A label-free purification method of influenza a virus neuraminidase, recombinant protein and application

Abstract

The application discloses a label-free purification method of influenza virus neuraminidase, a recombinant protein and application. The application provides a set of mild, stable and repeatable purification route aiming at the technical problems of difficult purification, low purity and easy conformation change of the label-free neuraminidase. The supernatant of the label-free neuraminidase expressed by CHO-k1 suspension cells is concentrated and liquid changed through ultrafiltration, and then is sequentially subjected to multistage purification through hydrophobic interaction chromatography, anion exchange chromatography and molecular sieve chromatography, so that the recombinant neuraminidase protein with a purity of greater than or equal to 94% is finally obtained. The obtained protein is a label-free natural tetramer conformation, is stable in the range of pH 5.0-9.0 and has neuraminidase activity, and can be directly used for efficient screening of influenza virus neuraminidase inhibitors. The process condition of the application is mild, the repeatability is good, the affinity label is not needed, the natural structure of the protein is retained to the maximum extent, and the application is suitable for use in scientific research and drug screening platform.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and antiviral technology, specifically relating to a method for eukaryotic expression, label-free purification, and application of influenza A virus neuraminidase (NA). Background Technology

[0002] Influenza A virus is characterized by rapid transmission, high mutation rate, and wide host range, easily triggering seasonal epidemics and even pandemics, posing a continuous threat to public health security. Neuraminidase (NA) is a key envelope glycoprotein on the surface of influenza A virus, capable of specifically catalyzing the hydrolysis of sialic acid residues on the surface of host cells and the virus, promoting the release and spread of progeny viruses from infected cells, and is one of the most important targets in the development of anti-influenza drugs. High-purity, native-conformation recombinant NA protein plays an irreplaceable role in high-throughput screening of neuraminidase inhibitors, enzymatic mechanism research, drug resistance mechanism analysis, and structural biology elucidation.

[0003] Currently, recombinant natural natriuretic (NA) proteins are mostly prepared using prokaryotic or eukaryotic expression systems. Prokaryotic expression systems lack glycosylation modification and a proper folding environment, leading to the easy formation of inclusion bodies in the product. After refolding, the protein exhibits low activity and a conformation that differs significantly from the native state, making it difficult to meet the requirements for drug screening. While eukaryotic expression systems can yield NA proteins with structures closer to the native state, affinity tags such as His and GST are often introduced to facilitate purification. These tags alter the protein's surface charge and spatial conformation, interfering with the active site microenvironment and affecting the accuracy of inhibitor binding evaluation. Therefore, tag-free expression better reflects the true physiological state.

[0004] However, tag-free recombinant NA proteins lack specific affinity purification sites, possess complex physicochemical properties, and exist in natural tetramer form. Existing purification processes generally suffer from low separation efficiency, incomplete impurity removal, low protein recovery rates, and purity levels that fail to meet research requirements. Conventional single-chromatographic methods such as ion exchange and gel filtration are insufficient for effective purification, while multi-step purification processes are cumbersome, unstable, and have low reproducibility, hindering the acquisition and application of high-quality tag-free NA proteins. Therefore, developing a mild, efficient, stable, and affinity-free NA protein purification method to obtain high-purity, highly active, native-conformation tag-free NA proteins has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] This invention provides a mild, efficient, stable, and reproducible tag-free purification method for influenza A virus neuraminidase. Through a combined purification strategy of ultrafiltration concentration, hydrophobic interaction chromatography, anion exchange chromatography, and molecular sieve chromatography, high-purity, natural tetrameric conformation, and high enzyme activity recombinant neuraminidase protein can be stably obtained without the use of affinity tags. This provides high-quality protein raw materials for screening influenza virus neuraminidase inhibitors, studying enzymatic mechanisms, and performing structural biological analysis.

[0006] The objective of this application is achieved through the following technical solution:

[0007] A label-free purification method for influenza A virus neuraminidase includes the following steps:

[0008] The supernatant of unlabeled neuraminidase expressed in CHO-k1 cells was concentrated by ultrafiltration using an ultrafiltration device with a molecular weight cutoff of 80kDa to 120kDa and the medium was changed. The concentrate was then purified by hydrophobic interaction chromatography, anion exchange chromatography and molecular sieve chromatography to obtain recombinant neuraminidase protein with a purity ≥94%.

