a compact structure. By adding an interacting protein to the target protein, when the target protein interacts with the interacting protein, its conformation becomes more compact (from a loose spherical structure to a solid spherical structure), thereby causing the cross-linking agent backbone to emit a stronger red fluorescent signal (fluorescence signal reaching 10). 3 (The above) Further analysis using fluorescence resonance energy transfer technology can screen out target proteins that interact with each other for protein function studies.

[0063] Example 2

[0064] 1) Construction of chassis cells expressing the target protein: The SIRT2 gene was transfected into HEK 293T cells via the NcmDH5-α vector (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the cells were cultured. The chassis cells transfected with lentivirus were continuously screened using puromycin (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the chassis cells stably expressing the target protein were cultured to a density of 80%-90%.

[0065] 2) Design of a targeted fluorescent indicator crosslinking agent 2: The reactive groups of the crosslinking agent were selected as benzenesulfonyl fluoride group and benzenesulfonyl chloride group, the reaction time was 5 min, and the reaction efficiency was 40%; the length of the spacer arm of the crosslinking agent was 150 Å; the water solubility of the crosslinking agent was achieved by introducing polyethylene glycol group into the crosslinking agent backbone, and the membrane permeability of the crosslinking agent was achieved by introducing amide group; the targeting of the crosslinking agent backbone was achieved by introducing antibody of the target protein into the crosslinking agent backbone, and the fluorescence properties of the crosslinking agent backbone were achieved by introducing the fluorescent molecule rhodamine; the preparation process and structure of crosslinking agent 2 are described in the section "Synthesis of a Targetable Fluorescent Crosslinking Agent";

[0066] 3) Harvesting chassis cells: Gently place the scraper tip on the cell culture dish and scrape along the surface to collect chassis cell samples. Centrifuge to collect chassis cell samples. Wash chassis cells with 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution at pH 7.0 to remove impurities such as culture medium during chassis cell culture. Collect washed chassis cell samples by centrifugation.

[0067] 4) Preparation of crosslinking solution: Prepare a 300mM solution of the crosslinking agent described in 2) with deionized water, and add it to a 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with a pH of 7.0, so that the final concentration of the crosslinking agent is 30mM.

[0068] 5) Crosslinking reaction: Add the crosslinking solution obtained in 4) to the chassis cell sample collected in 3). The volume ratio of crosslinking agent solution to chassis cells is 8:1. Use a pipette to blow and disperse the chassis cells evenly in the crosslinking solution. Carry out the crosslinking reaction in a constant temperature mixer at 25℃ for 5 min.

[0069] 6) Harvest the cross-linked chassis cells: Collect the cross-linked chassis cell samples by centrifugation, and wash the chassis cells with a 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution at pH 7.0 to remove unreacted cross-linking agents.

[0070] 7) Protein conformation detection in chassis cells: Fluorescence resonance energy transfer (FRET) was used to monitor the fluorescence signals emitted by the cross-linking agent backbone in different structural states (Reference: Erika Cecon et al., SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell Chem. Biol. 2022, 29, 74-83), thereby obtaining information on the structural state of the target protein in chassis cells. Red fluorescence signals indicate that the target protein is in a compact structural state (the protein structure is similar to a sphere), while green fluorescence signals indicate that the target protein is in an extended structural state (the protein structure is similar to a line). No fluorescence signal indicates that the cross-linking agent has not reacted with the target protein.

[0071] 8) Screening and separation of chassis cells based on conformational analysis results: Fluorescence signal emission from the cross-linking agent backbone is detected using fluorescence resonance energy transfer (FRET) technology to determine the protein expression level and structural state in different chassis cells. Stronger fluorescence signals indicate higher target protein expression levels. Using FRET, chassis cells emitting red fluorescence signals are sorted into a collection pool, while those emitting green fluorescence signals are discarded, thus separating chassis cells with a compact structure containing the target protein. Next, using FRET, chassis cells emitting strong red fluorescence signals (reaching 10⁻⁶) are sorted into a collection pool. 2 (Above), cells with weak red fluorescence signals are discarded, thereby isolating chassis cells with high expression of the target protein and a compact structural state. The number of cells obtained in this step is 13% of the initial total number of cells.

[0072] 9) Application: Collect and select chassis cells emitting red fluorescent signals. The target protein in these chassis cells is in a compact structure. By adding an interacting protein to the target protein, when the target protein interacts with the interacting protein, its conformation becomes more compact (from a loose spherical structure to a solid spherical structure), thereby causing the cross-linking agent backbone to emit a stronger red fluorescent signal (fluorescence signal reaching 10). 3 (The above) Further analysis using fluorescence resonance energy transfer technology can screen out target proteins that interact with each other for protein function studies.

