Expression and purification method of human recombinant interleukin-15
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI WUXI BIOLOGIC TECH CO LTD
- Filing Date
- 2024-08-21
- Publication Date
- 2026-05-08
AI Technical Summary
The prior art is difficult to efficiently and stably express and purify recombinant human interleukin 15 (rhIL-15) in mammalian cells, resulting in low expression and unstable protein structure.
The fusion protein containing maltose binding protein (MBP) and recombinant human IL-15 was purified by co-transfection of mammalian cells and purified by specific cleavage sites and tags, including His tag affinity chromatography and enterokinase cleavage, and finally purified by ion exchange chromatography to obtain high purity rhIL-15.
Efficient and stable rhIL-15 expression and purification were achieved, and the obtained protein has form and high purity in natural state, and has significant cell proliferation effect.
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Abstract
Description
Expression and purification method of human recombinant interleukin-15 (1) Technical field
[0001] The present invention relates to the field of genetically engineered recombinant protein production, and in particular to a method for constructing a vector for expressing recombinant human interleukin 15 (rhIL-15), expressing it in cells, and purifying rhIL-15 on a large scale from cell fermentation broth. (2) Background technology
[0002] Interleukin (IL)-15 is a pleiotropic cytokine that plays a key role in innate and adaptive immunity. It primarily regulates the activation and proliferation of T cells and natural killer (NK) cells. Similar in structure to interleukin 2 (IL2), IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor β chain (CD122) and the common γ chain (γ-C, CD132). After viral infection, IL-15 is primarily secreted by mononuclear phagocytes in the body, inducing the proliferation of natural killer cells, thereby killing virus-infected host cells. In addition, it can maintain memory T cell responses to invading pathogens, inhibit apoptosis, and activate dendritic cells.
[0003] IL15 has a quadruple helical structure, typically a short helical cytokine, consisting of four helices arranged in an "up-up-down-down" pattern. The structure of IL15 is most similar to that of IL2, with the two proteins sharing a 39.5% sequence similarity. IL15 and IL-2 are most similar in the A, C, and D helical regions, which form a complex with IL-2Rb and the gc subunit of IL-2R to transmit signals.
[0004] Currently, recombinant human interleukin-15 (rhIL-15) has significant biological functions in promoting the activation and proliferation of NK cells and T cells, and enhancing CD8+ T cell anti-tumor immunity. However, most studies have primarily used Escherichia coli expression to obtain IL-15. Because the prokaryotic E. coli system lacks the corresponding translation and modification systems, the expressed protein structure differs from the natural structure. IL-15 expressed in mammalian cells may represent the natural form of the cytokine, with a more stable structure, a longer half-life, lower immunogenicity in the body, and better maintenance in vivo. IL-15 is approximately 14 kDa in size. Due to its small molecular weight and poor protein structure stability, IL-15 expression in mammalian cells is extremely low. Literature reports that IL-15 is immediately degraded after expression in HEK293 cells (Bergamaschi C, Rosati M, Jalah R, Valentin A, Kulkarni V, Alicea C, et al. Intracellular interaction of interleukin-15 with its receptor a during production leads to mutual stabilization and increased bioactivity. J Biol Chem 2008; 283: 4189-99). Therefore, there is still a need in the art to develop methods for the efficient, stable expression and purification of rh-IL-15.
[0005] (3) Summary of the invention
[0006] Therefore, the present invention provides a method for efficiently expressing recombinant human interleukin 15 (rhIL-15) and purifying the same.
[0007] In one aspect of the present invention, a fusion protein comprising maltose binding protein and recombinant human IL-15 is provided, characterized in that the fusion protein comprises the following elements from the 5' end to the 3' end: 5'-MBP-Furin cleavage site-His tag-enterokinase cleavage site (EK)-rhIL-15-3'.
[0008] In one embodiment of this aspect, the amino acid sequence of maltose binding protein (MBP) is SEQ ID NO: 3; or the amino acid sequence of the Furin cleavage site is SEQ ID NO: 4; or the amino acid sequence of the EK cleavage site is SEQ ID NO: 6.