[0009] Preferably, the ultrafiltration concentration uses a hollow fiber column, the buffer solution is 10mM to 30mM phosphate buffer containing 0.4M to 0.6M ammonium sulfate, and the pH is 7.5 to 8.5; the feed flow rate is controlled at 150mL / min to 300mL / min and the transmembrane pressure is 0.3bar to 0.8bar during the ultrafiltration process.

[0010] Preferably, the hydrophobic interaction chromatography uses butyl hydrophobic packing material, the equilibration solution is 10mM to 30mM phosphate buffer containing 0.4M to 0.6M ammonium sulfate, pH 7.5 to 8.5; step elution is used, and the 60% to 100% low salt gradient elution peak is collected as the target protein component.

[0011] Preferably, the anion exchange chromatography uses a strongly basic anion exchange packing material, the equilibration solution is 10mM to 30mM phosphate buffer, pH 7.5 to 8.5; linear salt gradient elution is used, and the elution fraction containing neuraminidase is collected.

[0012] Preferably, the molecular sieve chromatography uses dextran or agarose gel packing material, and the mobile phase is 10mM to 30mM phosphate buffer at pH 7.0 to 8.0; the elution peak of the target protein is collected to obtain high-purity neuraminidase protein.

[0013] This invention also provides a recombinant NA protein and its application in screening influenza virus neuraminidase inhibitors.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention provides a complete and mild tag-free purification route that achieves efficient separation of neuraminidase without affinity tags, maximizing the preservation of the protein's native tetrameric conformation and enzyme activity, and avoiding tag interference with structure and activity. The process conditions of this invention are all within a controllable range, exhibiting good reproducibility, high stability, and easy scale-up, solving the technical challenges of high difficulty, low purity, and poor recovery rates in existing tag-free protein purification methods. This invention uses a mild buffer system and chromatographic conditions throughout the process, without the use of denaturing agents, strong acids, or strong bases, effectively maintaining the structural stability of the protein within the pH range of 5.0–9.0, significantly improving the applicability and reliability of the protein in drug screening and structural analysis. The tag-free, high-purity, native conformation NA protein obtained by this invention can be directly used for neuraminidase inhibitor screening, drug resistance mechanism research, enzymatic analysis, and structural biology research, providing a stable and efficient protein tool for anti-influenza drug development. The method of this invention is simple to operate and highly versatile, suitable for the direct purification of CHO-k1 eukaryotic expression supernatant, requiring no complex pretreatment, reducing experimental costs and equipment requirements, and is suitable for large-scale application in scientific research and drug screening platforms. Attached Figure Description

[0016] Figure 1 Determination of in vitro enzyme activity at different concentrations of G13 .

[0017] Figure 2 This is a schematic diagram of the active sites of (a) wild-type and (b) mutant NA proteins (obtained through homology modeling). Note: The active site regions are marked with purple boxes.

[0018] Figure 3 The quality assessment of the model prediction model for (a) wild-type and (b) mutant NA protein structure is shown in the figure. Note: The three color bands in the figure are divided according to the absolute value of the Z-score, representing the confidence interval of the model quality. Dark area: high quality; gray area: medium quality; light area: low quality.

[0019] Figure 4 The diagram shows the Laplace diagrams of the structure models of (a) wild-type and (b) mutant NA proteins. Note: Darker areas indicate more stable conformations (allowed regions), while white areas indicate unstable conformations (unallowed regions). A dot falling in a different region represents an amino acid residue.

[0020] Figure 5 The binding models of (a) wild-type and (b) mutant NA proteins to HNC042 are shown.

[0021] Figure 6The diagram shows the interaction between (a) wild-type and (b) mutant NA proteins and HNC042. Note: Green rod-shaped models represent amino acid residues of NA protein; pink and yellow rod-shaped models represent HNC042; yellow dashed lines represent hydrogen bonding; and purple dashed lines represent salt bridging.

[0022] Figure 7 This is a schematic diagram of the salt bridge interaction between (a) wild-type and (b) mutant NA proteins and HNC042. Detailed Implementation

[0023] The technical solutions of the present invention will be described in detail and comprehensively below with reference to specific embodiments. It should be noted that the provided embodiments represent only a part of the present invention, and not all of it. 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.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods; unless otherwise specified, the materials and reagents used are commercially available reagents and materials, and the commercially available raw materials are used after removing insoluble matter and other impurities by general filtration methods.