[0073] Example 3

[0074] 1) Construction of chassis cells expressing the target protein: The FGL1 gene was transfected into HEK 293T cells via the NcmDH5-α vector (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the cells were cultured. The chassis cells transfected with lentivirus were continuously screened using puromycin (Reference: Mingen Lin, et al., J Clin Invest. 2023,133,e164528), and the chassis cells stably expressing the target protein were cultured to a density of 80%-90%.

[0075] 2) Design of a targeted fluorescent indicator crosslinking agent 3: The reactive groups of the crosslinking agent were all selected as benzenesulfonyl fluoride groups and benzenesulfonyl chloride groups. The reaction time was 5 min and the reaction efficiency was 55%. The length of the spacer arm of the crosslinking agent was 150 Å. Water solubility of the crosslinking agent was achieved by introducing polyethylene glycol groups into the crosslinking agent backbone, and membrane permeability was achieved by introducing amide groups into the crosslinking agent backbone. Targeting of the crosslinking agent backbone was achieved by introducing an antibody of the target protein into the crosslinking agent backbone, and fluorescence properties of the crosslinking agent backbone were achieved by introducing the fluorescent molecule rhodamine. The preparation process and structure of crosslinking agent 3 are described in the section "Synthesis of Targetable Fluorescent Crosslinking Agents".

[0076] 3) Harvesting chassis cells: Gently place the scraper tip on the cell culture dish and scrape along the surface to collect chassis cell samples. Centrifuge to collect chassis cell samples. Wash chassis cells with 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution at pH 7.0 to remove impurities such as culture medium during chassis cell culture. Collect washed chassis cell samples by centrifugation.

[0077] 4) Preparation of crosslinking solution: Prepare a 500mM solution of the crosslinking agent described in 2) with deionized water, and add it to a 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with a pH of 7.0, so that the final concentration of the crosslinking agent is 50mM.

[0078] 5) Crosslinking reaction: Add the crosslinking solution obtained in 4) to the chassis cell sample collected in 3). The volume ratio of crosslinking agent solution to chassis cells is 5:1. Use a pipette to blow and disperse the chassis cells evenly in the crosslinking solution. Carry out the crosslinking reaction in a constant temperature mixer at 37℃ for 5 min.

[0079] 6) Harvest the cross-linked chassis cells: Collect the cross-linked chassis cell samples by centrifugation, and wash the chassis cells with a 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution at pH 7.0 to remove unreacted cross-linking agents.

[0080] 7) Protein conformation detection in chassis cells: Fluorescence resonance energy transfer (FRET) was used to monitor the fluorescence signals emitted by the cross-linking agent backbone in different structural states (Reference: Erika Cecon et al., SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell Chem. Biol. 2022, 29, 74-83), thereby obtaining information on the structural state of the target protein in chassis cells. Red fluorescence signals indicate that the target protein is in a compact structural state (the protein structure is similar to a sphere), while green fluorescence signals indicate that the target protein is in an extended structural state (the protein structure is similar to a line). No fluorescence signal indicates that the cross-linking agent has not reacted with the target protein.

[0081] 8) Screening and separation of chassis cells based on conformational analysis results: Fluorescence signal emission from the cross-linking agent backbone is detected using fluorescence resonance energy transfer (FRET) technology to determine the protein expression level and structural state in different chassis cells. Stronger fluorescence signals indicate higher target protein expression levels. Using FRET, chassis cells emitting red fluorescence signals are sorted into a collection pool, while those emitting green fluorescence signals are discarded, thus separating chassis cells with a compact structure containing the target protein. Next, using FRET, chassis cells emitting strong red fluorescence signals (reaching 10⁻⁶) are sorted into a collection pool. 2 (Above), cells with weak red fluorescence signals are discarded, thereby isolating chassis cells with high expression of the target protein and a compact structural state. The number of cells obtained in this step is 14% of the initial total number of cells.

[0082] 9) Application: Collect and select chassis cells emitting strong red fluorescence signals. The target protein in these chassis cells is in an extended structural state, which is associated with T cell dysfunction. By adding a therapeutically effective targeted drug, the conformation of the target protein can be made more compact (from a loose spherical structure to a solid spherical structure), thereby causing the cross-linking agent backbone to emit a stronger red fluorescence signal (fluorescence signal reaching 10). 3 (The above) Further analysis using fluorescence resonance energy transfer technology can screen out drugs with therapeutic effects, thereby achieving the purpose of drug screening analysis.