[0009] In another embodiment of this aspect, the recombinant human IL-15 (rhIL-15) is selected from the amino acid sequence of SEQ ID NO: 6, conservative variants thereof, or homologous substitutions.
[0010] In a preferred embodiment of this aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence of SEQ ID NO: 1.
[0011] In another aspect of the present invention, a method for expressing and purifying recombinant human interleukin-15 in vitro in mammalian cells is disclosed, comprising the steps of:
[0012] a. Providing a first expression vector comprising a nucleic acid encoding a fusion protein of the present invention comprising maltose binding protein (MBP) and recombinant human IL-15, wherein the fusion protein comprises the following elements from the 5' end to the 3' end:
[0013] 5'-MBP-Furin cleavage site-His tag-enterokinase cleavage site (EK)-rhIL-15-3';
[0014] b. providing a second expression vector comprising a nucleic acid encoding furin;
[0015] c. co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing the cells to express recombinant human IL-15 protein;
[0016] d. Centrifugation to obtain the fermentation supernatant from step c above;
[0017] e. Obtain His-EK-rhIL-15 fragment by His tag affinity chromatography;
[0018] f. The fragment was digested with enterokinase and again purified by His tag affinity to collect the flow-through, which contained rhIL-15;
[0019] g. Purify the flow-through from step f by anion exchange chromatography to obtain purified rhIL-15 protein.
[0020] In one embodiment of this aspect, the amino acid sequence of maltose binding protein (MBP) is SEQ ID NO: 3; or the amino acid sequence of the Furin cleavage site is SEQ ID NO: 4; or the amino acid sequence of the EK cleavage site is SEQ ID NO: 6.
[0021] In another embodiment of this aspect, the recombinant human IL-15 (rhIL-15) is selected from the amino acid sequence of SEQ ID NO: 6, conservative variants thereof, or homologous substitutions.
[0022] In a preferred embodiment of this aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence of SEQ ID NO: 1.
[0023] In one embodiment of the method, the mammalian cell is a CHO-K1 cell or a HEK293 cell.
[0024] In another embodiment of the method, the His tag is a (His)6 tag.
[0025] In another preferred embodiment of the method, the nucleic acid of the fusion protein is cloned into the SalI / NotI site of the first expression vector.
[0026] In another embodiment of this aspect, the Furin protein is cloned into the SalI / NotI site of the second expression vector.
[0027] In a preferred embodiment of the method, the fermentation culture is transient transfection, cultured in a shaking incubator at 150 rpm for 6-7 days, and the culture temperature is lowered from 36.5° C. to 33° C. 24 hours after the completion of the transfection.
[0028] In another embodiment of the method, the fermentation culture supernatant is centrifuged at 4° C. and 10,000 revolutions (rpm), and the supernatant is collected.
[0029] In another preferred embodiment of the method, the affinity chromatography is performed using a Ni-Excel chromatography column, preferably an AKTA system.
[0030] In another embodiment of this aspect, the wash buffer of the affinity chromatography comprises 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0; and the elution buffer comprises: 50 mM PB, 200 mM NaCl, 400 mM imidazole, pH 8.0.
[0031] In a preferred embodiment of this aspect, the fusion protein product is digested with 0.05% (W / W) enterokinase in 50 mM Tris, 100 mM NaCl, pH 8.0 buffer, preferably by incubation at 4°C overnight.
[0032] In a preferred embodiment of step f, the impurity protein with the His tag is affinity adsorbed under the same conditions as step e, and the flow-through is collected to obtain the rhIL-15 product without the tag.
[0033] In a preferred embodiment of step g, the ion exchange chromatography column is a Q-HP column, and the equilibration buffer of the ion exchange chromatography column is preferably 20 mM Tris, pH 8.0; and the elution buffer of the ion exchange chromatography column is preferably 20 mM Tris, 1 M NaCl, pH 8.0.
