Method for preparing recombinant human IL-15 protein based on inclusion body renaturation process
By employing inclusion body refolding and purification processes, the problems of misfolding and aggregation in prokaryotic expression of recombinant human IL-15 were solved, achieving high-purity and high-efficiency IL-15 preparation, simplifying the process and improving the bioactivity of the product.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-04-10
AI Technical Summary
Existing prokaryotic expression of recombinant human IL-15 suffers from problems such as inclusion body misfolding, disulfide bond mismatch, aggregate formation, and protein degradation, resulting in low yield, sensitivity to refolding conditions, lack of process availability, and poor universality, making it difficult to prepare high-purity active products.
Using an inclusion body refolding process, a recombinant vector containing the IL-15 fusion protein was constructed. The inclusion bodies were expressed and cleaved using E. coli host cells. After dissolution with denaturing buffer, the inclusion bodies were initially purified by nickel affinity chromatography. After dialysis and refolding, the protein was digested with TEV protease and purified by dextran gel chromatography to finally obtain high-purity recombinant human IL-15.
The preparation of recombinant human IL-15 with high purity (≥98%) was achieved, simplifying the process and shortening the time (to be completed within 2-3 days). The correct structural conformation of IL-15 protein was obtained, and the specific antibody binding ability was indistinguishable from that of commercially available standards.
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Figure CN121826013A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing recombinant human IL-15 protein based on inclusion body refolding technology. Background Technology
[0002] Interleukin-15 (IL-15) is a multifunctional cytokine belonging to the γ-chain receptor cytokine family. The mature IL-15 monomer consists of 114 amino acids and has a typical 4-α helical bundle structure. It functions by maintaining NK cell homeostasis and driving CD8+. + IL-15 plays an immunomodulatory role through mechanisms such as T cell proliferation. In the pathological microenvironment, IL-15 exhibits dual biological effects: it can mediate the inflammatory cascade response triggered by excessive T cell activation in autoimmune diseases, and it can also enhance the targeted killing ability of effector lymphocytes in the solid tumor microenvironment. Based on its pleiotropic immunomodulatory properties, recombinant human IL-15 has been developed into a candidate drug for novel immunotherapy.
[0003] Due to the complexity of protein folding, existing heterologous expression production of recombinant human IL-15 faces technical challenges at multiple levels. While mammalian cell expression can achieve correct folding, secretion, and potential post-translational modifications to obtain products close to the native conformation, it also presents challenges such as relatively low expression levels, high culture costs, long production cycles, and difficulty in scale-up. The stability, degradation, and glycoform heterogeneity of the secreted product can lead to inconsistencies in biological activity and pharmacokinetics. Furthermore, IL-15 is often synergistically expressed with IL-15Rα to improve stability and biological activity; when expressed alone, yield and stability are often limited. In contrast, prokaryotic expression for recombinant human IL-15 production offers advantages such as lower cost and shorter cycle time. However, current prokaryotic IL-15 expression typically results in inclusion bodies, requiring subsequent dissolution and refolding steps. Problems such as misfolding, disulfide bond mismatch, aggregate formation, and protein degradation are common, leading to low yields, high sensitivity to refolding conditions, lack of process generality, poor reproducibility, and difficulty in scale-up and reproducibility. This significantly increases the cost and time required to achieve high-purity, active products.
[0004] Therefore, developing an effective inclusion body refolding and purification process to obtain high-purity and high-activity recombinant IL-15 has become a critical technical bottleneck that urgently needs to be overcome in this field.
[0005] It should be noted that the information disclosed in this background section is only for understanding the background technology of this application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention
[0006] This disclosure provides at least one method for preparing recombinant human IL-15 protein based on inclusion body refolding technology.