Claims

1. A method for detecting and / or screening cell lines expressing target proteins based on fluorescence indicators, characterized in that: By targeting and fluorescent cross-linking agents to target proteins in chassis cells for chemical cross-linking reactions, and utilizing the differences in the conformation and / or chemical microenvironment of the target proteins themselves, changes in the skeletal structure of the cross-linking agent are caused, resulting in the cross-linking agent emitting fluorescence of different colors. This is used for rapid detection and / or efficient screening of the expression level and / or structural state of target proteins in different chassis cells. The cross-linking agent has reactive groups and is water-soluble and membrane-permeable. The reactive groups of the crosslinking agent are one or more of the following: benzenesulfonyl fluoride group, phenoxysulfonyl fluoride group, N-hydroxysuccinimide group, o-phenylenedialdehyde group, and bisacrylidine group; and the length of the spacer arm of the crosslinking agent is 150 Å. Water solubility of the crosslinking agent is achieved by introducing one or more of the following groups into the crosslinking agent backbone: hydroxyl groups, amide groups, sulfonic acid groups, amino groups, carboxyl groups, and polyethylene glycol groups. The membrane permeability of the crosslinking agent is achieved by introducing one or more of amino groups, positively charged groups, amide groups, and polyethylene glycol groups into the crosslinking agent backbone. The targeting of the cross-linking agent is achieved by adding one or more of the following to the spacer arm backbone: an inhibitor of the target protein, an affinity group of the target protein, an antibody of the target protein, and the opposite charge of the target protein. Fluorescent properties of the crosslinking agent backbone can be achieved by introducing one or more of the fluorescent molecules rhodamine, fluorescein, and Alexa Fluor series molecules and their derivatives into the crosslinking agent backbone. After the cross-linking agent cross-links the target protein in different chassis cells, based on the specific influence of the target protein's own conformation and chemical microenvironment on the fluorescence of the cross-linking agent backbone, the cross-linking agent exhibits the corresponding conformational state and emits fluorescence signals of specific colors and intensities, with colors being one or more of red, yellow, or blue. The detection of target protein conformation in chassis cells employs one or more of the following methods: fluorescence resonance energy transfer technology, fluorescence-activated cell sorting technology, fluorescence microscopy sorting, and fluorescence live cell imaging sorting. It is used to monitor the fluorescence signals emitted by the cross-linking agent backbone in different conformational states, thereby obtaining information on the structural state of the target protein in the chassis cell.

2. The method according to claim 1, characterized in that: The chassis cells are one or more of HEK 293T, HeLa, Escherichia coli, yeast, or Bacillus subtilis; Alternatively, gene editing and / or modification technologies can be used to modify and alter the genome of the chassis cells to add the desired target gene, thereby overexpressing the target protein and obtaining a large amount of the target protein.

3. The method according to claim 1, characterized in that: The cross-linking agent reacts with intracellular proteins in 1 s to 1 h with a reaction efficiency of 20% to 100%. It can target the target protein and also has fluorescent properties.

4. The method according to claim 1 or 2, characterized in that: Cells are obtained from the culture dish by scraping or digesting with trypsin, followed by centrifugation to collect and wash the cell sample from the chassis. The solution used to wash the chassis cells is one or more of the following: ammonium bicarbonate buffer solution, phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with a pH of 7.0-10.

0.

5. The method according to claim 1, 2 or 3, characterized in that: The chemical cross-linking reaction process involves preparing the cross-linking agent into a solution with a concentration of 0.1 mM to 1 M using an organic solvent, and then mixing it with a buffer solution containing target protein cells to achieve a final concentration of 0.1 to 100 mM for the cross-linking agent. The organic solvent is one or more of organic alcohols, aprotic polar solvents, organic bases, and organic acids. The buffer solution is one or more of the following: ammonium bicarbonate buffer solution, phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, with a pH of 7.0-10.

0.

6. The method according to claim 1, characterized in that: A cell buffer solution containing a cross-linking agent was added to chassis cells overexpressing the target protein for co-incubation. The volume ratio of the cross-linking agent solution to the chassis cells was 100:1 to 1:

1. The temperature was set at 20-50℃, and the cross-linking reaction time was 1s to 1 h to complete the cross-linking reaction.

7. The method according to claim 1, characterized in that: The screening of chassis cells utilizes one or more of the following techniques: fluorescence resonance energy transfer (FRET), fluorescence-activated cell sorting (FIRS), fluorescence microscopy sorting, and fluorescence live cell imaging sorting. These techniques are used to detect the intensity of fluorescence signals emitted by the cross-linking agent backbone. This allows for the acquisition of protein expression levels and / or structural states in different chassis cells, thereby accurately screening chassis cells with different functional states.

Images