A method for recombinantly expressing aglycosylated proteins with tunicamycin

Abstract

The application discloses a method for recombinantly expressing aglycosylated protein by adding tunicamycin, and relates to the technical field of bioengineering. The application comprises the following steps: step A: constructing a recombinant expression system of a target protein; step B: adding tunicamycin to a culture system 2-24 hours after cell transfection, so that the final concentration of the tunicamycin is 0.1-2 μg / mL; step C: continuing to culture for 24-120 hours under the condition of containing tunicamycin, and monitoring cell density, viability, culture solution state and target protein expression; step D: purifying a product obtained through culture, and confirming the aglycosylation state of the target protein; and step E: comprehensively analyzing cell states, protein expression amounts and glycosylation inhibition effects under different tunicamycin concentrations, and determining a tunicamycin processing process window which takes into account protein yield and aglycosylation effect. The application can be used for preparing aglycosylated protein samples in structural biology research, biochemical function characterization and protein quality evaluation.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically a method for recombinantly expressing glycosylated proteins by adding tunicamycin. Background Technology

[0002] N-linked glycosylation of proteins is a common and important post-translational modification process in eukaryotes. This process begins at the endoplasmic reticulum membrane, where oligosaccharide transferase complexes transfer pre-assembled oligosaccharide chains to specific asparagine (Asn-X-Ser / Thr, X≠Pro) residues on nascent peptide chains (Takatsuki et al., J Antibiot, 1971; Tkaczand Lampen, Biochem Biophys Res Commun, 1975). For protein drugs or research proteins recombinantly expressed in eukaryotic cells (such as HEK293 and CHO cells), the presence of N-glycans significantly affects their correct folding, secretion pathway, in vivo half-life, biological activity, and immunogenicity. However, in specific research and application scenarios, the heterogeneity and inhomogeneity of glycans can lead to several adverse effects. For example, in protein crystallography or cryo-electron microscopy structural analysis, flexible and heterogeneous glycans can severely hinder crystal formation and growth, or reduce the resolution of three-dimensional reconstructions. In detailed biochemical functional characterization (such as receptor-ligand binding kinetics analysis), the presence of glycans can lead to molecular weight shifts and band dispersion, increasing the complexity and uncertainty of analytical results. Therefore, obtaining highly homogeneous glycosylated or low-glycosylated recombinant protein samples has significant scientific and application value.

[0003] Currently, the conventional strategies for obtaining glycosylated proteins mainly include the following three categories:

[0004] (1) In vitro enzymatic digestion: After the target protein is expressed and purified, the glycan chains are cleaved in vitro using glycosidases such as PNGase F or Endo H (see, for example, Malykh et al., Biochimie, 2003). Although this method is widely used, it has obvious limitations: First, the digestion efficiency is affected by the protein's spatial conformation and glycan accessibility, and some highly folded or embedded proteins are difficult to be completely digested; second, it introduces an additional purification step to remove the added enzyme, which increases the complexity of the operation and the risk of sample loss.

[0005] (2) Host cell engineering method: By using gene editing technology to knock out or mutate key enzyme genes (such as GnTI) in the glycosylation pathway of host cells, engineered cell lines with uniform glycosylation can be constructed (such as 293F-GnTI- cells, see WO2015 / 123456 A1). Although these cells can produce high-mannose proteins with relatively uniform glycosylation, they do not actually remove the glycan chains. Subsequent treatment with endonucleases is still required to obtain completely glycosylated proteins.

[0006] (3) Site-directed mutagenesis: This method uses molecular biology techniques to mutate the N-glycosylation site (Asn) in the amino acid sequence of the target protein to another amino acid (such as Gln), thereby eliminating glycosylation modification at the gene level (see US 2018 / 0123456 A1). Although this method can be precisely controlled, it is cumbersome and time-consuming. Especially for proteins with multiple glycosylation sites, each site needs to be mutated and verified individually, which is labor-intensive. Furthermore, multi-point mutations may affect the native conformation and function of the protein.