[0025] 1. Construction of pMGT-PR8-NA recombinant eukaryotic expression vector

[0026] 1.1 Viral RNA Extraction

[0027] Collect influenza virus allantoic fluid and follow the instructions in the RNA extraction kit:

[0028] Add lysis buffer and proteinase K to a centrifuge tube, add viral allantoic fluid and mix well, then lyse at room temperature; add ethanol to precipitate nucleic acid, and wash, centrifuge, and dry sequentially, finally elute with elution buffer to obtain RNA.

[0029] 1.2 Reverse transcription to synthesize cDNA

[0030] Using RNA as a template, a 10 μL reaction system was prepared using a reverse transcription kit, and the reaction was carried out at 37°C for 15 min, followed by incubation at 85°C for 5 s to obtain cDNA.

[0031] 1.3NA gene amplification

[0032] Using cDNA as a template, PCR amplification was performed using NA-specific primers.

[0033] The reaction system was 25 μL, and the reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 30 s, for 32 cycles; and 72℃ final extension for 5 min.

[0034] 1.4 Agarose Gel Electrophoresis and Gel Recovery

[0035] Prepare a 1.0% agarose gel, separate the PCR products by electrophoresis, cut the target band under UV light, and purify the DNA fragments using a gel extraction kit.

[0036] 1.5 Vector digestion

[0037] The pMGT vector was double-digested with HindIII and BamHI in a 50 μL system and reacted at 37 °C for 1–2 h to completely linearize the vector. The linearized vector was recovered after verification by electrophoresis.

[0038] 1.6 Seamless Cloning for Recombinant Plasmid Construction

[0039] The purified NA fragment was mixed with the linear vector in a certain proportion, and a seamless cloning enzyme system was added. The mixture was reacted at 50°C for 30 min to obtain the recombinant plasmid pMGT-PR8-NA.

[0040] 1.7 Escherichia coli transformation and positive clone screening

[0041] The ligation product was transformed into DH5α competent cells, and after ice bath, heat shock, and recovery, the cells were plated; cultured overnight at 37°C, and single clones were selected for colony PCR identification, with positive clones sent for sequencing.

[0042] 2. Construction of CHO-k1 stable cell pool

[0043] 2.1 Cell resuscitation, passage and culture

[0044] CHO-k1 suspension cells were cultured in serum-free medium at 37°C, 5% CO2, and 140 rpm; the cell density reached 2.0 × 10⁻⁶ cells / year. 6 Passage the cells / mL at a proportional rate.

[0045] 2.2 PEI-mediated co-transfection

[0046] Collect cells in the logarithmic growth phase by centrifugation and resuspend them in transfection medium. Mix the recombinant plasmid with the transposase plasmid, add PEI transfection reagent, incubate at room temperature, add cells, and culture for 24 hours after transfection.

[0047] 2.3GS / MSX pressure screening

[0048] After transfection, cells were screened in a glutamine-free selection medium with MSX added to a final concentration of 15 μM or 25 μM for 12 consecutive days.

[0049] MSX was added every 3 days, and cell density and viability were monitored to obtain a stable expression cell pool.

[0050] 3. Expression of tagless NA protein

[0051] The stable cell pool was set at 0.5 × 10⁻⁶. 6 Seed at a density of cells / mL for fed-batch culture; starting from day 3, feed culture medium is added daily, and glucose is added when glucose is below 4 g / L; culture is terminated when cell viability is below 85% or after 14 days; culture supernatant is centrifuged at 1400×g for 10 min at 4℃, filtered through a 0.22μm filter membrane, and used for later use.

[0052] 4. Tag-free NA protein purification (100% reproducible)

[0053] 4.1 Ultrafiltration Concentration and Liquid Replacement

[0054] Collect the clear supernatant and perform ultrafiltration using an 80kDa–120kDa hollow fiber column; the buffer solution is 10mM–30mM phosphate buffer containing 0.4M–0.6M ammonium sulfate, pH 7.5–8.5; control the flow rate at 150–300mL / min and the transmembrane pressure at 0.3–0.8bar to complete the concentration and replacement.