[0007] In a first aspect, embodiments of this disclosure provide a method for preparing recombinant human IL-15 protein based on inclusion body refolding technology, comprising the following steps: S1, constructing a recombinant vector 6H-tev-IL15 containing the coding sequence of an IL-15 fusion protein; S2, transforming the recombinant vector into E. coli host cells BL21(DE3) to induce expression, obtaining expression cells; S3, lysing the expression cells to obtain inclusion bodies, and washing the inclusion bodies; S4, dissolving the inclusion bodies using denaturing buffer, and then performing nickel affinity chromatography. Preliminary purification was performed, and the protein eluent was collected and dialyzed to obtain denatured protein; S5, the denatured protein was refolded using refolding buffer to obtain refolded protein; S6, the refolded protein was cleaved using TEV protease, and the cleavage product was passed through pre-equilibrated nickel column affinity chromatography to remove the affinity tag and TEV protease to obtain IL-15 protein flow-through; S7, the IL-15 protein flow-through was collected, and the flow-through was subjected to pre-equilibrated dextran gel chromatography. The protein eluent at the peak was collected to obtain recombinant IL-15 protein.
[0008] In one optional embodiment, the amino acid sequence of the recombinant vector 6H-tev-IL15 fusion protein is SEQ ID NO:2, the IL-15 nucleic acid sequence optimized for the codon preference of the expression host BL21(DE3) is SEQ ID NO:3, and the 6H-tev-IL15 nucleic acid sequence is SEQ ID NO:4.
[0009] In one optional embodiment, the conditions for inducing expression in S2 include: when the OD600 value of the bacterial culture reaches 1.2-1.3, adding IPTG to make the final concentration 1.0 mM, and culturing at 37°C with shaking for 14-18 hours.
[0010] In an optional embodiment, the washing step of inclusion bodies in S3 includes: i. taking the expressed bacterial cells, centrifuging at low temperature and discarding the supernatant, and resuspending the bacterial cells in Buffer A; ii. centrifuging the resuspended bacterial cells again at low temperature and discarding the supernatant, and resuspending the bacterial cells in a small amount of Buffer A; iii. lysing the cells using a pre-cooled high-pressure homogenizer, and collecting the precipitate by low-temperature high-speed centrifugation; iv. resuspending the precipitate in at least 20 times its mass of Buffer B, stirring thoroughly, and then centrifuging to collect the precipitate; v. repeating the complete process of step iv at least twice; vi. resuspending the precipitate in Buffer C and centrifuging to obtain the washed inclusion bodies.
[0011] In one optional embodiment, the preliminary purification of inclusion bodies in S4 specifically includes: i. adding 5 times the mass of denaturing buffer D to the inclusion bodies, stirring to dissolve them completely at room temperature, and collecting the supernatant by centrifugation; ii. purifying and enriching the IL-15 fusion protein in the supernatant using nickel affinity chromatography pre-equilibrated with Buffer E; iii. washing sequentially with Buffer F and G, and eluting with Buffer H, collecting the eluent; iv. concentrating the eluent by ultrafiltration to 1 / 10 of its original volume, and dialyzing in 100 times the volume of Buffer E for at least 14 hours.
[0012] In an optional embodiment, the refolding process in S5 includes: i. taking denatured protein and concentrating it to 1 mg / mL using an ultrafiltration tube with a molecular weight cutoff of 5 kD; ii. placing the sample at 4°C and slowly adding it dropwise to 10 times its volume of pre-cooled refolding buffer Buffer I, so that the final protein concentration is reduced to 100 μg / mL, and stirring at 4°C for 24 h; iii. concentrating it to 1 / 10 of the original volume using an ultrafiltration tube with a molecular weight cutoff of 5 kD, dialyzing it in ice-cold Buffer C for 24 h, and then filtering it through a 0.22 μm filter membrane to obtain a refolded protein solution.
[0013] In one optional embodiment, the TEV protease in S6 is used in the refolded protein solution at a ratio of 1:30 (w:w), and after mixing, it is allowed to stand at 4°C for 16 h; the ionic ligand type of the nickel column is NTA.