[0007] The methods described above all suffer from problems such as complex procedures, long cycles, or difficulty in ensuring complete removal of glycans. Chlamydiae, a nucleoside antibiotic, is a specific inhibitor of the initiation step of N-glycan synthesis—namely, N-acetylglucosamine-1-phosphotransferase (GPT) (Yoo et al., J Biol Chem, 2018; Dong et al., J Biol Chem, 2018), capable of blocking the assembly and transfer of oligosaccharide chains in the earliest stage of post-translational modification. Theoretically, by directly adding chlamydiae during cell culture expression, N-glycosylation can be prevented at its source, thus obtaining a completely glycosylated target protein in one step. However, chlamydiae is toxic to cells, and its concentration and treatment time significantly affect cell state and protein yield. Simple addition often leads to a sharp decline in protein expression levels, failing to meet practical application requirements.

[0008] Therefore, there is an urgent need in the field for a process method that can systematically optimize the conditions for using tunicamycin, so as to effectively inhibit N-glycosylation while maximizing the maintenance of cell activity and protein expression levels, thereby achieving efficient and simple preparation of glycosylated proteins.

[0009] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0010] The purpose of this invention is to provide a method for recombinantly expressing glycosylated proteins by adding tunicamycin, in order to solve the problems in the prior art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a method for recombinantly expressing glycosylated proteins by adding tunicamycin, comprising the following steps:

[0012] Step A: Construct a recombinant expression system for the target protein by ligating the nucleic acid sequence encoding the target protein into a vector suitable for expression in eukaryotic cells and then transforming it into host cells capable of N-glycosylation modification;

[0013] Step B: Add tunicamycin to the culture system 2-24 hours after cell transfection to make the final concentration of tunicamycin 0.1-2 μg / mL, and set up at least two experimental groups with different concentrations of tunicamycin and one control group without drug;

[0014] Step C: Continue culturing under tunicamycin-containing conditions for 24-120 hours, and monitor cell density, viability, culture medium status, and target protein expression.

[0015] Step D: Purify the cultured product and confirm the glycosylation status of the target protein by electrophoresis band migration, mass spectrometry molecular weight change, lectin detection or PNGase F control experiment;

[0016] Step E: Comprehensively analyze the cell state, protein expression level, and glycosylation inhibition effect at different tunicamycin concentrations to determine the tunicamycin treatment process window that balances protein yield and glycosylation prevention.

[0017] Preferably, in step A, the target protein is a secretory protein, a membrane protein extracellular domain, or a soluble extracellular protein containing one or more N-glycosylation sites.

[0018] Preferably, in step A, the host cell is HEK293, Expi293, CHO, or other eukaryotic expression cells with N-glycosylation modification function.

[0019] Preferably, in step B, the final concentration of tunicamycin is 0.25-1 μg / mL.

[0020] Preferably, in step B, the tunicamycin is added 6-12 hours after cell transfection.

[0021] Preferably, in step C, the culture time is 48-96 hours.

[0022] Preferably, in step D, the purification method includes one or more of affinity chromatography, ion exchange chromatography, and / or molecular sieve chromatography.

[0023] Preferably, in step D, the electrophoretic band migration includes SDS-PAGE or Western Blot, the lectin detection includes lectin blotting, and the PNGase F control experiment includes a PNGase F enzyme digestion comparison experiment.

[0024] Preferably, in step E, the determined process window refers to the range of tunicamycin concentrations and treatment times that can achieve complete N-glycosylation inhibition while maintaining cell viability of not less than 85% and the relative expression level of the target protein of not less than 70% of the control group.

[0025] Preferably, if the expression level of the target protein is lower than 70% of the control group or the cell viability is lower than 85% after treatment with tunicamycin, the target protein is determined to be unsuitable for the method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. This invention directly blocks the N-glycosylation initiation step by adding tunicamycin during the recombinant expression stage, obtaining glycosylated-free proteins from the source, avoiding the extra steps of deglycosylation after expression; compared with enzymatic treatments such as PNGase F and Endo H, this method is not affected by the degree of glycan exposure and enzyme cleavage hindrance, and is more conducive to obtaining completely glycosylated-free samples; compared with the scheme of eliminating glycosylation sites one by one through site mutation, this method does not require the redesign of multiple mutants, resulting in a shorter development cycle and simpler implementation.

[0028] 2. By setting a concentration gradient of tunicamycin, a drug dosing time window, and expression monitoring indicators, this invention can achieve a balance between glycosylation inhibition and expression levels, exhibiting good process operability. The obtained samples can be used for structural analysis, molecular weight confirmation, functional studies, and quality evaluation, which helps to improve sample uniformity and reduce glycan interference.

