An expression system, cell strain and application thereof for improving the expression amount and / or stability of a target protein in mammalian cells

Abstract

The present application relates to the technical field of biotechnology, in particular to an expression system for improving the expression amount and / or stability of target proteins in mammalian cells, a cell strain and application thereof. The expression system comprises one or more vectors and a bifunctional coupling molecule, wherein the one or more vectors comprise a first nucleic acid and a second nucleic acid; and the bifunctional coupling molecule comprises a first ligand part and a second ligand part. Experiments show that the system can increase the yield of three recombinant proteins (TPO, OPG and VN) in HEK293F cells by 2.6-3.1 times, and the natural biological activity is retained. The present application does not depend on specific small molecule ligands of target proteins, has the advantages of strong universality, good modularity, high safety, etc., and can be widely applied to the fields of recombinant protein drug production, diagnostic reagent development and engineering cell strain construction, etc.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an expression system, cell line, and its application for improving the expression level and / or stability of target proteins in mammalian cells. Background Technology

[0002] Recombinant protein technology is a cornerstone of modern biomedical research and pharmaceuticals. Mammalian cell expression systems have become the primary platform for producing therapeutic recombinant proteins, thanks to their ability to produce human proteins with high fidelity, post-translational modifications (PTMs), correct folding, and assembly processes. However, challenges often arise when mammalian cells are responsible for expressing large quantities of exogenous proteins, as this typically exceeds the protein folding capacity of the endoplasmic reticulum (ER). This overload leads to protein accumulation, triggering the unfolded protein response (UPR) and ER stress (ERS), subsequently activating the ubiquitin-proteasome system (UPS). Misfolded proteins are ubiquitinated and tagged for degradation via the ER-associated protein degradation (ERAD) pathway. Consequently, this results in reduced yields of the target protein (POI) and may even induce apoptosis. Furthermore, challenges such as oxidative stress, protein aggregation, and transcriptional bottlenecks further hinder the efficiency of recombinant protein expression. Traditional approaches typically involve optimizing the expression host and culture conditions or modifying protein structure to increase yield. However, fundamentally inhibiting ubiquitination-induced POI degradation and maintaining its intracellular stability remains a critical scientific problem that urgently needs to be solved.

[0003] Deubiquitinase-targeted chimera (DUBTAC) technology is a representative technique that maintains the levels of specific proteins within cells by actively blocking the ubiquitination degradation pathway. DUBTAC functions as a heterobifunctional small molecule, with one end acting as a ligand for deubiquitinases (DUBs) and the other end as a specific ligand for protein ions (POIs). By reducing the spatial distance between the DUB and the POI, DUBTAC catalyzes the cleavage of the ubiquitin chain on the POI, thereby preventing its degradation via the ubiquitin-proteasome system (UPS) and achieving stable upregulation at the protein level. Currently, DUBTAC technology has been successfully applied to various disease-related proteins, including tumor suppressor factors, transcription factors, and metabolic regulators. However, traditional DUBTAC methods are limited to POIs with high-affinity small molecule ligands, thus excluding many proteins lacking known ligands.

[0004] Therefore, it is of great significance to develop a DUBTAC-enhanced expression system that does not depend on target protein-specific ligands and can be widely applied to a variety of recombinant proteins. Summary of the Invention

[0005] This invention aims to solve the technical problem of low yield of exogenous recombinant proteins due to ubiquitination degradation in mammalian cell expression systems. It provides an expression system, method, and applications based on DUBTAC technology, independent of target protein-specific small molecule ligands, that can universally improve the stability and yield of various recombinant proteins. Figure 1 As shown, the target protein was fused with the CRABP1 tag, and a UCHL1-SNAPTag deubiquitinating enzyme fusion protein was constructed. A bifunctional small molecule, BG-ATRA, was also designed and synthesized. One end of this molecule is benzylguanine, the ligand of SNAPTag, and the other end is all-trans retinoic acid, the specific ligand of CRABP1. Through the bridging effect of BG-ATRA, UCHL1-SNAP was precisely recruited to the vicinity of the CRABP1-target protein fusion protein, achieving deubiquitination of the target protein and thus improving its stability and expression level in cells.

[0006] This invention is achieved through the following technical solutions: One objective of this invention is to provide an expression system that enhances the expression level and / or stability of a target protein in mammalian cells. The expression system comprises one or more vectors and a bifunctional conjugate molecule, wherein the one or more vectors comprise: (1) A first nucleic acid comprising a separate polynucleotide encoding a first fusion protein comprising a target protein and a tag protein; (2) A second nucleic acid, which contains a polynucleotide encoding a second fusion protein, the second fusion protein containing a deubiquitinase and a binding protein; The tag protein has specific ligand-binding ability, and its ligand-binding ability is independent of the ligand-binding ability of the target protein itself. The bifunctional conjugate molecule includes a first ligand portion and a second ligand portion, wherein the first ligand portion is configured to bind to the tag protein and the second ligand portion is configured to bind to the binding protein; The bifunctional conjugate molecule recruits the deubiquitinating enzyme to the vicinity of the target protein by conjugating the tag protein and the binding protein to perform deubiquitination of the target protein, thereby inhibiting the ubiquitin-proteasome system-mediated degradation of the target protein.

[0007] In some implementations, the first nucleic acid and the second nucleic acid are present in the same or different vectors.

[0008] In some embodiments, the tag protein includes a member of the cellular retinoic acid-binding protein family or a protein with all-trans retinoic acid binding capacity.

[0009] In some preferred embodiments, the tag protein is cell retinoic acid binding protein 1 (CRABP1).

[0010] As an optional implementation, the tag protein includes an enzyme cleavage site.

[0011] As an optional implementation, the tag protein includes a TEV restriction site.

[0012] In some embodiments, the deubiquitinating enzyme is selected from members of the ubiquitin C-terminal hydrolase (UCH) subfamily, including but not limited to: UCHL1, UCHL3, UCHL5, and BAP1.

[0013] In some preferred embodiments, the deubiquitinating enzyme is UCHL1.

[0014] In some embodiments, the binding protein is a self-labeling protein tag configured to covalently bind to a specific small molecule substrate.

[0015] In some embodiments, the self-labeled protein tag is selected from one or more of tetracysteine ​​tags, SNAP-tags, eDHFR-tags, Halo-tags, or CLIP-tags.

[0016] In some preferred embodiments, the self-labeled protein tag is a SNAP-tag.

[0017] As an alternative implementation, it is possible to transfer the labeling group on the substrate (such as benzylguanine BG) to the cysteine ​​residue of its own active site under conditions of spatial proximity, forming an irreversible covalent bond.

[0018] In some embodiments, the binding protein is attached to the N-terminus or C-terminus of the deubiquitinating enzyme.

[0019] In some preferred embodiments, the binding protein is attached to the N-terminus of the deubiquitinating enzyme.

[0020] In some embodiments, the first ligand portion comprises a retinoic acid ligand structure configured to covalently bind to the tag protein.

[0021] In some embodiments, the second ligand portion comprises a benzylguanine ligand structure configured to covalently bind to the binding protein.

[0022] In some preferred embodiments, the bifunctional coupling molecule is BG-ATRA, whose chemical structure is shown below: .

[0023] In some embodiments, the target protein is selected from one or more of cytokines, growth factors, secretory receptor proteins, extracellular matrix proteins, hormones, enzymes, coagulation factors, antibodies or fusion proteins, and vaccine antigens.

[0024] In some embodiments, the mammalian cells are selected from one or more of CHO cells, HEK293 cells, NSO cells, Vero cells, BHK-21 cells, MDCK cells, COS-7 cells, HeLa cells, SKBR3 cells, BXPC3 cells, Jurkat cells, MO7e cells, and B16-F1 cells.