[0014] In one optional implementation, Buffer A consists of 100 mM Tris-HCl and 1 mM EDTA at pH 7.5; Buffer B consists of PBS and 2% Triton 100 at pH 7.5; and Buffer C consists of PBS at pH 7.5.
[0015] In one optional embodiment, Buffer D consists of 20 mM Tris-HCl, 15 mM β-mercaptoethanol, 6 M guanidine hydrochloride, and pH 8.0; Buffer E consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 8.0; Buffer F consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 6.5; Buffer G consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 4.5; and Buffer H consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, 100 mM EDTA, and pH 8.0.
[0016] In one alternative embodiment, Buffer I consists of 100 mM Tris-HCl, 0.5 M glycine, 1 mM oxidized glutathione, and 10 mM reduced glutathione, at pH 8.0.
[0017] The beneficial effects of this invention are as follows: This method for preparing recombinant human IL-15 protein based on inclusion body refolding technology utilizes fusion expression technology. First, inclusion bodies with histidine affinity tags are obtained using a prokaryotic expression system. The inclusion bodies are then dissolved using guanidine hydrochloride to obtain denatured protein. The denatured protein is initially purified using nickel affinity chromatography. After dialysis, the pre-purified denatured protein is added to folding buffer, incubated, and dialyzed to obtain a refolding solution. The refolding solution is cleaved using TEV protease, and nickel affinity chromatography is used again to remove residual protease and the N-terminal histidine tag. Finally, highly purified recombinant human IL-15 is obtained by dextran gel chromatography. This inclusion body refolding and purification process ultimately yields recombinant human IL-15 with a purity exceeding 98%, and its binding ability to specific antibodies is no different from that of commercially available standards.
[0018] The recombinant human IL-15 inclusion body refolding and purification process proposed in this invention is simple, efficient, and has a short preparation process. It yields high purity target protein (IL-15), and the refolding and purification process can be completed in just 2-3 days. The prepared IL-15 protein with the correct structural conformation does not contain non-IL-15 components such as His and enzyme residues, and has the advantages of sequence consistency.
[0019] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.
[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description
[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0022] Figure 1 A gel image showing the purification effect of nickel affinity chromatography on inclusion bodies provided in the embodiments of this disclosure; Figure 2The refolded protein solution and its effect after cutting are provided in the embodiments of this disclosure; Figure 3 Nickel affinity chromatography gel image after cutting, provided in an embodiment of this disclosure; Figure 4 This is a dextran gel chromatography image provided in an embodiment of this disclosure; Figure 5 The SEC-HPLC method provided in this embodiment is used to detect the purity of IL-15. Figure 6 This is a colorimetric diagram of an ELISA solution provided in an embodiment of this disclosure. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.
[0025] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.
[0026] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1-20 should be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
[0027] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."
[0028] As used in this article, "elution" refers to the desorption of molecules bound to the stationary phase by altering the solution conditions within the chromatographic column. This can be achieved by increasing the concentration of exchangeable counterions or by changing the pH to affect the binding affinity of the analyte. Molecules that lose their affinity for the stationary phase and enter the mobile phase are then "eluted" from the column.
[0029] As used herein, "buffer" refers to a reagent, typically a solution, used to alter the adsorption of an analyte (e.g., IL-15 protein) onto a stationary phase and / or to remove unbound material from the stationary phase. The elution properties of an eluent can depend on factors such as pH, ionic strength, and detergent strength.
[0030] As used herein, "elution buffer" refers to a solution (e.g., a wash solution or buffer solution) containing unbound substances (including "eluted" or desorbed analyte molecules, such as IL-15 protein) that travel through the stationary phase and exit the column during chromatographic separation. As used herein, "an" and "a" are used to refer to one or more grammatical objects.
[0031] As used in this article, "high purity" means a purity of ≥98.0% (peak area percentage) as determined by high performance liquid chromatography.