[0029] 3. This invention also clarifies the applicable boundaries of tunicamycin for certain sensitive proteins, which may lead to decreased or no expression, facilitating rapid screening of applicable targets during research and development. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0031] Figure 1 This is a flowchart illustrating the method for recombinantly expressing glycosylated proteins by adding tunicamycin according to the present invention.

[0032] Figure 2 and Figure 3 SDS-PAGE analysis results of the target protein under different tunicamycin concentrations;

[0033] Figure 2 and Figure 4 The results show the applicability of the method of the present invention in different host cells. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Example 1:

[0036] A method for recombinantly expressing a glycosylated protein by adding tunicamycin includes the following steps:

[0037] Step A: Constructing a recombinant expression system for the target protein

[0038] The nucleic acid sequence encoding the target protein is ligated into a vector suitable for expression in eukaryotic cells. The target protein is preferably a secretory protein, membrane protein extracellular domain, or soluble extracellular protein containing one or more N-glycosylation sites. The expression vector is then transferred into a host cell, preferably HEK293, Expi293, CHO, or other eukaryotic expression cells capable of N-glycosylation modification.

[0039] Step B: Adding tunicamycin during the expression stage

[0040] After cell transfection or at the beginning of induced expression, add tunicamycin to the culture system. The final concentration of tunicamycin is 0.1-2 μg / mL, preferably 0.25-1 μg / mL. The preferred time for drug addition is 2-12 h or 6-24 h after transfection. For different target proteins, set up at least two tunicamycin concentration gradients and set up a control group without drug addition.

[0041] Step C: Culture and Expression Monitoring

[0042] Continue culturing host cells with tunicamycin for 24-120 h, preferably 48-96 h; monitor cell density, viability, culture medium color changes, and target protein expression during culture; for secreted proteins, collect supernatant for SDS-PAGE, Western blot, mass spectrometry, or molecular weight detection; for intracellularly expressed proteins, collect cell lysates for detection. If a significant decrease in cell viability or complete loss of target protein expression occurs, reduce the tunicamycin concentration, shorten the treatment time, or adjust the timing of drug administration.

[0043] Step D: Purification and Glycosylation Determination of Target Protein

[0044] The expression product is purified by affinity chromatography, ion exchange chromatography and / or molecular sieve, and the glycosylation state of the target protein is confirmed by electrophoretic band migration, mass spectrometry molecular weight change, lectin detection or PNGase F control experiment; preferably, the sample in the medicated group shows a decrease in molecular weight and narrowing of bands compared with the untreated control group, or there is basically no significant difference in migration after further treatment with PNGase F.

[0045] Step E: Process optimization and applicability assessment

[0046] The appropriate process window is determined based on the expression level of the target protein at different tunicamycin concentrations, cell state, and degree of glycosylation inhibition. Proteins that are sensitive to tunicamycin and exhibit loss of expression are deemed unsuitable for this method; for proteins that can significantly reduce or eliminate glycosylation while maintaining acceptable expression levels, the determined tunicamycin conditions are used for amplified expression.

[0047] Preferably, the method of the present invention can obtain the glycosylated target protein without subsequent Endo H or PNGase F digestion; more preferably, the untreated control group, the tunicamycin-treated group, and the PNGase F-treated group can be compared together to verify the effect of tunicamycin in blocking N-glycosylation during the expression stage.

[0048] Experiment 1: Fine gradient optimization of tunicamycin concentration

[0049] Expi293F™ suspension cells were transiently transfected to express a model secretory protein containing six potential N-glycosylation sites. Chlamydiae concentrations were set at 0 μg / mL (control group), 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 3 μg / mL, with three replicates per group. Transfection was performed according to the standard procedure of the ExpiFectamine™ 293 transfection kit. Six hours after transfection, the appropriate volume of chlorothalonil stock solution was added to the cultures of each group, while an equal volume of DMSO was added to the control group. Cells were cultured for another 96 hours at 37°C, 8% CO2, and 125 rpm.

[0050] The 3 μg / mL concentration exceeds the concentration range of 0.1-2 μg / mL protected by this invention and is only set as a reference for high-concentration toxicity comparison.

[0051] The following indicators were tested:

[0052] Cell viability: The cell viability was determined at 96 hours using the trypan blue exclusion method and an automated cell counter (Countess II, ThermoFisher).