[0025] A second objective of this invention is to provide an engineered cell line comprising the aforementioned expression system, wherein the engineered cell line stably expresses the first fusion protein and the second fusion protein, and wherein the engineered cell line is configured to enhance the expression level and / or stability of the target protein in the presence of a bifunctional conjugate molecule.

[0026] A third objective of this invention is to provide at least one of the following uses for the aforementioned expression system or engineered cell line: (a) Inhibit the ubiquitin-proteasome system-mediated degradation of target proteins in mammalian cells; (b) Increase the expression level and / or stability of target proteins in mammalian cells; (c) Prepare reagents or kits for improving the expression level and / or stability of target proteins in mammalian cells.

[0027] The fourth objective of this invention is to provide a method for increasing the expression level and / or stability of a target protein in mammalian cells, comprising: After the aforementioned expression system is transferred into mammalian cells and cultured under suitable conditions, or the aforementioned engineered cell lines are cultured under suitable conditions, the ubiquitin-proteasome system-mediated degradation of the target protein is inhibited in mammalian cells, thereby increasing the expression level and / or stability of the target protein in mammalian cells.

[0028] In some embodiments, the method further includes the steps of recovering and / or purifying the target protein and removing the tag protein after expression.

[0029] Compared with the prior art, the beneficial effects of the present invention are: (1) Significantly increased recombinant protein yield: In HEK293F cells, the yields of three different types of recombinant proteins, TPO, OPG, and VN, were increased to 3.1 times, 2.8 times, and 2.9 times that of the control group, respectively; (2) Preservation of protein bioactivity: The purified recombinant TPO showed increased EC50 in MO7e cell proliferation experiments. 50The concentration was 3.489 ng / mL; recombinant OPG showed positive EC in Jurkat cell protection assays. 50 The concentration was 77.48 ng / mL; recombinant VN showed positive results in B16-F1 cell adhesion assays. 50 The concentration was 298.5 ng / mL, and all of them retained good biological activity; (3) Strong versatility: It is a small molecule ligand that does not depend on the target protein and can be widely used in the expression of various types of recombinant proteins such as cytokines (TPO), secretory receptors (OPG), and extracellular matrix proteins (VN); (4) Modular design: The CRABP1 tag and BG-ATRA molecules constitute a standardized module, which is convenient to promote to different expression systems and different target proteins; (5) Low cytotoxicity: BG-ATRA has no significant toxic effect on HEK293F cells in the concentration range of 0-10 μM, and the cell survival rate is >90%; (6) Specific binding: Immunoprecipitation experiments have confirmed that BG-ATRA can specifically bind to CRABP1 protein, and free ATRA can competitively inhibit this binding; (7) Can be used in combination with other technologies: This system can be used in combination with existing technologies such as gene amplification and culture medium optimization to further improve industrial production efficiency. Attached Figure Description

[0030] Figure 1 : Schematic diagram of the DUBTAC enhanced mammalian expression system of this invention; Figure 2 : Compound AMPK-N3-10 1 H NMR spectrum; Figure 3 LCMS spectrum of compound AMPK-N3-10; Figure 4 Compound 13 1 H nuclear magnetic resonance spectrum; Figure 5 Compound 7 1 H nuclear magnetic resonance spectrum; Figure 6 Compound 14 1 H nuclear magnetic resonance spectrum; Figure 7 Compound 19 1 H nuclear magnetic resonance spectrum; Figure 8 Compound 20-1 1 H nuclear magnetic resonance spectrum; Figure 9 Compound 20-3 1 H nuclear magnetic resonance spectrum; Figure 10 Compound 23 1 H nuclear magnetic resonance spectrum; Figure 11 Compound 1 1 H nuclear magnetic resonance spectrum; Figure 12 Compound 2 1 H nuclear magnetic resonance spectrum; Figure 13 Compound 3 1 H nuclear magnetic resonance spectrum; Figure 14 Compound BG-ATRA 1 H nuclear magnetic resonance spectrum; Figure 15 LCMS spectrum of compound BG-ATRA; Figure 16 Schematic diagram of the DUB-SNAP fusion protein; Figure 17 Expression status of the DUB-SNAP fusion protein; Figure 18 Characterization of the ability of the DUB-SNAP fusion protein to stabilize target proteins; Figure 19 Determination of the relationship between AMPK α1 protein expression level and AMPK-N3-10 concentration; Figure 20 The effect of BG-ATRA on cell viability; Figure 21 Determination of the binding ability of BG-ATRA to CRABP1 protein; Figure 22 Construction of stable DUB-SNAP expression cell lines; where A is the Western blot verification of 293F-DUB-SNAP cells expressing DUB-SNAP fusion protein, B is the Western blot verification of SKBR3-DUB-SNAP cells expressing DUB-SNAP fusion protein, and C is the Western blot verification of BXPC3-DUB-SNAP cells expressing DUB-SNAP fusion protein. Figure 23 Assay: Determination of the ability of the BG-ATRA system to stabilize CRABP1 protein in 293F-DUB-SNAP cells; Figure 24 Assay: Determination of the ability of the BG-ATRA system to stabilize CRABP1 protein in SKBR3-DUB-SNAP cells; Figure 25 Assay: Determination of the ability of the BG-ATRA system to stabilize CRABP1 protein in BXPC3-DUB-SNAP cells; Figure 26 The concentration-dependent effect of BG-ATRA; Figure 27BGATRA's time-dependent effect; Figure 28 The DUBTAC-enhanced mammalian expression system improved the expression level of recombinant proteins. Among them, A and B represent the expression of recombinant protein TPO, C and D represent the expression of recombinant protein OPG, and E and F represent the expression of recombinant protein VN. Figure 29 Purification of recombinant proteins; Figure 30 Yield analysis of recombinant protein TPO; Figure 31 : Yield analysis of recombinant protein OPG; Figure 32 : Yield analysis of recombinant protein VN; Figure 33 Bioactivity assay of recombinant protein TPO; Figure 34 Bioactivity assay of recombinant protein OPG; Figure 35 Bioactivity assay of recombinant protein VN. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0032] Experimental Materials and Methods 1. Experimental Materials BG-DBCO was purchased from Xi'an Kangfuluoer Co., Ltd., China (catalog number BCD-302-100mg). The genes encoding the DUB-SNAP fusion protein, the gene encoding UCHL1-SN with the mCherry fluorescent reporter sequence, and the genes encoding TPO, OPG, and VN proteins containing the CRABP1 protein sequence were all synthesized by Anhui General Biotechnology Co., Ltd., China, and cloned into the pcDNA3.1(+) vector using the BamHI restriction endonuclease site.

[0033] 2. Cell Culture Human HEK293F, HEK293FT, and JurKat cells were purchased from Wuhan Precella Biotechnology Co., Ltd.; human Hela, SKBR-3, BxPC-3, and mouse B16-F1 cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.; and human MO7e cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd. All cell lines were confirmed to be free of mycoplasma contamination by PCR testing, and their species origin was also verified by PCR and confirmed by STR analysis.