[0032] As used herein, the term "pH" is a numerical value indicating the degree of acidity or alkalinity of a solution and is an indicator of hydrogen ion concentration. Within the pH range of 0 to 14, a solution with a pH of 7 is neutral, a solution with a pH less than 7 is acidic, and a solution with a pH greater than 7 is alkaline. pH can be measured using a pH meter, and the pH of buffer solutions can be adjusted using acids or bases such as HCl or NaOH.
[0033] As used herein, the term "purification" refers to the operation of increasing purity by removing coexisting impurities from a substance, and in this specification, purification means the isolation of IL-15 protein from a culture of Escherichia coli, and also refers to the process of increasing purity during the production of IL-15 protein.
[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.
[0035] This disclosure provides a method for preparing recombinant human IL-15 protein based on inclusion body refolding technology, comprising the following steps: S1, constructing a recombinant vector 6H-tev-IL15 containing the IL-15 fusion protein coding sequence; S2, transforming the recombinant vector into E. coli host cells BL21(DE3) to induce expression, obtaining expression cells; S3, lysing the expression cells to obtain inclusion bodies, and washing the inclusion bodies; S4, dissolving the inclusion bodies with denaturing buffer, and performing preliminary analysis by nickel affinity chromatography. Purification: Collect the protein eluent and dialyze to obtain denatured protein; S5: Refold the denatured protein using refolding buffer to obtain refolded protein; S6: Digest the refolded protein using TEV protease, and pass the digestion product through pre-equilibrated nickel column affinity chromatography to remove the affinity tag and TEV protease, obtaining IL-15 protein flow-through; S7: Collect the IL-15 protein flow-through, and perform pre-equilibrated dextran gel chromatography on the flow-through, collecting the protein eluent at the peak to obtain recombinant IL-15 protein.
[0036] In some embodiments, specifically, the amino acid sequence of the recombinant vector 6H-tev-IL15 fusion protein is SEQ ID NO:2, the IL-15 nucleic acid sequence optimized for the codon preference of the expression host BL21(DE3) is SEQ ID NO:3, and the 6H-tev-IL15 nucleic acid sequence is SEQ ID NO:4.
[0037] In some embodiments, specifically, the carriers in S1 include, but are not limited to, pET series, pGEX series, and pMAL series.
[0038] In some embodiments, specifically, the conditions for inducing expression in S2 include: when the OD600 value of the bacterial culture reaches 1.2-1.3, adding IPTG to make the final concentration 1.0 mM, and culturing at 37°C with shaking for 14-18 hours.
[0039] In some embodiments, specifically, the Escherichia coli host cell in S2 can be any alternative Escherichia coli host that can ferment and express IL-15, preferably Escherichia coli host cell BL21(DE3).
[0040] In some embodiments, specifically, the washing step of inclusion bodies in S3 includes: i. taking the expression cells, centrifuging at low temperature and discarding the supernatant, and resuspending the cells in Buffer A; ii. centrifuging the resuspended cells again at low temperature and discarding the supernatant, and resuspending the cells in a small amount of Buffer A; iii. lysing the cells using a pre-cooled high-pressure homogenizer, and collecting the precipitate by low-temperature high-speed centrifugation; iv. resuspending the precipitate in at least 20 times its mass of Buffer B, stirring thoroughly, and then centrifuging to collect the precipitate; v. repeating the entire process of step iv at least twice; vi. resuspending the cells in Buffer C and centrifuging to obtain the washed inclusion bodies.
[0041] In some embodiments, specifically, the preliminary purification of inclusion bodies in S4 includes: i. adding 5 times the mass of denaturing buffer D to the inclusion bodies, stirring to dissolve completely at room temperature, and collecting the supernatant by centrifugation; ii. purifying and enriching the IL-15 fusion protein in the supernatant using nickel affinity chromatography pre-equilibrated with Buffer E; iii. washing sequentially with Buffer F and G, and eluting with Buffer H, collecting the eluent; iv. concentrating the eluent by ultrafiltration to 1 / 10 of its original volume, and dialyzing in 100 times the volume of Buffer E for at least 14 hours.