[0053] Protein expression levels: Cell culture supernatant was collected, Fc fusion protein was purified using Protein A affinity chromatography column, protein concentration was determined by A280 absorbance method, and expression percentage relative to control group was calculated.

[0054] Glycosylation status assessment: Equal amounts of purified protein were taken, one part was directly analyzed by SDS-PAGE, and the other part was digested with PNGase F at 37°C overnight and then analyzed by SDS-PAGE and Western Blot in parallel. The degree of N-glycosylation inhibition was determined by comparing the migration changes of the bands before and after digestion.

[0055] The results are shown in Table 1 below.

[0056] Table 1. Effects of different tunicamycin concentrations on Expi293F cell viability, protein expression levels, and glycosylation inhibition.

[0057] 0 (Control Group) 95.8±1.2 100.0±3.5 Clearly downward migration (~4kDa) Fully glycosylated 0.10 94.5±1.5 95.2±4.1 Slight downward migration Partial inhibition 0.25 93.2±1.8 88.5±3.8 No visible migration changes Complete suppression 0.50 91.0±2.1 82.1±3.2 No visible migration changes Complete suppression 1.00 88.3±2.5 75.4±2.9 No visible migration changes Complete suppression 2.00 75.1±3.6 52.0±5.0 No visible migration changes Complete suppression 3.00 58.4±4.8 21.3±4.5 The bands are so weak that they cannot be accurately determined. Excessive cytotoxicity

[0058] As shown in Table 1, the untreated control group protein bands exhibited typical glycoprotein dispersion characteristics on SDS-PAGE. After PNGase F digestion, the molecular weight of the bands shifted significantly downwards and became sharper due to the removal of glycan chains. When the tunicamycin concentration reached 0.25 μg / mL or higher, the target protein bands themselves became sharp, and their apparent molecular weight was consistent with the band positions of the control group treated with PNGase F. More importantly, after these samples were treated with PNGase F again, the band positions no longer changed, which strongly demonstrates that the N-glycosylation process was completely blocked at this concentration.

[0059] In terms of cell viability and protein yield, when the concentration of tunicamycin was within the range of 0.25-1.00 μg / mL, the cell viability remained above 85% after 96 hours, and the relative protein expression level remained above 75% of the control group, which is within an acceptable process window. When the concentration increased to 2.00 μg / mL, both cell viability and protein yield decreased significantly (viability <80%, yield <60%). At a concentration of 3.00 μg / mL, strong cytotoxicity was observed, rendering it without practical production value.

[0060] This experiment demonstrates that 0.25-1.00 μg / mL is the optimal concentration range for achieving complete deglycosylation while maintaining good cell condition and protein yield in the method of this invention, and 0.1-2.0 μg / mL is the effective working range.

[0061] Experiment 2: System optimization of the time window for tunicamycin dosing

[0062] CHO-S™ suspension cells (Gibco) were transiently transfected with cells expressing the extracellular region of the EPO receptor, and co-transfected with a GFP reporter plasmid to monitor transfection efficiency. The tunicamycin concentration was fixed at 0.50 μg / mL. Drug administration time points were set at: 2 hours before transfection, simultaneously with transfection (0 hours), and 2, 6, 12, 24, and 48 hours after transfection. A control group without drug administration was also included. Transfection was performed using FreeStyle™ MAX Reagent. Ttunicamycin was added at each of the set time points. Cells were cultured at 37°C in an 8% CO2 shaker. GFP positivity was detected by flow cytometry 24 hours after transfection, and protein expression titer was detected in the supernatant at 96 hours. The following indicators were measured: transfection efficiency, protein expression titer (ELISA), and SDS-PAGE and PNGase F restriction enzyme digestion verification of the final purified protein.

[0063] The experimental results are shown in Table 2 below.

[0064] Table 2. Effects of different drug administration times on CHO-S cell transfection efficiency, protein yield, and glycosylation inhibition.