[0034] Human HEK293F, HEK293FT, and HeLa cells were cultured in DMEM complete medium, which was prepared by adding 5% fetal bovine serum (Catalog No. A5256701, Gbico), 1×NEAA (Catalog No. 60707ES60, YEASEN), 1×GlutaMAX™ (Catalog No. 35050061, Gbico), and 1×penicillin-streptomycin solution (Catalog No. 60162ES76, YEASEN) to DMEM medium (catalog No. 11995065, Gbico). SKBR-3 cells were cultured using McCoy's 5A complete medium, which consists of McCoy's 5A medium (catalog number AC507736, OPM) supplemented with 5% fetal bovine serum (catalog number A5256701, Gbico), 1×NEAA (catalog number 60707ES60, YEASEN), 1×GlutaMAX™ (catalog number 35050061, Gbico) and 1×penicillin-streptomycin solution (catalog number 60162ES76, YEASEN). BXPC-3 and JurKat cells were cultured in RPMI 1640 complete medium, which was prepared by adding 5% fetal bovine serum (FBS) (A5256701, Gbico), 1×NEAA (60707ES60, YEASEN), 1×GlutaMAX™ (35050061, Gbico), and 1×penicillin-streptomycin solution (60162ES76, YEASEN) to RPMI 1640 medium (catalog number 11875093, Gbico). MO7e cells were cultured in RPMI 1640 complete medium, which consisted of RPMI 1640 medium (catalog number 11875093, Gbico), 5% fetal bovine serum (catalog number A5256701, Gbico), 10 μg / μL GM-CSF (catalog number G329704, Aladdin Reagent (Shanghai) Co., Ltd.), 1×NEAA (catalog number 60707ES60, YEASEN), and 1×GlutaMAX. TMThe solution consisted of RPMI 1640 (catalog number 35050061, Gbico) and 1× penicillin-streptomycin solution (catalog number 60162ES76, YEASEN). Mouse B16-F1 cells were cultured in RPMI 1640 complete medium, which included RPMI 1640 medium (catalog number 11875093, Gbico), 10% fetal bovine serum (catalog number A5256701, Gbico), and 1× penicillin-streptomycin solution (catalog number 60162ES76, YEASEN). All cells were cultured in an incubator at 37°C and 5% CO2.

[0035] 3. Plasmid transfection To screen for high-performance DUB-SNAP fusion proteins, plasmids encoding these proteins were transfected into cells. Plasmid transfection was performed using Lipofectamine 3000 transfection reagent (catalog number L3000015, Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were treated overnight with complete medium containing 1 μM BG-DBCO, followed by replacement with medium containing 500 nM AMPK-N3-10 (or a specified concentration). Cells were cultured for another 24 hours before being collected for analysis.

[0036] To assess the impact of this system on the stability of the recombinant protein, the plasmid encoding the recombinant protein was transfected into cells. Plasmid transfection was performed using Lipofectamine 3000 transfection reagent (catalog number L3000015, Invitrogen) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were treated with complete medium containing 1 μM BG-ATRA for 24 hours, after which cells were collected for analysis.

[0037] 4. Construction of stable cell lines This invention utilizes lentiviral transfection to construct a stable cell line. Using Lipofectamine 3000 transfection reagent (catalog number L3000015, Invitrogen), plasmids (DUBRFP2 plasmid [containing UCHL1-SN expression plasmid and mCherry fluorescent reporter sequence], pMD2.G plasmid, and psPAX2 plasmid in a 3:2:1 ratio) were transfected into HEK293FT cells according to the manufacturer's instructions. After culturing for 4-6 hours, the medium was replaced with fresh medium free of penicillin and streptomycin, and culturing continued. The supernatant was collected every 24 hours, and the medium was replaced with fresh medium, repeating this step three times. The collected supernatant was centrifuged at 2000 rpm for 10 minutes and filtered through a 0.45 μm filter to remove cell debris. The filtered lentiviral solution was then concentrated using lentiviral concentrator reagent (catalog number 41101ES50, YEASEN) according to the manufacturer's instructions. Add 200 μL of lentivirus (or a specified volume) and 8 μL of polyamine (catalog number 40804ES76, YEASEN) to complete culture medium and continue culturing cells for 24 hours. After changing the culture medium, observe the cells using a fluorescence microscope during 24-48 hours to assess the lentivirus transfection effect. Successfully transfected cells are sorted using a flow cytometer (FACSDiscover S8, BD) to obtain stable cell lines. The sorted stable cells can be re-examined by flow cytometry and Western blotting (WB) to verify the effectiveness of the stable cell line.

[0038] 5. Cell viability assay Cells were seeded at a density of 300-500 cells per well in 96-well plates (catalog number TCP011096, Biolfil). After 24 hours, the medium was replaced with different concentrations of BG-ATRA. Each compound concentration was tested in quadruple replicates. After 48 hours of treatment, cell viability was assessed using the CCK8 assay (catalog number 40203ES80, YEASEN) according to the manufacturer's instructions. Data analysis was performed using Excel (Microsoft) and GraphPad Prism 10 (Dotmatics).

[0039] 6. Immunoprecipitation 100 μL of SNAP-modified magnetic beads (10 mg / mL) were washed three times with PBST, resuspended in 100 μL of 1 mMMBG-ATRA, and incubated overnight at room temperature. The beads were then washed three more times with PBST. Next, 500 μL of 5% BSA (catalog number 36100ES60, YEASEN) was added and incubated for 1 hour at room temperature to block the bead surface. The supernatant was then discarded. 500 μL of cell lysis buffer was added, and the mixture was incubated for 2 hours at room temperature. The beads were washed three more times with PBST and resuspended in 1× sample loading buffer (catalog number 20315ES05, ​​YEASEN), and heated at 100 °C for 10 minutes to denature the proteins. The target proteins captured by the magnetic beads were identified by Western blotting. The first control experiment omitted the BG-ATRA incubation step; the second control experiment added 5mM ATRA (catalog number A9120, Solarbio) during the cell lysis step to competitively bind to the binding site on the target protein.

[0040] 7. Western blotting Wash cells twice with pre-chilled PBS. Add appropriate amounts of PMSF (catalog number 20104ES03, YEASEN) and lysis buffer (catalog number E1531, Promega) and incubate on ice for 10 minutes. Collect the supernatant and lyse cells using an ultrasonic homogenizer. Centrifuge at 4°C and 14,000 rpm for 15 minutes to collect the supernatant. The resulting protein supernatant is separated by SDS-PAGE electrophoresis and then transferred to an NC membrane. After blocking non-specific binding sites with 1×TBST solution containing 5% skim milk powder (catalog number 36120ES60, YEASEN), the membrane was incubated with the corresponding primary antibodies (AMPK α1 rabbit pAb, catalog number A18855, ABclonal; CRABP1 rabbit mAb, catalog number A5434, ABclonal; anti-SNAP tag antibody, catalog number P9310S, NEB; rabbit anti-His tag mAb, catalog number AE086, ABclonal; β-actin rabbit mAb, catalog number 30102ES80, YEASEN) at room temperature with continuous shaking for 1 hour. The membrane was then washed three times with 1×TBST solution (5 minutes each time), and incubated with HRP-labeled secondary antibody (HRP-labeled goat anti-rabbit IgG antibody, catalog number 33101ES60, YEASEN) at room temperature for 1 hour, followed by three more washes. Finally, HRP chromogenic substrate (catalog number 36222ES76, YEASEN) was added, and the signal was detected by chemiluminescence using a GelView 6000Plus gel imaging system (Guangzhou Borui Biotechnology Co., Ltd.).

[0041] 8. Purification of recombinant proteins To evaluate the yield and bioactivity of the recombinant protein expressed by this system, the recombinant protein was purified using HisSep Ni-NTA Mag beads (catalog number 20561ES08, YEASEN) according to the manufacturer's instructions. The purification effect was then assessed using Coomassie Brilliant Blue rapid staining solution (catalog number 20308ES72, YEASEN). The target protein was quantified using a BCA protein quantification kit (catalog number 20201ES76, YEASEN) to determine its concentration and assess yield. Data analysis was performed using Excel (Microsoft) and GraphPad Prism 10 (Dotcom) software.