[0034] In another preferred embodiment of step g, step g includes the following sub-steps:
[0035] The product from step f was diluted ten-fold with the equilibration buffer to a conductivity of less than 8 ms, and then loaded onto a 5 mL Q-HP column at a flow rate of 5 mL / min. After loading, the column was gradient eluted with the elution buffer, with the elution buffer ratio increasing from 0 to 50% within 50 column volumes at an elution flow rate of 2.5 mL / min, and the eluted protein was collected.
[0036] The method of the present invention has the following advantages:
[0037] The method offers simple operating conditions and excellent reproducibility, yielding hrIL-15 in its native form with high purity. Mass spectrometry analysis indicates that the purified IL-15 is over 95% pure, with a molecular weight of 12,770.26 Da. Cell proliferation assays demonstrate that recombinantly expressed rhIL-15 in mammalian cells from various sources exhibits potent cell proliferation-promoting effects, confirming its functional activity, if not superior, to that of its native form. (4) Description of the accompanying drawings
[0038] Figure 1 depicts the plasmid maps of the recombinant expression vectors pWX4.1 / IL-15 ( Figure 1 a ) and pWX155 / Furin ( Figure 1 b );
[0039] Figures 2a and 2b respectively depict schematic diagrams of SDS-PAGE analysis of IL-15 expressed in CHO-K1 (2a) and HEK293 cells (2b) after one-step affinity purification using Ni-Excel; the symbols represent: S: supernatant; FT: flow-through; E: eluate; M: standard protein; NR: non-reduced state; R: reduced state.
[0040] Figures 3a and 3b show the protease cleavage profiles after Ni-Excel purification. Figure 3a shows an SDS-PAGE of IL-15 expressed in CHO-K1 cells before and after enterokinase digestion; Figure 3b shows an SDS-PAGE of IL-15 expressed in HEK293 cells before and after enterokinase digestion. Symbols indicate: M: standard protein; NR: non-reduced state; R: reduced state; B: before digestion; A: after digestion.
[0041] Figures 4a and 4b describe the SDS-PAGE images of the flow-through after the His-tag was adsorbed by Ni-Excel affinity chromatography after enzyme cleavage; Figure 4a is an SDS-PAGE image of the flow-through after the His-tag was adsorbed by Ni-Excel affinity chromatography; Figure 4-2 is an SDS-PAGE image of the flow-through after the His-tag was adsorbed by Ni-Excel affinity chromatography; the symbols represent: S: supernatant; FT: flow-through; E: eluate; M: standard protein; NR: non-reduced state; R: reduced state.
[0042] Figures 5a and 5b depict SDS-PAGE images of IL-15 purified using Q-HP ion exchange chromatography. Figure 5a depicts IL-15 expressed in CHO-K1 cells; Figure 5b depicts IL-15 expressed in HEK293 cells. Figure 5c depicts SDS-PAGE images of the purified protein samples from CHO-K1 cells and HEK293 cells after deglycosylation. Symbols denote M: standard protein; NR: non-reduced state; and R: reduced state, respectively.
[0043] Figures 6a and 6b are LC-MS mass spectrometry analysis patterns; Figure 6a is the LC-MS analysis of IL15 expressed from CHO-K1; Figure 6b is the LC-MS analysis of IL15 expressed from HEK293.
[0044] FIG7 shows the cell activity detection of the positive control and hrIL-15 produced in Example 1. (5) Specific implementation methods
[0045] definition
[0046] As used herein, unless the context clearly dictates otherwise, singular forms beginning with "a," "an," and "the" include plural references. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0047] As used herein, the terms "about," "approximately," or "approximately," when preceding a numerical value, refer to a range defined by approximately 1%, 2%, 3%, 4%, 5%, 10% or more around the specified value.
[0048] In the present disclosure, one or more features in one embodiment may be combined with any one or more features in another embodiment without departing from the spirit and concept of the present invention.
[0049] In this disclosure, unless otherwise specified, all ranges, including ranges defined as between two specified endpoints, include the specified endpoints. For example, a range between 1 and 10 means a range between 1 and 10 (inclusive).