[0055] 4.2 Hydrophobic Interaction Chromatography (HIC)

[0056] Butyl-based hydrophobic packing material was used; the equilibration buffer was 10mM–30mM phosphate buffer containing 0.4M–0.6M ammonium sulfate, pH 7.5–8.5; after loading the sample, the sample was washed until the baseline stabilized, and step elution was performed, using 50%, 70%, and 100% low-salt buffers in sequence; the 60%–100% elution peak was collected as the target protein fraction.

[0057] 4.3 Anion Exchange Chromatography (AEX)

[0058] Strongly basic anion exchange packing material was used; the equilibration solution was 10mM–30mM phosphate buffer, pH 7.5–8.5; after loading and rinsing, linear salt gradient elution was used, and the elution fraction containing NA was collected.

[0059] 4.4 Molecular sieve chromatography (SEC)

[0060] Gel filtration was used; the mobile phase was 10mM–30mM phosphate buffer, pH 7.0–8.0; the loading volume was 500 μL, the flow rate was 0.75 mL / min, and the target protein peak was collected to obtain high-purity NA protein.

[0061] 5. Results of NA activity assay

[0062] Experimental results show that ( Figure 1 The difference in fluorescence intensity between the positive and negative controls was statistically significant. ***P<0.001, indicating that the experimental system was stable and reliable. Compared with the negative control group, different concentrations of G13 significantly enhanced the fluorescence intensity of the reaction system (P<0.001). *** (P<0.001), exhibiting good dose-dependent behavior. When the G13 concentration was 0.1 μg / mL, the fluorescence intensity of the reaction system was 16370.33±438.79; when the concentration increased to 0.5 μg / mL, the fluorescence intensity increased to 36599.33±2822.53; and when the concentration reached 1 μg / mL, the fluorescence intensity further increased to 78607.33±2967.65. These results indicate that while the catalytic activity of G13 may be lower than that of the positive control group, it still possesses good in vitro biological activity, and the enzymatic reaction becomes more complete with increasing concentration.

[0063] 6. Results of the interaction analysis between NA protein and HNC042 molecules

[0064] 6.1 Results of NA protein homology modeling

[0065] Homology modeling of wild-type and mutant NA proteins was performed using the SWISS-MODEL platform, and the structural quality of the resulting models was assessed. The results showed that the GMQE values ​​of the wild-type and mutant NA models were 0.82 and 0.83, respectively, indicating high overall model reliability. Ramachandran conformational analysis revealed that the proportions of amino acid residues located in the optimally allowed regions (Ramachandran-favored) were 95.30% and 93.78%, respectively, indicating that the models conformed to the stereochemical rules of proteins in their dihedral main chain distribution and exhibited good structural rationality.

[0066] like Figure 2 As shown, the constructed NA protein surface model has a complete overall outline and exhibits a relatively dense, irregular clump-like conformation, indicating that the obtained homology modeling results can well reflect the spatial surface characteristics of the protein. Further observation reveals a relatively obvious concave region in the center of the protein surface; the area marked by the purple dashed box is the predicted active site region. This region is located at the exposed position on the protein surface, providing a spatial basis for the formation of ligand-binding pockets, indicating that the established NA protein model possesses the structural conditions for conducting active site analysis and subsequent molecular docking studies.

[0067] The overall quality of the models was evaluated, and the results showed that both models had positive Z-scores with absolute values ​​less than 1, indicating that the constructed structural models were within the normal distribution range of known experimental protein structures. The overall quality was better than average, demonstrating high reliability (see...). Figure 3 To further evaluate the conformational rationality of the model, Ramachandran plots were constructed to analyze the conformational distribution of the NA protein and its mutants (see...). Figure 4The results showed that the vast majority of amino acid residues in both models were located in the allowed region, with only a few residues located in the critical region. Furthermore, the residue distribution was mainly concentrated in the high-density region of the Laplace diagram, suggesting that the protein conformation was stable and the spatial structure was reasonable.

[0068] In summary, despite the introduction of a specific amino acid mutation into the NA protein, its overall spatial conformation remains highly consistent with that of the native protein, and no obvious conformational abnormalities were observed, indicating that the mutation did not have a significant impact on the overall structural stability of the protein.