[0042] To maximize the preservation of the recombinant protein's bioactivity and minimize the impact of the protein tag, we used TEV protease (Catalog No. S23581-1KU, Shanghai Yuye Biotechnology Co., Ltd.) to remove the CRABP1 tag protein from the target recombinant protein. First, a 10×TEV buffer was prepared: a mixture of 500 mM Tris-HCl (Catalog No. R27430, Shanghai Yuye Biotechnology Co., Ltd.), 500 mM sodium chloride (Catalog No. C111535, Aladdin Reagent (Shanghai) Co., Ltd.), 5 mM EDTA (Catalog No. B540625-0500, Sanggong Biotechnology (Shanghai) Co., Ltd.), and 10 mM DTT (Catalog No. D104861, Aladdin Reagent (Shanghai) Co., Ltd.) was added, and the pH was adjusted to 8.0. 20 μg of the target protein was added, along with 10 μL of TEV protease and 5 μL of 10×TEV buffer, diluted with water to 50 μL, and incubated at 30°C for 4 hours. Finally, the product was purified again using the same purification steps as described above.

[0043] 9. Cell proliferation experiment To determine the bioactivity of recombinant TPO protein, MO7e cells were cultured for 24 hours in GM-CSF-free medium. Subsequently, samples were collected at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well in 96-well plates (catalog number TCP011096, Biolfil). Different concentrations of recombinant TPO protein were added to the cells, and the plates were incubated for 48 hours. The absorbance at 450 nm was then measured using the CCK8 assay (catalog number 40203ES80, YEASEN) on a microplate reader (Varioskan LUX, Thermo) according to the manufacturer's instructions. Data analysis was performed using Excel (Microsoft) and GraphPad Prism 10 (Dotmatics) software, and the half-maximum effective concentration (EC50) was calculated.

[0044] 10. Cytotoxicity test To determine the bioactivity of recombinant OPG protein, Jurkat cells in logarithmic growth phase were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL of culture medium in 96-well plates (Catalog No. TCP011096, Biolfil). Different concentrations of recombinant OPG protein and 50 ng / mL control solution were added to the cells. After culturing for 24 hours, the absorbance at 450 nm was measured using the CCK8 method (Catalog No. 40203ES80, YEASEN) on a microplate reader (Varioskan LUX, Thermo) according to the manufacturer's instructions. Data analysis was performed using Excel (Microsoft) and GraphPad Prism 10 (Dotmatics) software, and the half-maximum effective concentration (EC50) was calculated.

[0045] 11. Cell adhesion experiment To determine the bioactivity of recombinant VN protein, we diluted it to different concentrations with PBS. 100 μL of VN solution was added to each well of a 96-well plate (Catalog No. TCP011096, Biolfil) and incubated at 37°C for 2 hours. The plate was then washed three times with PBS to remove unbound VN protein, followed by blocking with 200 μL of 1% BSA (Catalog No. 36100ES60, YEASEN) and incubation at 37°C for 1 hour. The plate was then washed three times with PBS again. B16-F1 cells in the logarithmic growth phase were collected and washed twice with PBS to eliminate serum protein interference. The cells were resuspended in serum-free medium containing 5% BSA (Catalog No. 36100ES60, YEASEN) at a density of 5 × 10⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per well into treated 96-well plates and cultured for 2 hours, followed by washing three times with PBS to remove non-adherent cells. Absorbance at 450 nm was measured using a microplate reader (Varioskan LUX, Thermo) according to the manufacturer's instructions (catalog number 40203ES80, YEASEN). Data were analyzed using Excel (Microsoft Corporation) and GraphPad Prism 10 (Dotcom) software, and the half-maximum effective concentration (EC50) was calculated.

[0046] 12. Evaluate the ability of the DUBTAC-enhanced mammalian expression system to stabilize proteins. To evaluate the ability of the DUBTAC-enhanced mammalian expression system to stabilize proteins, 1 μM BG-ATRA was added to untreated cells and corresponding stable transfected cells, and the cells were collected after 24 hours of culture for analysis.

[0047] 13. Optimization of BG-ATRA concentration and treatment time To determine the concentration dependence of BG-ATRA, different concentrations of BG-ATRA were added to 293F-DUB-SNAP cells, and the cells were collected after 24 hours of culture for analysis.

[0048] To determine the time dependence of BG-ATRA, 10 μM BG-ATRA was added to 293F-DUB-SNAP cells, and cells were collected at different time points for analysis.

[0049] 14. General process for the preparation of AMPK-N3-10

[0050] NMM (58.6 mg, 579 μmol), EDCI (27.7 mg, 145 μmol), and HOAt (15.8 mg, 116 μmol) were added to a DMF solution (0.50 mL) of compound 2 (50.0 mg, 115 μmol) and compound 10A (34.4 mg, 173 μmol). The mixture was stirred at 25°C for 12 hours. LC-MS analysis showed that the target compound was detected in 33% of the solution. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 × 30 mm × 10 μm; mobile phase: [water (containing 10 mM ammonium bicarbonate)]-ACN; gradient elution: phase B concentration from 65% to 95% over 8.0 min). AMPK-N3-10 (6.75 mg, 10.9 μmol, yield 9.47%, purity 99.4%) was finally obtained as a pale white solid.

[0051] ¹H NMR spectrum: (400MHz, DMSO-d6): δ 8.31 (t-type signal, J = 5.63Hz, ¹H); 7.54 (s-type signal, ¹H); 7.44–7.51 (m-type signal, ²H); 7.28–7.38 (m-type signal, ²H). 5 H); 7.18 (dd type signal, J=8.44, 1.19Hz, ¹H); 6.45 (d type signal, J=3.13Hz, ¹H); 3.82 (s type signal, ³H); 3.26–3.29 (m type signal, ²H); 3.20 (q type signal, J=6.63Hz, ²H); 2.34 (s type signal, ³H); 1.48–1.50 (m type signal, 4 H); 1.22–1.33 (m-type signal, ¹²H).

[0052] C 34 H 38LCMS of ClN7O2[M+H]: Calculated value: 612.2, Measured value: 612.2.

[0053] The compound AMPK-N3-10 1 H nuclear magnetic resonance spectrum as follows Figure 2 As shown, the LCMS spectrum is as follows Figure 3 As shown.

[0054] 15. General preparation process for BG-ATRA Compound BG-ATRA was prepared via a 14-step synthetic route, the specific steps of which are as follows: Step 1: To a 20.0 mL solution of DMF containing compound 11 (3.00 g, 11.1 mmol, 1.00 equivalent), HATU (5.49 g, 14.4 mmol, 1.30 equivalent) and DIEA (2.15 g, 16.6 mmol, 2.90 mL, 1.50 equivalent) were added. The mixture was then stirred at 25°C for 10 hours. Thin-layer chromatography (mobile phase: commercial hexane / ethyl acetate = 0 / 1, eluent Rf(product) = 0.20) showed that the target compounds were obtained. The reaction mixture was concentrated under vacuum, and the residue was purified by prep-HPLC column chromatography (column: WePure Biotech XP tC18 250×70 7μm; mobile phase: [water (containing 10mM ammonium bicarbonate)]-ACN; gradient elution: phase B concentration increased from 20% to 50% over 17.0 minutes), finally yielding compound 13 (5.00 g, 8.09 mmol, yield 72.9%) as a colorless solid. The reaction formula is as follows:

[0055] ¹H NMR spectrum of compound 13: (400 MHz, DMSO-d6) δ 8.34 (peak shape, J = 5.7 Hz, ¹H), 7.89–7.75 (peak shape, ¹H), 7.44 (peak shape, J = 7.9 Hz, ²H), 7.26 (peak shape, J = 8.0 Hz, ²H), 6.74 (peak shape, J = 5.0 Hz, ¹H), 6.26 (peak shape, ²H), 5.45 (peak shape, ²H), 4.27 (peak shape, J = 5.8 Hz, ²H), 3.63 (peak shape, J = 6.4 Hz, ²H), 3.40–3.29 (peak shape, ¹H). 6 H), 3.05 (peak shape is sharp, J=5.9Hz,²H), 2.37 (peak shape is sharp, J=6.3Hz,²H), 1.36 (peak shape is single peak), 9H). The ¹H NMR spectrum of compound 13 is shown below. Figure 4 As shown.