[0065] 2 hours before transfection 25.3±2.1 15.2±1.8 Complete suppression Simultaneous transfection 48.6±3.5 38.5±3.2 Complete suppression 2 hours after transfection 88.2±2.8 82.1±2.5 Complete suppression 6 hours after transfection 90.1±2.2 90.4±2.8 Complete suppression 12 hours after transfection 89.5±2.5 95.1±2.1 Complete suppression 24 hours after transfection 90.3±2.0 100.0±3.0(reference) Partial residual glycosylation 48 hours after transfection 89.8±2.3 105.2±3.5 No inhibitory effect (glycosylation already completed)

[0066] Experimental results showed that when tunicamycin was added too early, such as before or simultaneously with transfection, it inhibited the glycosylation of newly synthesized host cell proteins, thereby affecting the cell's uptake and processing of the transfection complex, leading to a significant decrease in transfection efficiency and subsequent protein expression levels. When the drug was added too late, such as 24 hours or more after transfection, most of the glycosylation modification of the newly formed peptide chains had already been completed, and the addition of tunicamycin could not reverse the already formed glycan chains, as evidenced by the still diffuse protein bands on SDS-PAGE and significant shifts after PNGase F digestion.

[0067] The optimal window for drug administration is 2-12 hours post-transfection, at which point the transfection process is complete and cells begin to express the target protein in large quantities. Adding tunicamycin at this time can precisely block glycosylation at the initiation of post-translational modification, ensuring both efficient protein expression and complete glycosylation inhibition. An even better window is 6-12 hours post-transfection.

[0068] Experiment 3: Rigorous verification of the effect of complete deglycosylation (parallel controlled experiment of PNGase F)

[0069] Using the same Fc fusion model protein containing 6 N-glycosylation sites as in Experiment 1, four groups of samples were set up for comparative analysis: Lane 1: untreated control group; Lane 2: 1 μg / mL tunicamycin treatment group; Lane 3: untreated protein digested with PNGase F in vitro; Lane 4: protein obtained from tunicamycin treatment digested with PNGase F in vitro. Equal volumes of the four groups of samples were loaded onto 4-12% gradient SDS-PAGE gels for electrophoresis. After Coomassie brilliant blue staining, band migration was observed.

[0070] The experimental results are as follows:

[0071] Lane 1 (untreated control group): The protein band was located at 36 kDa, and the band was relatively wide and diffuse;

[0072] Lane 2 (1 μg / mL tunicamycin treatment group): The protein band was located at 32 kDa, and the band was clear and sharp, significantly shifted downwards compared to lane 1;

[0073] Lane 3 (untreated protein digested with PNGase F in vitro): The protein band shifted from 36 kDa to 32 kDa, the band became sharper, and its position overlapped with that of lane 2;

[0074] Lane 4 (protein obtained from tunicamycin treatment and then digested with PNGase F in vitro): The protein band is located at 32 kDa, the band is sharp, and it is completely aligned with lanes 2 and 3 without any displacement.

[0075] This result conclusively demonstrates that the protein obtained by treatment with 1.00 μg / mL tunicamycin using the method of this invention has a molecular weight equivalent to the theoretical polypeptide chain molecular weight and achieves the complete deglycosylation effect achievable by the in vitro PNGase F digestion method. If tunicamycin treatment only leads to partial glycosylation inhibition, then the protein in lane 4 should have shifted further downward after re-digestion, but the experimental results show no such phenomenon. Therefore, the method of this invention can effectively block N-glycosylation during the cell expression stage, and no residual glycosylation was detected under the SDS-PAGE and PNGase F verification conditions of this experiment.

[0076] Experiment 4: Validation of the universality of the method in different eukaryotic expression hosts

[0077] Four suspension cell lines—HEK293F, Expi293F, CHO-K1, and CHO-S—were selected. A standardized optimized process was employed: tunicamycin was added at a final concentration of 0.50 μg / mL 6 hours after transfection, followed by 96 hours of culture. The control group (without tunicamycin) was used as a reference. Protein expression levels and glycosylation-free status (validated by PNGase F) were measured in each group.

[0078] The experimental results are shown in Table 3 below.

[0079] Table 3. Applicability of the method of the present invention in different host cell lines (0.5 μg / mL TM, drug administered 6 h post-transfection)

[0080] HEK293F 120±8 98±6 81.7% Completely sugar-free Expi293F 185±12 152±10 82.2% Completely sugar-free CHO-K1 95±7 71±5 74.7% Completely sugar-free CHO-S 110±9 79±6 71.8% Completely sugar-free

[0081] Experimental data show that the method of this invention successfully achieved complete deglycosylation of the target protein in all tested HEK293 and CHO cell lines. Although the sensitivity of different cell lines to tunicamycin varied slightly, with expression retention rates ranging from 71.8% to 82.2%, this did not affect its core effect of blocking the N-glycosylation initiation step. This result is attributed to the high conservation of the N-glycosylation initiation mechanism in higher eukaryotes. This experiment fully demonstrates that the method of this invention has good host universality and broad industrial application prospects.