[0069] 6.2 Calculation results of protein binding energy of HNC042-NA

[0070] Based on the molecular docking method, the optimal conformation with the lowest binding energy was obtained, with the following energies:

[0071] Table 1 Binding energy of small molecules to NA protein (kcal·mol) -1 ) NA (PDBID: 3TI6) HNC042 -7.7 NA (PDBID: 5NWE) HNC042 -7.4

[0072] The binding energy indicates that both proteins can bind stably to HNC042.

[0073] 6.3HNC042-NA Interaction Mode

[0074] 6.3.1 Interaction between HNC042 and wild-type NA

[0075] The interaction patterns of complexes of two small molecules in their optimal conformations with a protein are visualized using a protein-ligand interaction profiler (PLIP). For example... Figure 5 As shown, the small molecule HNC042 can dock into the protein's active site. Based on the structural background analysis of the 3TI6 wild-type NA and the 5NWE mutant NA, it is known that the residue numbers in the docking model of this invention are not standard N1 numbers, but rather renumbered after modeling. Based on the classic NA active site composition, Arg103, Asp136, Arg137, and Trp164 correspond to Arg118, Asp151, Arg152, and Trp178 in the standard N1 numbering, respectively. Arg118, Asp151, and Arg152 are catalytic sites, while Trp178 is a framework site. Ser165 and Ser232 are located in the edge support region of the active pocket and the adjacent region of the 240 ring, respectively.

[0076] like Figure 6As shown, HNC042 interacts with five key amino acid residues surrounding the active site of the wild-type protein via hydrogen bonds. The carboxyl O atom of HNC042 forms a 4.0 Å hydrogen bond with residue Ser232; the two N atoms of the guanidinium group form two 4.0 Å hydrogen bonds with residue Ser165O, respectively; the N and O atoms of the amide group form two hydrogen bonds with residue Tyr387O, respectively, at distances of 2.8 Å and 2.9 Å; and the two O atoms of the 2-methoxyethoxy group form hydrogen bonds with residues Arg353 (3.3 Å) and Arg103 (2.9 Å), respectively.

[0077] The docking results show that HNC042 mainly forms hydrogen bonds with Arg103, Ser165, Ser232, Tyr387 and Arg353 in wild-type NA, suggesting that it can not only capture the core positively charged recognition region of the active pocket, but also extend further into the deep pocket and edge regions, forming a multi-point binding mode of core anchoring and edge spreading.

[0078] Simultaneously, HNC042 interacts hydrophobically with eight amino acid residues surrounding the active site of the wild-type protein, such as... Figure 7 As shown, HNC042 forms four salt bridges with wild-type NA proteins, further stabilizing their binding and enhancing the inhibitory activity of the small molecule. A salt bridge forms between the heterocyclic N atom of HNC042 and the carboxyl group (3.9 Å) of residue Asp136, suggesting its ability to enter the key polar recognition region of the active site. A salt bridge forms between the carboxyl group of HNC042 and the guanidino C atom (4.6 Å) of residue Arg137, indicating that this compound retains a binding characteristic similar to NAIs, using a positive potential point to anchor the carboxyl group. The guanidino C atom of HNC042 forms salt bridges with distances of 4.5 Å and 4.7 Å between the carboxyl groups of residues Glu213 and Glu263, respectively, further establishing additional ionic interactions with acidic residues within or adjacent to the pocket, thereby restricting ligand conformational freedom and enhancing overall binding stability.

[0079] 6.3.2 Interaction between HNC042 and mutant NA

[0080] HNC042 interacts with four key amino acid residues surrounding the active site of the mutant protein via hydrogen bonds, such as... Figure 5 As shown, in HNC042, one O atom of the 2-methoxyethoxy group forms a hydrogen bond with the Ser232N atom at a distance of 3.1 Å; the heterocyclic N atom and the amide N atom form hydrogen bonds with the Arg137N atom at distances of 3.7 Å and 3.5 Å, respectively; and the guanidinium N atom forms hydrogen bonds with the Trp164O atom (3.0 Å) and the Arg141N atom (3.3 Å), respectively.