[0056] Step 2: Compound 13 (5.00 g, 8.09 mmol, 1.00 equivalent) was dissolved in HFIP (150 mL) to obtain a solution, which was then pumped into flow reactor 1 (FLR1, SS type coil reactor, tube diameter 3.175 (1 / 8) mm, volume 36.59 × 10⁻⁶) via pump 1 (S1, P1, 3.659 mL / min). 5585 The pressure was adjusted to 5 MPa. The residence time in flow reactor 1 was 10.0 min. The reaction mixture was concentrated under vacuum, and the residue was purified by prep-HPLC (column: WePure Biotech XP T-C18 250×70×10 μm; mobile phase: water (containing 10 mM ammonium bicarbonate)-ACN; elution gradient: 1%-35% B, duration: 20.0 min). Compound 7 (2.00 g, 3.86 mmol, yield 47.7%) was finally obtained as a pale white solid. The reaction formula is as follows:

[0057] The ¹H NMR spectrum of compound 7 (400 MHz, DMSO-d6) shows the following peaks: δ 8.35 (single, ¹H), 7.82 (single, ¹H), 7.44 (doublet, J = 8.0 Hz, ²H), 7.27 (doublet, J = 8.0 Hz, ²H), 6.25 (single, ²H), 5.45 (single, ²H), 4.27 (doublet, J = 5.7 Hz, ²H), 3.63 (doublet, J = 6.3 Hz, ³H), 3.49 (single, ¹²H), 3.40–3.31 (multiplet, ³H), 3.09–3.03 (multiplet, ¹H), 2.64 (doublet, J = 5.7 Hz, ¹H), and 2.37 (singlet, J = 6.3 Hz, ²H). 1 H nuclear magnetic resonance spectrum as follows Figure 5 As shown.

[0058] Step 3: 18934-81-1 (25.0 g, 79.2 mmol, 1.00 equivalent) and N-methoxymethylamine hydrochloride (9.28 g, 95.1 mmol, 1.20 equivalent) were dissolved in DCM (180 mL), and HOBt (11.7 g, 87.2 mmol, 1.10 equivalent), EDCI (18.2 g, 95.1 mmol, 1.20 equivalent), and DIEA (40.9 g, 317 mmol, 55.2 mL, 4.00 equivalent) were added. The mixture was stirred at 25°C for 10 hours. Thin-layer chromatography (commercial hexane / ethyl acetate = 1 / 1, elution (product) 0.30) indicated that the reaction was complete. The reaction mixture was quenched with ammonium chloride aqueous solution (300 mL) and extracted with ethyl acetate (100 mL × 3 times). The combined organic layers were washed with brine (150 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (silica, commercial hexane / ethyl acetate = 30 / 1 to 0 / 1) to give compound 14 as a white solid (25.0 g, 69.7 mmol, yield 87.9%). The reaction formula is as follows:

[0059] ¹H NMR spectrum of compound 14: (400 MHz, CDCl3) δ 4.50 (single peak, ¹H), 3.69 (single peak, ³H), 3.19 (single peak, ³H), 3.15–3.06 (multiple peaks, ²H), 2.41 (doublet, J = 7.5 Hz, ²H), 1.67–1.60 (multiplets, ²H), 1.45 (single peak, ¹¹H), 1.35–1.23 (multiplets, ¹¹H). 4 H). Compound 14 1 H nuclear magnetic resonance spectrum as follows Figure 6 As shown.

[0060] Step 4: Compound 14 (4.00 g, 11.2 mmol, 1.00 equivalent) was dissolved in THF (40.0 mL) and cooled to 0 °C. Then, under nitrogen protection, compound 14a (0.5 M, 78.10 mL, 3.50 equivalent) was added dropwise to the mixture, and the mixture was stirred at 0 °C for 2 hours. Thin-layer chromatography (commercial hexane / ethyl acetate = 1 / 1, product Rf = 0.60) indicated that the reaction was complete. The reaction mixture was quenched with 100 mL of water and extracted with ethyl acetate (80.0 mL × 3 times). The combined organic phases were washed with brine (80.0 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (silica, commercial hexane / ethyl acetate = 50 / 1 to 0 / 1) to give compound 19 (4.00 g, 9.67 mmol, yield 86.6%) as a white solid. The reaction formula is as follows:

[0061] ¹H NMR spectrum of compound 19: (400 MHz, CDCl3) δ 4.50 (single, ¹H), 3.10 (quartet, J = 6.1 Hz, ²H), 2.42 (triplet, J = 7.4 Hz, ²H), 2.14 (single, ³H), 1.62–1.54 (multiplet, ²H), 1.45 (single, ¹H) 0 H), 1.26 (single peak, ¹ 5 H). Compound 19 1 H nuclear magnetic resonance spectrum as follows Figure 7 As shown.

[0062] Step 5: Compound 19 (3.00 g, 7.25 mmol, 1.00 equivalent) was dissolved in a mixture of hydrochloric acid / ethyl acetate (4 M, 30.0 mL, 16.5 equivalent) in ethyl acetate (10.0 mL) and stirred at 25°C for 1 hour. Thin-layer chromatography (commercial hexane / ethyl acetate = 1 / 1, elution (product) = 0.60) showed that the target compound was obtained. The reaction mixture was concentrated under vacuum without purification to obtain compound 20-1 (2.50 g, crude product, hydrochloric acid). The product was a pale white solid. The reaction formula is as follows:

[0063] ¹H NMR spectrum of compound 20-1: (400 MHz, DMSO-d6) δ 4.57 (t-type signal, J = 5.0 Hz, ¹H), 4.14 (t-type signal, J = 6.3 Hz, ¹H), 4.03 (double peak signal, J = 5.0, 10.8 Hz, ¹H), 3.82–3.70 (single peak signal, ¹H), 2.91 (strong t-type signal, J = 7.6 Hz, ²H), 2 0.57–2.49 (single-peak signal, ¹H), 2.48–2.40 (single-peak signal, ²H), 1.98 (strong d-type signal, J=12.8Hz, ¹H), 1.79 (double-peak signal, J=5.4, 7.4Hz, ²H), 1.65 (quadruple-peak signal, J=7.4Hz, ²H), 1.58–1.48 (single-peak signal, ²H), 1.37–1.28 (single-peak signal, ¹²H). Compound 20-1 1 H nuclear magnetic resonance spectrum as follows Figure 8 As shown.

[0064] Step 6: Cpd 20-1 (2.50 g, 7.14 mmol, 1.00 equivalent, hydrochloric acid) and FmocOSu (2.89 g, 8.57 mmol, 1.20 equivalent) were dissolved in THF (10.0 mL) and water (2.00 mL), and sodium bicarbonate (1.50 g, 17.86 mmol, 694.95 μL, 2.50 equivalent) was added. The mixture was then stirred at 25°C for 10 hours. The reaction mixture was quenched with water (50.0 mL) and extracted with ethyl acetate (50.0 mL × 3 times). The combined organic layers were washed with brine (80.0 mL), dried over sodium sulfate, filtered, and concentrated under vacuum without purification. The product compound 20-2 (3.00 g, crude) was given as a yellow solid. The reaction formula is as follows:

[0065] Step 7: Compound 20-2 (3.00 g, 5.24 mmol, 1.00 equivalent, hydrochloric acid) was dissolved in dichloromethane (21.0 mL) with Jones' reagent (2.5 M, 20.97 mL, 10.0 equivalent) and stirred at 25°C for 24 hours. The reaction mixture was quenched with an aqueous sodium sulfite solution (100 mL) and extracted with dichloromethane (100 mL × 3 times). The combined organic layers were washed with brine (100 mL), dried over anhydrous ammonium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica, dichloromethane / ethyl acetate = 80 / 1 to 1 / 1) to give the pale white solid product compound 20-3 (1.10 g, 1.29 mmol, yield 24.6%, purity 58.0%). The reaction formula is as follows:

[0066] ¹H NMR spectrum of compound 20-3: (400 MHz, CDCl3) δ 7.77 (d peak, J = 7.5 Hz, ²H), 7.66–7.57 (m peak, ²H), 7.46–7.37 (m peak, ²H), 7.36–7.29 (m peak, ²H), 5.93–5.82 (m peak, ¹H), 4.86–4.68 (m peak, ¹H), 4.41 (strong d peak, J = 6.9 Hz, ²H), 4.30–4.20 (m peak, ¹H), 3.27–3.07 (m peak, ²H), 2.76–2.60 (m peak, ²H). 4 H), 2.45 (t peak, J=7.3Hz,²H), 1.70–1.39 (m peak, 4 H), 1.28 (strong s peak, ¹ 4 H). Compound 20-3 1 H nuclear magnetic resonance spectrum as follows Figure 9 As shown.