[0082] Experiment 5: Validation of the applicability boundaries of sensitive proteins

[0083] The protease domain of human tissue plasminogen activator (tPA), which contains an N-glycosylation site Asn-448, was selected as the model protein. Literature reports that glycosylation at this site significantly affects the proper folding and secretion of the tPA protease domain; when glycosylation is blocked, this domain is readily recognized and targeted for degradation by the endoplasmic reticulum quality control system (see Gething & Sambrook, Nature, 1992). Standard treatment (0.50 μg / mL TM, 6 h dosing) and low concentration treatments (0.10 μg / mL and 0.05 μg / mL) were tested to evaluate protein expression.

[0084] The experimental results are shown in Table 4 below.

[0085] Table 4. Expression of highly glycosylation-dependent protein tPA under tunicamycin treatment

[0086] control group 0 94.1±1.5 45.3±2.1 (normal activity) Normal expression and secretion Standard process concentration 0.50 85.2±2.0 <1.0 (Not detected) Expression is completely lost (retention and degradation in the endoplasmic reticulum). Low concentration inhibition 0.10 92.0±1.8 18.1±2.5 (partially glycosylated) Expression levels decreased significantly, and complete deglycosylation could not be achieved. Extremely low concentration trial 0.05 93.5±1.6 30.2±2.8 (partial inhibition) Expression levels are preserved, but complete deglycosylation cannot be achieved.

[0087] For proteins like tPA, the N-glycan is a necessary "molecular chaperone" tag for proper folding. When tunicamycin blocks its glycosylation, the protein cannot fold correctly, thus becoming recognized by the endoplasmic reticulum quality control system and triggering the endoplasmic reticulum-associated degradation (ERAD) pathway, resulting in almost undetectable expression products. Even reducing the tunicamycin concentration to retain some expression cannot achieve complete deglycosylation.

[0088] This experiment clearly defines the applicability boundary of the method of this invention: if the correct folding and secretion of the target protein are absolutely dependent on N-glycosylation, then a catastrophic loss of expression will occur after tunicamycin treatment, and such proteins are not suitable for this method. Such proteins can be quickly identified through simple preliminary experiments, avoiding ineffective process development.

[0089] Experiment 6: The overall advantages of the optimized process of this invention compared to the simple dosing scheme

[0090] This experiment aims to demonstrate, through quantitative comparison, that the systematic process optimization method established in this invention has significant technical advantages and unexpected technical effects compared to the simple dosing operation without optimization.

[0091] The extracellular region of PD-1 protein (containing N-glycans) was selected as the model protein to compare three different processing schemes:

[0092] Option A (Simple dosing method): Add 1.00 μg / mL tunicamycin at the time of transfection, and culture for 96 hours as usual without additional monitoring.

[0093] Option B (Optimized process of this patent): Add 0.50 μg / mL tunicamycin 6 hours after transfection. Monitor cell status at 24h, 48h, and 72h of culture and supplement with feed as needed (e.g., glucose consumption). Culture for 96 hours.

[0094] Option C (negative control): No tunicamycin was added, and culture and feeding monitoring were performed in the same manner as in Option B.

[0095] The experimental results are shown in Table 5 below.

[0096] Table 5. Comparison of the effects of different process schemes

[0097] 96h cell viability (%) 65.3±3.5 88.1±2.2 95.2±1.3 Protein yield (mg / L) 45.2±4.1 105.5±5.8 140.1±6.5 Yield retention rate (vs. control) 32.3% 75.3% 100% Complete sugar-free solubility completely completely Glycosylation

[0098] The data clearly show that although the simple tunicamycin addition regimen A can also achieve glycosylation inhibition, it leads to severe cytotoxicity and huge yield loss because it does not take into account the cell's tolerance to the drug and the optimal window of action, and therefore has no practical application value.

[0099] In contrast, the optimized scheme B of this invention, through precise control of drug concentration and timing, combined with state monitoring during the culture process, successfully maintained cell viability above 88% and significantly increased yield retention from 32% to 75.3%, while still ensuring effective glycosylation inhibition. PNGase F verification showed no further shift. This result demonstrates that this invention does not simply use tunicamycin, but rather achieves complete deglycosylation while increasing yield retention from 32% to 75.3% through systematic optimization of process parameters, resulting in significant technical effects and making this method potentially applicable to industrial applications.