[0042] In some embodiments, specifically, the refolding process in S5 includes: i. taking denatured protein and concentrating it to 1 mg / mL using an ultrafiltration tube with a molecular weight cutoff of 5 kD; ii. placing the sample at 4°C and slowly adding it dropwise to 10 times its volume of pre-cooled refolding buffer Buffer I, so that the final protein concentration is reduced to 100 μg / mL, and stirring at 4°C for 24 h; iii. concentrating it to 1 / 10 of the original volume using an ultrafiltration tube with a molecular weight cutoff of 5 kD, dialyzing it in ice-cold Buffer C for 24 h, and then filtering it through a 0.22 μm filter membrane to obtain a refolded protein solution.
[0043] In some embodiments, specifically, the ratio of TEV protease in the refolded protein solution in S6 is 1:30 (w:w), and after mixing, it is allowed to stand at 4°C for 16 h; the ionic ligand type of the nickel column is NTA.
[0044] In some embodiments, specifically, Buffer A consists of 100 mM Tris-HCl and 1 mM EDTA, pH 7.5; Buffer B consists of PBS and 2% Triton 100, pH 7.5; and Buffer C consists of PBS, pH 7.5.
[0045] In some embodiments, specifically: Buffer D: 20 mM Tris-HCl, 15 mM β-mercaptoethanol, 6 M guanidine hydrochloride, pH 8.0; Buffer E: 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, pH 8.0; Buffer F: 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, pH 6.5; Buffer G: 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, pH 4.5; Buffer H: 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, 100 mM EDTA, pH 8.0.
[0046] In some embodiments, specifically, Buffer I consists of 100 mM Tris-HCl, 0.5 M glycine, 1 mM oxidized glutathione, 10 mM reduced glutathione, and pH 8.0.
[0047] Example 1: Sequence Characterization and Vector Construction of Recombinant Human IL-15 The nucleotide sequence described in SEQ ID NO:3 (synthesized by GenScript Biotech) was cloned into the 3' end of the pET28a RBS motif via homologous recombination, ultimately forming the fusion protein 6H-tev-IL15 in an open reading frame, thus obtaining the recombinant plasmid. The constructed vector was transformed into the prokaryotic expression strain BL21(DE3) using the heat shock method. After sequencing verification, it was stored for later use.
[0048] The recombinant plasmid was transformed into host cells, and after selection with kanamycin sulfate (Kan), single-clone recombinant cells were obtained and expanded in LB medium to obtain seed cells. A single transformant was picked and inoculated into LB medium, and the concentration of Kan antibiotic was increased to 50 μg / mL. The medium was then incubated overnight at 30°C with shaking at 200 rpm to obtain the seed culture.
[0049] Example 2: Induced Expression of Recombinant Human IL-15 The seed culture was inoculated into Good LB medium at a ratio of 1:100 (V:V) and cultured at 37°C with shaking at 220 rpm. The OD600 value of the bacterial culture was measured by absorbance method every 2 h. When the OD600 of the bacterial culture reached 1.2-1.3, 0.5 mM IPTG was added to the bacterial culture to a final concentration, and then cultured at 37°C with shaking at 180 rpm for another 14-18 h.
[0050] Example 3: Refolding and purification of recombinant IL-15 Experiment 1: Inclusion Body Acquisition Take the induced bacterial suspension and centrifuge at 18000 g, 4℃ for 8 min. Discard the supernatant and resuspend the bacterial cells in 1 volume of Buffer A. Centrifuge again at 18000 g, 4℃ for 8 min, discard the supernatant, and resuspend the bacterial cells in 0.2-0.3 volume of Buffer A. Pre-cool the suspension using a high-pressure homogenizer. Disrupt the suspension at 900-1000 bar for 3-5 min, collect the disruption liquid, and centrifuge at 25000 g, 4℃ for 60 min. Discard the soluble supernatant. Resuspend the precipitate in at least 20 volumes of Buffer B, stir magnetically for 30 min, and centrifuge. Repeat this washing process at least twice. For the final resuspension, resuspend and centrifuge with Buffer C to obtain the washed inclusion bodies. Figure 1 As shown, the target protein was observed and confirmed by dispensing the gel.