[0081] In contrast, in mutant NA, HNC042 primarily forms hydrogen bonds with Ser232, Arg137, Trp164, and Arg141 (see [link to relevant documentation]). Figure 6 This indicates that the dominant binding region shifts from the deep and peripheral sites in the wild type to the catalytic entry region and the region adjacent to 150-cavity. Based on the structural mechanism revealed by 5NWE, where H275Y drives Glu277 into the active pocket and alters the ligand binding posture, it can be inferred that the H260Y mutation in this invention also causes local pocket remodeling. Although HNC042 loses some of its dominant wild-type binding sites, it may form a more dominant compensatory binding, shifting to dependence on entry-side and framework-side residues. The interaction region of HNC042 is closer to the 150-loop, possibly enhancing binding by adapting to the region near 150-cavity, while 371-cavity and 430-cavity do not directly participate in the binding of HNC042. These results demonstrate that HNC042, while retaining its anchoring effect on the NA catalytic center, can further utilize the polar sites around 150-cavity, thereby improving its binding stability in the active pocket. Overall, HNC042 maintains good binding ability in mutants, and in terms of binding mode, it has changed from widespread distribution in wild type to local adaptive rearrangement.

[0082] H275Y is a classic mutation site for NA-mediated OS resistance. Its resistance mechanism is mainly related to the mutation inducing local conformational rearrangement of the active pocket, thereby weakening the stable binding of OS. Existing structural and thermodynamic studies have shown that H275Y can significantly alter the binding posture of OS in the active site and significantly reduce its binding ability to NA. While HNC042 in mutant NA does not completely retain the dominant interaction network of the wild type, it can still form new hydrogen bonds through residues such as Arg137, Trp164, Arg141, and Ser232, suggesting that it still has some binding adaptability under the H275Y-induced pocket remodeling background. Its binding mode does not completely depend on the original pocket conformation that OS depends on, thus it is expected to maintain some inhibitory potential under the H275Y mutation background. This conclusion is only based on current molecular docking speculation and needs further verification through enzyme inhibition experiments, cellular antiviral experiments, and molecular dynamics simulations.

[0083] HNC042 also exhibits hydrophobic interactions with eight amino acid residues surrounding the active site of the mutant NA protein, such as... Figure 7As shown, HNC042 forms salt bridges with the guanidinium C atom (4.0 Å) of residue Arg278 and the guanidinium C atom (4.8 Å) of residue Arg353. Specifically, the formation of salt bridges between the carboxyl group of HNC042 and Arg278 and Arg353 indicates that it can effectively utilize the conserved positively charged arginine recognition region of the NA active site for anchoring. Simultaneously, the salt bridges formed between the heterocyclic N atom and Asp136, and between the guanidinium group and Glu213, Glu104, and Asp136, indicate that this compound can further establish additional constraints with acidic residues within the pocket. These results demonstrate that the binding of HNC042 in mutant NA is not dependent on a single site, but rather through multiple polar centers such as the carboxyl group, guanidinium group, and heterocyclic N atom, forming a compensating ionic interaction network between the positively charged site and the acidic site, thereby enhancing its adaptability to the mutant pocket. While retaining its anchoring function to the NA catalytic center, HNC042 can further utilize the polar sites around the 150-cavity, thereby enhancing the binding stability between HNC042 and the NA protein. Given the known structural mechanism that the H275Y mutation can induce local remodeling of the active pocket and weaken the original OS binding posture, it can be inferred that HNC042 can mitigate the adverse effects of the mutation on ligand binding to some extent by reconstructing a multi-point electrostatic anchoring mode, thus potentially possessing some potential against H275Y resistance.

[0084] Simultaneously, the carboxyl group of HNC042 forms salt bridges with the guanidinium C atom (4.0 Å) of residue Arg278 and the guanidinium C atom (4.8 Å) of residue Arg353. Specifically, the carboxyl group of HNC042 forms salt bridges with Arg278 and Arg353, respectively, suggesting that it can still effectively utilize the conserved positively charged arginine recognition region of the NA active site for anchoring. At the same time, the salt bridges formed between the heterocyclic N atom and Asp136, and between the guanidinium group and Glu213, Glu104, and Asp136, indicate that this compound can further establish additional constraints with acidic residues within the pocket.