[0067] Step 8: Compound 7 (524 mg, 1.01 mmol, 1.00 equivalent), compound 20-3 (500 mg, 1.01 mmol, 1.00 equivalent), HOBt (164 mg, 1.22 mmol, 1.20 equivalent), DIEA (392 mg, 3.04 mmol, 529 μL, 3.00 equivalent), and EDCI (233 mg, 1.22 mmol, 1.20 equivalent) were dissolved in DMF (5.00 mL) and stirred at 25°C for 2 hours. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (30.0 mL × 5 times). The combined organic layers were washed with brine (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum without purification. Compound 22 (2.00 g, crude product) was finally obtained as a yellow oil.

[0068]

[0069] Step 9: Compound 22 (1.00 g, 1.01 mmol, 1.00 equivalent) was dissolved in DMF (5.00 mL) with piperidine (428 mg, 5.03 mmol, 497 μL, 5.00 equivalent) and stirred at 25°C for 5 hours. The reaction mixture was concentrated under vacuum, and the residue was purified by prep-HPLC column chromatography (column: WePure Biotech XP tC18 250×70×10 μm; mobile phase: water (containing 10 mM ammonium bicarbonate)-ACN; elution gradient: from 15% to 45% B over 20.0 min). Compound 23 (300 mg, 389 μmol, yield 19.3%) was finally obtained as a pale white solid. The reaction formula is as follows:

[0070] ¹H NMR spectrum of compound 23: (400 MHz, DMSO-d6) δ 8.34 (peak shape, J = 5.6 Hz, ¹H), 7.81 (single peak, ¹H), 7.44 (doublet, J = 8.0 Hz, ²H), 7.26 (doublet, J = 8.0 Hz, ²H), 6.24 (single peak, ²H), 5.45 (single peak, ²H), 4.27 (doublet, J = 5.9 Hz, ²H), 3.62 (peak shape, J = 6.4 Hz, ²H), 3.48–3.24 (multiplet, ¹H) 63.18–3.06 (multiplet, ¹H), 2.88 (singlet, J=6.3Hz, ¹H), 2.60–2.51 (multiplet, ¹H), 2.41–2.32 (multiplet, ³H), 2.32–2.24 (multiplet, ¹H), 2.19–1.97 (multiplet, ²H), 1.85–1.72 (multiplet, ¹H), 1.72–1.61 (multiplet, ¹H), 1.55–1.40 (multiplet, ¹H), 1.37–1.14 (multiplet, ¹H), ...¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, ¹H, 8 H). Compound 23 1 H nuclear magnetic resonance spectrum as follows Figure 10 As shown.

[0071] Step 10: Potassium carbonate (4.60 g, 33.2 mmol, 2.00 equivalent) and methyl iodide (3.54 g, 24.9 mmol, 1.55 mL, 1.50 equivalent) were added to a DMF solution (35.0 mL) of 302-79-4 (5.00 g, 16.6 mmol, 1.00 equivalent). The mixture was then stirred at 25 °C for 5 hours. Thin-layer chromatography (commercial hexane / ethyl acetate = 3 / 1, Rf (product) = 0.68) showed that the reaction was complete. The reaction mixture was quenched with 200 mL of water and extracted with ethyl acetate (50.0 mL, 3 times). The combined organic phases were unpurified. Compound 1 (5.00 g, 15.9 mmol, 95.5% yield) was given as a yellow solid. The reaction formula is as follows:

[0072] The ¹H NMR spectrum of compound 1 (400 MHz, CDCl₃) δ 7.08–6.95 (multiplet, ¹H), 6.29 (broad doublet, J = 16.1 Hz, ²H), 6.20–6.10 (multiplet, ²H), 5.78 (singlet, ¹H), 3.72 (doublet, J = 1.9 Hz, ³H), 2.37 (singlet, ³H), 2.09–1.98 (multiplet, ¹H). 5 1.68–1.58 (multiplet, ²H), 1.47 (broad doublet, J=3.4, 5.8Hz, ²H), 1.04 (doublet, J=1.9Hz, ⁻¹⁰) 6 H). The 1H NMR spectrum of compound 1 is as follows: Figure 11 As shown.

[0073] Step 11: To a DCM solution of compound 1 (3.00 g, 9.54 mmol, 1.00 equivalent), manganese dioxide (82.9 g, 954 mmol, 100 equivalent) was added, and the mixture was stirred at 25 °C for 72 hours. Thin-layer chromatography (commercial hexane / ethyl acetate = 3 / 1, product Rf value = 0.55) indicated that the reaction was complete. After filtering the reaction mixture, the filter cake was washed with DCM (100 mL × 2 times), the filtrates were combined and concentrated under vacuum. The residue was purified by column chromatography (silica, commercial hexane / ethyl acetate = 30 / 1 to 0 / 1) to give compound 2 (1.70 g, 5.18 mmol, yield 54.2%) as a yellow solid. The reaction formula is as follows:

[0074] ¹H NMR spectrum of compound 2: (400 MHz, DMSO-d6) δ 6.99 (dd, J = 11.4, 15.1 Hz, ¹H); 6.40–6.22 (m, 4H); 5.83 (s, ¹H); 3.78–3.68 (m, ³H); 2.52 (t, J = 6.9 Hz, ²H); 2.37 (d, J = 1.0 Hz, ³H); 2.07–2.01 (m, 4 H); 1.91–1.83 (m, 5 H); 1.20 (s, 6 H). Compound 2 1 H nuclear magnetic resonance spectrum as follows Figure 12 As shown.

[0075] Step 12: Compound 2a (152 mg, 1.67 mmol, 1.10 equivalent) was added to a solution of compound 2 (500 mg, 1.52 mmol, 1.00 equivalent) dissolved in pyridine (2.00 mL), and the mixture was stirred at 25°C for 10 hours. The reaction mixture was concentrated under vacuum to give an unpurified residue, ultimately yielding compound 3 (500 mg, 1.25 mmol, crude) as a yellow solid. The reaction formula is as follows:

[0076] The ¹H NMR spectrum of compound 3: (400 MHz, CDCl3) δ 6.96–6.85 (m, ¹H), 6.31–6.08 (m, ¹H), δ 6.96–6.85 (m, ¹H), δ 6.31–6.08 ...96–6.8 4H), 5.73 (s, ¹H), 4.64–4.57 (m, ²H), 3.68–3.60 (m, ³H), 2.64 (t, J=6.8Hz, ²H), 2 .29 (d, J=0.8Hz, ³H), 1.95 (s, ³H), 1.81 (s, ³H), 1.55 (t, J=6.8Hz, ²H), 1.02 (s, 6 H). Compound 3 1 H nuclear magnetic resonance spectrum as follows Figure 13 As shown.