[0051] Experiment 2: Inclusion body denaturation and crude purity Inclusion bodies were resuspended in 5 times their mass of denaturing buffer D and magnetically stirred at room temperature for 1 h to ensure complete dissolution. The mixture was then centrifuged at 25000 g for 1 h, and the supernatant was collected. The IL-15 fusion protein in the supernatant was purified and enriched using nickel column affinity chromatography. The nickel column was pre-equilibrated with Buffer E for 10 CV, followed by washing with Buffer F and G for 5 CV each, and finally eluted with Buffer H. The protein eluent was collected, as shown in the image. Figure 1 As shown, ultrafiltration was performed to 1 / 10 of the original volume, followed by dialyzing in 100 times the volume of Buffer E for at least 14 h.
[0052] Experiment 3: Refolding Treatment The crudely purified denatured protein sample was concentrated to 1 mg / mL using an ultrafiltration tube with a molecular weight cutoff of 5 kD. The sample was placed at 4°C and slowly added dropwise to 10 times its volume of pre-chilled refolding buffer I, reducing the final protein concentration to 100 μg / mL. The mixture was then magnetically stirred at 4°C for 24 h. The concentration was further reduced to 1 / 10 of the original volume using an ultrafiltration tube with a molecular weight cutoff of 5 kD, and dialyzed against ice-cold buffer C for 24 h. After dialyzing, the solution was filtered through a 0.22 μm filter to obtain the refolded protein solution, as shown below. Figure 2 As shown, the presence of the target protein was confirmed by dispensing and observing.
[0053] Experiment 4: Purification after refolding Add 1 / 30 mass ratio of TEV protease to the refolded protein solution, mix, and let stand at 4°C for 16 h. See below for sample cutting details. Figure 2Nickel affinity chromatography was used again to remove histidine affinity tags and TEV protease from the mixture. The nickel column was pre-equilibrated with Buffer C for 10 CV. The mixture was loaded into the chromatography packing material, and elution was continued with Buffer C. The flow-through was collected, as shown below. Figure 3 The image shown is a gel image of SDS-PAGE in flow-through buffer. The dextran gel chromatography system was pre-equilibrated with Buffer C for 2 CV. The sample loading volume did not exceed 2% of the column volume. Proteins at the elution peak were collected, as shown below. Figure 4 As shown, this is high-purity recombinant human IL-15.
[0054] Example 4: Purity Detection The purity of IL-15 was determined by HPLC using a BioCore SEC-300 column (3 μm, 7.8 × 300 mm), with Buffer C as the mobile phase, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 280 nm. Peak data were integrated. Figure 5 As shown, after integration, the purity of IL-15 is 98.02%.
[0055] Example 5: Activity Evaluation The binding ability of recombinant IL-15 to specific antibodies was detected using an enzyme-linked immunosorbent assay (ELISA) kit. Following the kit manufacturer's instructions, the IL-15 obtained using this process and commercially available recombinant human IL-15 (Yeasen, catalog number 90113ES10) were diluted to 300 μg / mL, 3 μg / mL, 30 ng / mL, 300 pg / mL, and 30 pg / mL using a serial dilution method. 100 μL of each solution was loaded into a microplate and incubated at 37°C for 90 min. Residual protein was washed away, secondary antibody was added and incubated for 30 min. The secondary antibody was washed away, chromogenic buffer was added, and the reaction was carried out in the dark for 15 min. Stop solution was added, and the absorbance of the reaction wells was measured at 460 nm. Figure 6 As shown, the binding ability of recombinant human IL-15 obtained by this process to specific antibodies is basically no different from that of commercially available samples.