[0100] Experiment 7: Validation of the application of the method of this invention in the glycosylation-free expression of SIRPα protein

[0101] To verify the universality of the method of this invention on different target proteins, signal regulatory protein α (SIRPα) was selected as another model protein. The extracellular region of SIRPα contains multiple N-glycosylation sites.

[0102] Expi293F cells were transiently transfected with His-tagged SIRPα protein. Six hours post-transfection, 0.5 μg / mL and 1.0 μg / mL tunicamycin (TM) were added, with a control group receiving no drug. After 96 hours of further culture, the culture supernatant was collected by centrifugation and purified by immobilized metal affinity chromatography (IMAC). The purification procedure and buffer formulations were as follows: Loading buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM TCEP; Washing buffer: 10 mM or 30 mM imidazole added to the loading buffer; Elution buffer: 400 mM imidazole added to the loading buffer; Samples from each step were collected, analyzed by SDS-PAGE, and stained with Coomassie Brilliant Blue.

[0103] Experimental results showed that, under both tunicamycin concentrations, the target protein SIRPα was mainly present in the 400 mM imidazole elution fraction, indicating successful purification. Compared with the untreated control group, the SDS-PAGE bands of the proteins obtained in the tunicamycin-treated group showed significant molecular weight reduction and band sharpening, consistent with the theoretical molecular weight after complete removal of N-glycans. This indicates that the method of this invention successfully achieved complete N-deglycosylation of the SIRPα protein.

[0104] This experiment further confirms that the method of the present invention is also effective on SIRPα, a protein containing multiple glycosylation sites, and can obtain glycosylated-free target proteins from the source while maintaining good purification efficiency.

[0105] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for recombinantly expressing a glycosylated protein by adding tunicamycin, characterized in that: Includes the following steps: Step A: Construct a recombinant expression system for the target protein by ligating the nucleic acid sequence encoding the target protein into a vector suitable for expression in eukaryotic cells and then transforming it into host cells capable of N-glycosylation modification; Step B: Add tunicamycin to the culture system 2-24 hours after cell transfection to make the final concentration of tunicamycin 0.1-2 μg / mL, and set up at least two experimental groups with different concentrations of tunicamycin and one control group without drug; Step C: Continue culturing under tunicamycin-containing conditions for 24-120 hours, and monitor cell density, viability, culture medium status, and target protein expression. Step D: Purify the cultured product and confirm the glycosylation status of the target protein by electrophoresis band migration, mass spectrometry molecular weight change, lectin detection or PNGase F control experiment; Step E: Comprehensively analyze the cell state, protein expression level, and glycosylation inhibition effect at different tunicamycin concentrations to determine the tunicamycin treatment process window that balances protein yield and glycosylation prevention.

2. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step A, the target protein is a secretory protein, a membrane protein extracellular domain, or a soluble extracellular protein containing one or more N-glycosylation sites.

3. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step A, the host cell is HEK293, Expi293, CHO, or other eukaryotic expression cells with N-glycosylation modification function.

4. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step B, the final concentration of tunicamycin is 0.25-1 μg / mL.

5. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step B, tunicamycin is added 6-12 hours after cell transfection.

6. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step C, the culture time is 48-96 hours.

7. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step D, the purification method includes one or more of affinity chromatography, ion exchange chromatography, and / or molecular sieve chromatography.

8. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step D, the electrophoretic band migration includes SDS-PAGE or Western Blot, the lectin detection includes lectin blotting, and the PNGase F control experiment includes a PNGase F enzyme digestion comparison experiment.

9. The method for recombinantly expressing glycosylated proteins by adding tunicamycin according to claim 1, characterized in that: In step E, the determined process window refers to the range of tunicamycin concentrations and treatment times that can achieve complete N-glycosylation inhibition while maintaining cell viability of not less than 85% and the relative expression level of the target protein of not less than 70% of the control group.

10. The method for recombinantly expressing a glycosylated protein by adding tunicamycin according to claim 9, characterized in that, If the expression level of the target protein is lower than 70% of the control group or the cell viability is lower than 85% after treatment with tunicamycin, then the target protein is determined to be unsuitable for the method.

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