[0077] Step 13: Compound 3 (160 mg, 398 μmol, 1.00 equivalent), TSTU (179 mg, 597 μmol, 1.50 equivalent), and DIPEA (155 mg, 1.20 mmol, 208 μmol, 3.00 equivalent) were dissolved in DMF (1.60 mL) and stirred at 25°C for 0.5 h. Compound 23 (215 mg, 278 μmol, 0.700 equivalent) was then added to the mixture, and the reaction was continued at 25°C for 2 h. The reaction mixture was filtered, and the filtrate was collected. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18 150×40×7 μm; mobile phase: [water (containing 0.05% NH3·H2O + 10 mM ammonium bicarbonate) - ACN]; elution gradient: 45% to 95% fraction B, 8.0 min). The final product was a yellow solid, compound 8 (50.0 mg, crude product). The reaction formula is as follows:

[0078] Step 14: Compound 8 (50.0 mg, 43.3 μmol, 1.00 equivalent), LiOH·H₂O (1 M solution, 2.17 mL, 50.0 equivalent), THF (1.00 mL), and MeOH (1.00 mL) were mixed and stirred at 25°C for 10 hours. The reaction mixture was concentrated under reduced pressure at 35°C to remove THF and MeOH. The residue was purified by pre-chromatographic purification (column: WePure Biotech XP T-C18 150×40×7 μm; mobile phase: water (containing 10 mM ammonium bicarbonate)-ACN; gradient elution: 25% to 70% B, 8.0 min) to obtain a yellow solid BG-ATRA (6.00 mg, 5.00 μmol, yield 11.5%, purity 95%). The reaction formula is as follows:

[0079] ¹H NMR spectrum of compound BG-ATRA: (400 MHz, DMSO-d6) δ 7.83 (s, ¹H), 7.49 (d, J = 7.8 Hz, ²H), 7.31 (d, J = 7.8 Hz, ²H), 6.90–6.73 (m, ¹H), 6.43–6.10 (m, ¹H). 4 H), 5.90 (s, ¹H), 5.80 (brs, ¹H), 5.54 (s, ²H), 4.49 (s, ²H), 4.40 (s, ²H), 3.75 (brt, J=5.9Hz, ³H), 3.62–3.52 (m, ¹ 5 H), 3.48 (brt, J=5.4Hz, ¹H), 3.25 (brt, J=6.8Hz, ³H), 2.81–2.69 (m, ³H), 2.55–2.37 (m, 5 H), 2.33–2.22 (m, ¹H), 2.20–2.10 (m, ³H), 2.03–1.85 (m, 8 H), 1.72–1.56 (m, 4 H), 1.50 (brd, J=1.5Hz, ³H), 1.27 (brs, ¹ 6 H), 1.15–1.08 (m, 6 H).

[0080] C 61 H 89 N9O 12 The LCMS value of [MH] is calculated to be 1138.9, and the measured value is also 1138.9.

[0081] Compound BG-ATRA 1 H nuclear magnetic resonance spectrum as follows Figure 14 As shown, the LCMS spectrum is as follows Figure 15 As shown.

[0082] Example 1: Construction and Screening of DUB-SNAP Fusion Protein To optimize the performance of DUB-SNAP fusion protein components, the N-terminus and C-terminus of 12 representative DUBs (including OTUB1, UCHL1, USP13, USP14, USP38, USP39, KLHL40, PRPS2, CDC37, HSPA1A, TANK, and CALM3) were fused with SNAPTag, generating 24 different DUBSNAP fusion proteins. A screening process was then conducted to identify the fusion protein with the most effective deubiquitination activity. Figure 16 Twenty-four DUB-SNAP expression plasmids were transfected into HEK293FT cells, and their expression levels were confirmed by Western blot analysis.

[0083] The results showed that 23 out of 24 DUB-type proteins successfully expressed the DUB-SNAP fusion protein (…). Figure 17 ).

[0084] The function of the DUB-SNAP system was evaluated using the established target protein AMPK α1 of DUBTAC. In cell lines expressing different DUB-SNAP fusion proteins, the self-synthesized AMPK α1 ligands AMPK-N3-10 and BG-DBCO were added. These two compounds underwent a click reaction within the cells, promoting the proximity of the deubiquitinating enzyme to the AMPK α1 protein. The relative content of AMPK α1 was assessed using the Jess automated blot system. Among the 23 DUB-SNAP fusion proteins, UCHL1-SN showed the most significant stabilizing effect on AMPK α1 protein, increasing its expression level to twice that of the control group. Figure 18 Furthermore, this invention found that the stabilizing effect of UCHL1-SN on the target protein is dependent on the concentration of the AMPK-N3-10 ligand. Within the 0-5 μM range, this effect increases with increasing AMPK-N3-10 concentration, reaching a peak of 3.8 times the initial level. Figure 19 Therefore, this invention selected UCHL1-SN as the DUB-SNAP fusion protein for subsequent experiments.

[0085] Example 2: Construction of a mammalian protein expression system based on DUB-SNAP After determining that UCHL1-SN is the optimal DUB-SNAP fusion protein for enhancing protein stability, a recombinant protein expression system was developed. Cellular retinoic acid binding protein 1 (CRABP1) was selected as the tag protein on the point of interest (POI). CRABP1, with a molecular weight of 15 kDa, exhibits specificity and high affinity for biocompatible all-trans retinoic acid (ATRA), making it an ideal universal tag protein for recombinant protein production systems. ATRA was used as the specific recognition ligand for CRABP1, and the bifunctional DUBTAC molecule BG-ATRA was synthesized. This invention evaluated the cytotoxicity characteristics and CRABP1 protein binding ability of BG-ATRA. The results showed that, within the concentration range of 0-10 μM, BG-ATRA had negligible cytotoxic effects on cells. Figure 20 BG-ATRA exhibits specific binding to the CRABP1 protein. Figure 21 Subsequently, three stable cell lines expressing UCHL1-SN were constructed: 293F-DUB-SNAP, SKBR3-DUB-SNAP, and BXPC3-DUB-SNAP. Figure 22Next, the stabilizing effect of the DUBTAC molecule BG-ATRA on endogenous CRABP1 protein levels was evaluated. In 293F-DUB-SNAP cells, CRABP1 expression was increased by 1.4-fold compared to wild-type cells (…). Figure 23 In SKBR3-DUB-SNAP cells, the expression level increased by 1.6 times. Figure 24 Notably, in BXPC3-DUB-SNAP cells, the expression level surged by approximately 2-fold. Figure 25 These results confirm the effectiveness of this expression system in enhancing protein stability across various mammalian cell types.

[0086] The effects of BG-ATRA on protein stability were comprehensively evaluated in a concentration- and time-dependent manner. Changes in CRABP1 protein expression levels at different BG-ATRA concentrations (from 0 μM to 10 μM) within a non-cytotoxic range were monitored to establish the correlation between stability effects and drug concentration. Results showed that within this concentration range, CRABP1 expression in 293FDUB-SNAP cells increased proportionally with increasing BG-ATRA concentration, peaking at 3.5 times that of the untreated group. Figure 26 Furthermore, over a 24-hour time span, the expression level of CRABP1 increased synchronously with the extension of BG-ATRA treatment time. Figure 27 ).

[0087] Example 3: DUBTAC-enhanced mammalian expression system improves the stability of recombinant proteins The CRABP1 protein was fused with target recombinant proteins to achieve efficient expression of these recombinant proteins. Based on structural type, biological function, and challenges of recombinant expression, three representative recombinant proteins were selected: thrombopoietin (TPO), osteoprotegerin (OPG), and vitreous-binding protein (VN). These proteins represent three different classes of proteins: cytokines, secreted receptor proteins, and extracellular matrix proteins.