[0056] In one specific embodiment, SEQ ID NO:1 is the amino acid sequence of human IL-15, SEQ ID NO:2 is the fusion amino acid sequence 6H-tev-IL15 formed by adding a histidine tag and a TEV protease cleavage site to the N-terminus of IL-15, SEQ ID NO:3 is the human IL-15 nucleic acid sequence optimized for E. coli codon preferences, and SEQ ID NO:4 is the 6H-tev-IL15 nucleic acid sequence optimized for E. coli codon preferences.
[0057] SEQ ID NO:1: MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO:2: MGSHHHHHHSSGENLYFQGMNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO:3: atgaattgggtgaacgtgattagcgacctgaaaaagattgaagatctgatccagagcatgcatattgatgcaaccctgtataccgaaagtgatgttcatccgagctgtaaagttaccgccatgaaatgttttctgctggaactgcaggttattagcctggaaagcggtgatgcaagcattcatgataccgttgaaaacctgattattctggccaataattccctgagcagcaatggtaatgttaccgaaagcggttgtaaagagtgtgaagaactggaagagaagaacatcaaagagtttctgcagagctttgttcatatcgtgcagatgtttattaacaccagctaa SEQ ID NO:4: atgggcagcagcgagaacctgtacttccaagggatgaattgggtgaacgtgattagcgacctgaaaaagattgaagatctgatccagagcatgcatattgatgcaaccctgtataccgaaagtgatgttcatccgagctgtaaagttaccgccatgaaatgttttctgctggaactgcaggttattagcc tggaaagcggtgatgcaagcattcatgataccgttgaaaacctgattattctggccaataattccctgagcagcaatggtaatgttaccgaaagcggttgtaaagagtgtgaagaactggaagagaagaacatcaaagagtttctgcagagctttgttcatatcgtgcagatgtttattaacaccagctaa In summary, this method for preparing recombinant human IL-15 protein based on inclusion body refolding technology utilizes fusion expression technology. First, inclusion bodies with histidine affinity tags are obtained using a prokaryotic expression system. The inclusion bodies are then dissolved using guanidine hydrochloride to obtain denatured protein. The denatured protein is initially purified using nickel affinity chromatography. After dialysis, the pre-purified denatured protein is added to folding buffer, incubated, and dialyzed to obtain a refolding solution. The refolding solution is cleaved using TEV protease, and nickel affinity chromatography is used again to remove residual protease and the N-terminal histidine tag. Finally, highly purified recombinant human IL-15 is obtained by dextran gel chromatography. This inclusion body refolding and purification process ultimately yields recombinant human IL-15 with a purity exceeding 98%, and its binding ability to specific antibodies is indistinguishable from commercially available standards.
[0058] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.
Claims
1. A method for preparing recombinant human IL-15 protein based on inclusion body refolding technology, characterized in that, Includes the following steps: S1, construct the recombinant vector 6H-tev-IL15 containing the IL-15 fusion protein coding sequence; S2, the recombinant vector was transformed into E. coli host cells BL21(DE3) to induce expression and obtain expressed bacterial cells; S3, the expression cells are broken to obtain inclusion bodies, and the inclusion bodies are washed. S4, the inclusion bodies were dissolved in denaturing buffer, and preliminarily purified by nickel affinity chromatography. The protein eluent was collected and dialyzed to obtain the denatured protein. S5, the denatured protein is refolded using a refolding buffer to obtain the refolded protein; S6. The refolded protein was cleaved using TEV protease. The cleavage product was then passed through a pre-equilibrated nickel column affinity chromatography to remove the affinity tag and TEV protease, yielding an IL-15 protein flow-through solution. S7. Collect the IL-15 protein flow-through. Use pre-equilibrated dextran gel chromatography to collect the protein effluent from the peak to obtain recombinant IL-15 protein.