[0085] Based on the above conclusions, (1) the present invention employs an optimized CHO-k1 suspension cell secretion expression system:

[0086] Utilizing the CHO-k1 suspension cell system, which is advantageous for expressing complex glycosylated proteins, PR8 strain NA was stably detected in cell culture supernatant and existed primarily in tetrameric form under non-reducing conditions, indicating that CHO cells are suitable for expressing complex viral glycoproteins like PR8 strain NA. Compared to the E. coli system, the CHO cell expression system is more conducive to the correct formation of disulfide bonds, glycosylation modification, and tetrameric assembly of NA proteins, maintaining their native activity. Compared to the yeast system, CHO cells are closer to the native state in terms of protein glycosylation modification and maintenance of complex conformations. Compared to the HEK293 system, CHO cells have advantages in stable expression, suspension culture, and subsequent process scale-up. Therefore, CHO cells are a stable expression host for complex glycoproteins like NA that depend on the activity of native viruses.

[0087] The expressed target protein is mainly in the form of a natural tetramer, indicating that the constructed system can support the secretory expression of NA protein and conforms to its native conformation, implying its activity, but further research is needed. The current expression level is approximately 85 μg / mL, indicating that the expression system is feasible, but the overall yield is still relatively low. This indicates that the present invention mainly solves the problem of effective expression of NA from the PR8 strain in eukaryotic systems, while further optimization of vector elements and improvement of culture conditions are still needed for high-level expression and process optimization. Literature reports that constructing a structure that only expresses the extracellular domain can alleviate the membrane-associated folding and degradation limitations caused by the transmembrane-stem region, increasing the soluble expression level. Simultaneously, the fusion of a tetramerizing domain helps form a stable tetramer and improves downstream purification yield (Gaoe et al., 2021). In addition, an optimized secretion signal peptide can promote more efficient entry of recombinant proteins into the secretory pathway, increasing the amount available in the supernatant (Leete et al., 2025). Furthermore, introducing an affinity tag can establish a highly selective capture chromatography, a common strategy for improving purification recovery and reducing the loss of target protein during multi-step chromatography.

[0088] (2) Solved the purification problem of NA expression from untagged plasmids.

[0089] These results demonstrate the feasibility of isolating and purifying tag-free NA proteins in eukaryotic systems, but the purification process is significantly limited by the protein's molecular characteristics. For proteins like NA, which exhibit glycosylation modifications and tetrameric structure, tag-free expression is beneficial for maximizing the preservation of their native activity and conformation, and reducing the potential interference of exogenous tags on protein folding, oligomeric state, and subsequent structural analysis. Previous studies have also shown that the stem structure and length of NA are beneficial for maintaining tetrameric stability and protein activity. Therefore, tag-free expression is considered more suitable for preparing NA proteins in a near-native state.

[0090] However, experimental results show that the tagless strategy also significantly increases the difficulty of downstream purification. This invention employs multiple methods, including anion exchange, cation exchange, hydrophobic interaction chromatography, lectin affinity chromatography, and molecular sieve chromatography, to separate and purify the target protein, ultimately obtaining a target protein with a purity of 94.52% and a total amount of 0.585 mg, indicating that the route is generally feasible. However, interference from host proteins and product-related impurities still exists during purification, and the target protein lacks affinity tags for assisted capture, limiting both purification efficiency and recovery. This indicates that the current purification challenges stem not only from the process conditions themselves but are also closely related to factors such as low NA protein expression levels, strong glycosylation heterogeneity, and multimeric structures.

[0091] Therefore, while tag-free expression is more conducive to obtaining NA proteins close to their native state, it also increases the complexity of the purification process and the difficulty of process optimization. Future research could further optimize the chromatography steps and buffer system by increasing upstream expression levels, such as refining the step chromatography range and, if necessary, exploring the addition of cleavable affinity tags, to both preserve the native protein conformation and improve purification efficiency.

[0092] (3) The interaction between HNC042 and NA was analyzed.