[0088] Three recombinant proteins, CRABP1-TPO, CRABP1-OPG, and CRABP1-VN, were introduced into wild-type 293F cells and 293F-DUB-SNAP cells, respectively. These proteins were designed with TEV protease cleavage sites to remove the CRABP1 tag during subsequent purification. The expression levels of the corresponding recombinant proteins were assessed after the addition of the DUBTAC functional molecule BG-ATRA. Western blot analysis showed that the expression levels of all three recombinant proteins were significantly increased in the 293F-DUB-SNAP system. Figure 28(A, C, E). Specifically, TPO expression increased approximately 3-fold ( Figure 28 OPG expression increased by 2.4 times (B). Figure 28 The expression of VN increased by 2.6 times (D). Figure 28 These results demonstrate that the expression system exhibits broad stability for a variety of heterologous recombinant proteins.

[0089] Example 4: The DUBTAC-enhanced mammalian expression system retains the biological activity of the recombinant protein. The biological functional integrity of the recombinant proteins was further investigated. First, three fusion recombinant proteins expressed after BG-ATRA molecule treatment were purified and recovered, followed by removal of the CRABP1 tag using TEV protease. SDS-PAGE analysis was performed... Figure 29 The results showed that all three recombinant proteins exhibited a single band, indicating good purification. Quantitative analysis of the target protein yield revealed that, compared to the control group, the expression level of TPO protein in 293F-DUB-SNAP cells was 3.1 times that in wild-type 293F cells. Figure 30 The expression level of OPG protein was 2.8 times higher. Figure 31 The expression level of VN protein was 2.9 times higher. Figure 32 These results further confirm that the DUBTAC-enhanced mammalian expression system can significantly increase the production yield of recombinant proteins.

[0090] Subsequently, the biological functions of these three recombinant proteins were evaluated using different bioactivity assays. TPO stimulates cell proliferation by activating the JAK2 / STAT5 signaling pathway via the c-Mpl receptor. The activation of this receptor was assessed using proliferation assays on the MO7e megakaryocyte cell line. OPG, as a TRAIL agonist receptor, inhibits cytotoxicity by competitively binding to TRAIL. Its potency was assessed by inhibiting TRAIL-treated Jurkat cells. VN promotes cell adhesion and spread by binding to integrin αvβ3 / αvβ5 via its RGD sequence. Its ability to promote adhesion was tested using B16-F1 cell adhesion assays. The activity analysis results indicated that the biological functions of these three recombinant proteins were unaffected. The measured half-maximum effective concentrations (EC50) were: TPO 3.489 ng / mL (… Figure 33 OPG was 77.48 ng / mL. Figure 34 VN was 298.5 ng / mL. Figure 35 These findings collectively support the view that the DUBTAC-enhanced mammalian expression system can increase the expression levels of recombinant proteins while preserving their biological activity.

[0091] Mammalian expression systems are limited by the ubiquitination and degradation system activated by exogenous proteins, leading to target protein degradation and ultimately insufficient yield. In this study, we developed a DUBTAC-enhanced mammalian expression system to improve the expression efficiency of recombinant proteins. We designed and screened the DUB-SNAP fusion protein UCHL1-SN, which exhibited robust expression in cells and demonstrated excellent ability to stabilize points of interest (POIs). In cells stably expressing UCHL1-SN, we achieved efficient expression of the target recombinant protein by transfecting a protein tagged with CRABP1 and introducing the DUBTAC molecule BG-ATRA. Furthermore, the biological activity of the recombinant protein remained unaffected after the CRABP1 protein tag was removed.

[0092] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. An expression system for increasing the expression level and / or stability of a target protein in mammalian cells, the expression system comprising one or more vectors and a bifunctional conjugate molecule, said one or more vectors comprising: (1) A first nucleic acid comprising a separate polynucleotide encoding a first fusion protein comprising a target protein and a tag protein; (2) A second nucleic acid, which contains a separate polynucleotide encoding a second fusion protein, the second fusion protein containing a deubiquitinase and a binding protein; The tag protein has specific ligand-binding ability, and its ligand-binding ability is independent of the ligand-binding ability of the target protein itself. The bifunctional conjugate molecule includes a first ligand portion and a second ligand portion, wherein the first ligand portion is configured to bind to the tag protein and the second ligand portion is configured to bind to the binding protein; in, The bifunctional conjugate molecule recruits the deubiquitinating enzyme to the vicinity of the target protein by conjugating the tag protein to the binding protein, thereby performing deubiquitination of the target protein and inhibiting the ubiquitin-proteasome system-mediated degradation of the target protein.

2. The expression system according to claim 1, characterized in that, The tag proteins include members of the cellular retinoic acid-binding protein family or proteins with all-trans retinoic acid binding capacity.

3. The expression system according to claim 2, characterized in that, The tag protein is cell retinoic acid-binding protein 1.

4. The expression system according to claim 3, characterized in that, The tag protein contains an enzyme cleavage site.

5. The expression system according to any one of claims 1-4, characterized in that, The deubiquitinating enzyme is a member of the ubiquitin C-terminal hydrolase subfamily, selected from UCHL1, UCHL3, UCHL5, or BAP1.

6. The expression system according to claim 5, characterized in that, The deubiquitinating enzyme is UCHL1.

7. The expression system according to claim 6, characterized in that, The binding protein is a self-labeled protein tag, which is configured to covalently bind to a specific small molecule substrate.

8. The expression system according to claim 7, characterized in that, The self-labeled protein tag is selected from one or more of the following: tetracysteine ​​tag, SNAP-tag, eDHFR-tag, Halo-Tag, or CLIP-tag.

9. The expression system according to claim 8, characterized in that, The self-labeled protein tag is a SNAP-tag.

10. The expression system according to any one of claims 7-9, characterized in that, The binding protein is attached to the N-terminus or C-terminus of the deubiquitinating enzyme.

11. The expression system according to claim 10, characterized in that, The binding protein is attached to the N-terminus of the deubiquitinating enzyme.

12. The expression system according to claim 10, characterized in that, The first ligand portion comprises a retinoic acid ligand structure configured to covalently bind to the tag protein; and / or The second ligand portion comprises a benzylguanine ligand structure configured to covalently bind to the binding protein.

13. The expression system according to claim 12, characterized in that, The bifunctional coupling molecule is BG-ATRA, and its chemical structure is shown below: 。 14. The expression system according to claim 12 or 13, characterized in that, The target protein is selected from one or more of the following: cytokines, growth factors, secretory receptor proteins, extracellular matrix proteins, hormones, enzymes, coagulation factors, antibodies or fusion proteins, and vaccine antigens.

15. The expression system according to claim 14, characterized in that, The mammalian cells are selected from one or more of the following: CHO cells, HEK293 cells, NSO cells, Vero cells, BHK-21 cells, MDCK cells, COS-7 cells, HeLa cells, SKBR3 cells, BXPC3 cells, Jurkat cells, MO7e cells, and B16-F1 cells.

16. An engineered cell line comprising the expression system of any one of claims 1-15, wherein the engineered cell line stably expresses the first fusion protein and the second fusion protein, and wherein the engineered cell line is configured to enhance the expression level and / or stability of the target protein in the presence of a bifunctional conjugate molecule.

17. The expression system according to any one of claims 1-15 or the engineered cell line according to claim 16, used in at least one of the following applications: (a) Inhibit the ubiquitin-proteasome system-mediated degradation of target proteins in mammalian cells; (b) Increase the expression level and / or stability of target proteins in mammalian cells; (c) Prepare reagents or kits for improving the expression level and / or stability of target proteins in mammalian cells.

18. A method for increasing the expression level and / or stability of a target protein in mammalian cells, characterized in that, The method includes: The expression system of any one of claims 1-15 is transferred into mammalian cells and cultured under suitable conditions, or the engineered cell line of claim 16 is cultured under suitable conditions to inhibit the ubiquitin-proteasome system-mediated degradation of the target protein in mammalian cells, thereby increasing the expression level and / or stability of the target protein in mammalian cells.

19. The method according to claim 18, characterized in that, The method further includes the steps of recovering and / or purifying the target protein and removing the tag protein after expression.

Images