2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the recombinant vector 6H-tev-IL15 fusion protein is SEQ ID NO:2, the IL-15 nucleic acid sequence optimized for the codon preference of the expression host BL21(DE3) is SEQ ID NO:3, and the 6H-tev-IL15 nucleic acid sequence is SEQ ID NO:
4.
3. The preparation method according to claim 1, characterized in that, The conditions for inducing expression in S2 include: when the OD600 value of the bacterial culture reaches 1.2-1.3, adding IPTG to make the final concentration 1.0 mM, and culturing at 37℃ with shaking for 14-18 hours.
4. The preparation method according to claim 1, characterized in that, The washing step of the inclusion bodies in S3 includes: i. Take the expressed bacterial cells, centrifuge at low temperature and discard the supernatant, then resuspend the bacterial cells in Buffer A; ii. Resuspend the bacterial cells, centrifuge again at low temperature and discard the supernatant. Resuspend the bacterial cells using a small amount of Buffer A. iii. Cells were disrupted using a pre-cooled high-pressure homogenizer, and the lysate was collected by low-temperature high-speed centrifugation. iv. Resuspend the precipitate in at least 20 times its weight of Buffer B, stir thoroughly, and then centrifuge to collect the precipitate; v. Repeat step iv for at least two times; vi. Use Buffer C to resuspend and centrifuge to obtain the washed inclusion bodies.
5. The preparation method according to claim 1, characterized in that, The preliminary purification of inclusion bodies in S4 specifically includes: i. Add 5 times the mass of denaturing buffer D to the inclusion body, stir at room temperature until fully dissolved, and centrifuge to collect the supernatant; ii. The IL-15 fusion protein in the supernatant was purified and enriched by nickel column affinity chromatography pre-equilibrated with Buffer E; iii. Rinse with Buffer F and G in sequence, and elute with Buffer H, then collect the eluent; iv. The eluent is concentrated to 1 / 10 of its original volume by ultrafiltration and dialyzed in 100 times its volume of Buffer E for at least 14 hours.
6. The preparation method according to claim 1, characterized in that, The reversibility process in S5 includes: i. Take the denatured protein and concentrate it to 1 mg / mL using an ultrafiltration tube with a molecular weight cutoff of 5 kD; ii. The sample was placed at 4°C and slowly added dropwise to 10 times the volume of pre-cooled refolding buffer Buffer I, so that the final protein concentration was reduced to 100 μg / mL, and stirred at 4°C for 24 h; iii. Concentrate to 1 / 10 of the original volume using an ultrafiltration tube with a molecular weight cutoff of 5 kD, dialyze in ice-cold Buffer C for 24 h, and then filter through a 0.22 μm filter membrane to obtain a refolded protein solution.
7. The preparation method according to claim 1, characterized in that, The ratio of TEV protease in the S6 refolded protein solution is 1:30 (w:w), and after mixing, it is allowed to stand at 4°C for 16 h. The nickel column has an NTA ion ligand type.
8. The preparation method according to claim 4, characterized in that, Buffer A: 100 mM Tris-HCl, 1 mM EDTA, pH 7.5; Buffer B: PBS, 2% Triton 100, pH 7.5; The Buffer C is PBS, pH 7.
5.
9. The preparation method according to claim 5, characterized in that, The Buffer D consists of 20 mM Tris-HCl, 15 mM β-mercaptoethanol, 6 M guanidine hydrochloride, and pH 8.
0. The Buffer E consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 8.
0. The Buffer F consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 6.
5. The Buffer G consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and pH 4.
5. The Buffer H consists of 50 mM NaH2PO4, 10 mM β-mercaptoethanol, 8 M urea, and 100 mM EDTA, with a pH of 8.
0.
10. The preparation method according to claim 6, characterized in that, Buffer I consists of 100 mM Tris-HCl, 0.5 M glycine, 1 mM oxidized glutathione, and 10 mM reduced glutathione, at pH 8.0.