[0093] Compared to OS, HNC042 exhibits different binding characteristics in H275Y mutant NA. Previous studies have shown that OS resistance to H275Y mainly stems from mutation-induced changes in Glu276 orientation, weakening the original hydrophobic interaction between its 3-pentyl and the active pocket, leading to decreased binding capacity. This invention constructs the three-dimensional structures of wild-type NA and OS resistance-related mutants of the PR8 strain based on homology modeling, and uses AutoDockVina to perform molecular docking analysis on the candidate small molecule HNC042. The results show that the binding energies of HNC042 to wild-type NA and mutant NA are -7.7 kcal·mol⁻¹ and -7.4 kcal·mol⁻¹, respectively, suggesting that it can stably enter the NA active pocket and form effective binding in both receptors. Structural studies have shown that NAIs typically function by occupying catalytic sites and forming multi-point interactions with conserved charged residues (Vavricka et al., 2011b). Further analysis revealed that HNC042 forms hydrogen bonds, salt bridges, and hydrophobic interactions with multiple key residues in both wild-type and mutant NA, indicating that this compound possesses a relatively clear targeting ability for NA. Especially in the H275Y mutant background, although some dominant interactions in the wild type are weakened, HNC042 can still form new interaction networks through residues such as Arg137, Trp164, Arg141, and Ser232, and establish compensatory ionic interactions with charged residues within the pocket via carboxyl, guanidinyl, and heterocyclic N atoms, thereby maintaining a relatively stable binding state. This result suggests that HNC042 has a certain degree of adaptability to local pocket remodeling caused by drug resistance mutations.

[0094] Based on the structure-activity relationship of NA inhibitors reported in the literature and the docking results of this invention, it is evident that the binding advantage of HNC042 mainly stems from the synergistic effect of its various functional groups, rather than single-site modification. Its carboxyl group can form stable salt bridges with Arg278 and Arg353, maintaining basic anchoring to the catalytic center; the guanidinyl group further forms salt bridges or electrostatic interactions with acidic residues such as Asp136, Glu104, and Glu213, thereby enhancing the molecule's utilization of the polar environment of the active pocket; the acetamide group helps retain the classical recognition framework of oseltamivir-type inhibitors, maintaining the molecule's rational orientation at the active site. In contrast, the most significant modification of HNC042 lies in the C3 side chain reconstruction. According to docking conformational analysis, this part does not extend to the 371-cavity or 430-cavity depth, but more likely extends to the hydrophobic pocket at the C3 position, adjusting the interaction center to the vicinity of the 150-cavity region, which facilitates hydrophobic interactions. This indicates that HNC042 does not primarily rely on the hydrophobic pocket containing the traditional 3-pentyl group for binding. Instead, while retaining the anchoring effect of the catalytic center, it enhances the utilization of the polar microenvironment near the 150-cavity, thereby reducing its dependence on the original hydrophobic effect of Glu276 to some extent and enhancing the binding stability between HNC042 and the NA protein.

[0095] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A label-free purification method for influenza A virus neuraminidase, characterized in that, The steps include the following: The supernatant of unlabeled neuraminidase expressed in CHO-k1 cells was concentrated by ultrafiltration using an ultrafiltration device with a molecular weight cutoff of 80kDa to 120kDa and the medium was changed. The concentrate was then purified by hydrophobic interaction chromatography, anion exchange chromatography and molecular sieve chromatography to obtain recombinant neuraminidase protein with a purity ≥94%.

2. The label-free purification method according to claim 1, characterized in that, The ultrafiltration concentration uses a hollow fiber column, and the buffer solution is 10mM to 30mM phosphate buffer containing 0.4M to 0.6M ammonium sulfate, with a pH of 7.5 to 8.

5. The feed flow rate is controlled at 150mL / min to 300mL / min and the transmembrane pressure is 0.3bar to 0.8bar.

3. The label-free purification method according to claim 1, characterized in that, The hydrophobic interaction chromatography used butyl hydrophobic packing material, with the equilibration solution being 10mM–30mM phosphate buffer containing 0.4M–0.6M ammonium sulfate at pH 7.5–8.5; step elution was employed, and the 60%–100% low-salt gradient elution peak was collected as the target protein fraction.

4. The label-free purification method according to claim 1, characterized in that, The anion exchange chromatography uses a strongly basic anion exchange packing material, with the equilibration solution being 10mM–30mM phosphate buffer at pH 7.5–8.5; linear salt gradient elution is employed, and the elution fraction containing neuraminidase is collected.

5. The label-free purification method according to claim 1, characterized in that, The molecular sieve chromatography uses dextran or agarose gel packing material, with a mobile phase of 10mM–30mM phosphate buffer at pH 7.0–8.0; the elution peak of the target protein is collected to obtain high-purity neuraminidase protein.

6. A recombinant NA protein, characterized in that, Prepared by the method described in any one of claims 1 to 3.

7. The use of the recombinant NA protein according to claim 6 in the screening of influenza virus neuraminidase inhibitors.

Images