[0050] In this document, when a duration, period or interval is expressed in days, and when a time point is expressed as a day or a certain day, it means that the duration or moment is measured or divided by days (days), and the numerical value is not required to represent a multiple of 24 hours.
[0051] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into the disclosure herein. The citations are intended solely to illustrate the level of skill of those skilled in the art and do not constitute prior art prior to the present invention.
[0052] As described herein, "human recombinant IL-15" can be any genetically engineered modified IL-15 protein that retains IL-15 biological activity. It may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to the naturally derived mature interleukin-15 (GenBank: CR542007.1), and retain its biological activity. The interleukin-15 proteins targeted by the methods of the present invention include various functional recombinant forms thereof. Commercially available examples include recombinant human IL-15 protein produced from HEK293 cells (T&L Bitechnology, GMP-TL202); recombinant human IL-15R alpha (I31-T172) & IL-15 (N49-S162) fusion protein produced from HEK293 cells (MedChemExpress, HY-P70655), and the like. The amino acid sequence of the recombinant IL-15 protein used in the examples is shown in SEQ ID NO: 6, and its conservative variants, homologous substitutions, or functional fragments may also be used.
[0053] The mammalian cells described herein can be derived from a variety of mammalian sources, examples of which include, but are not limited to, primates (e.g., humans and non-human primates such as chimpanzees, baboons, or monkeys), dogs, cats, pigs, sheep, rabbits, mice, and rats. In some cases, the mammal can be a human.
[0054] Preferred mammalian cells are CHO cells and HEK293 cells. Other mammalian cells can also be used, such as NSO, etc.
[0055] The term "expression vector" refers to a nucleic acid molecule when used herein, which can guide the expression of a gene operatively connected thereto after entering a host cell. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which other DNA fragments can be joined. Another type of vector is a viral vector, in which other DNA fragments can be joined to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated together with the host genome. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (for example, electroporation, lipofection). Enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions or this area generally realized or as described herein. The aforementioned techniques and procedures can generally be performed according to the conventional methods described in the various general and more specific references as well-known in the art and as cited and discussed throughout the specification of the present invention.
[0056] As used herein, the term "fusion protein" refers to a protein composed of at least two domains encoded by separate genes that have been linked together to be transcribed and translated as a single unit, thereby producing a polypeptide. Currently, almost all recombinant proteins in the art are prepared using a fusion domain (also known as a "tag"). Fusion proteins are also known as fusion-tagged proteins or chimeric proteins.
[0057] Preferably, the fusion protein tag for increasing expression includes maltose binding protein (MBP), which is a member of the Escherichia coli maltose transport system and is primarily responsible for the capture and transport of maltose. The present invention uses MBP for fusion expression of recombinant proteins because it has advantages such as high expression levels and ease of purification. MBP is widely used as a fusion tag. Those skilled in the art will appreciate that MBP can also be replaced by other fusion protein tags that increase expression, such as glutathione transferase GST, small ubiquitin-modified protein (SUMO), transcription termination anti-termination factor (NusA), etc.
[0058] The fusion protein of the present invention has the following structure from 5' to 3':
[0059] 5'-MBP-Furin cleavage site-His tag-enterokinase cleavage site (EK)-rhIL-15-3', the amino acid sequence (SEQ ID NO: 1) comprises:
[0060] in:
[0061] 1. The MBP tag (SEQ ID NO: 3) is in normal font.
[0062] 2. The Furin cleavage site (SEQ ID NO: 4) is in italics, not underlined, and not bold. The EK cleavage site (SEQ ID NO: 5) is in italics, underlined, and not bold. The amino acid sequence of recombinant IL-15 (SEQ ID NO: 6) is in bold and underlined.
[0063] 4. His tag (SEQ ID NO: 7) is in bold without underlining.
[0064] The fusion protein may optionally have an IgG signal peptide sequence at the 5' end, preferably, e.g.
[0065] The domains of the fusion protein are preferably connected by a short oligopeptide (linker), and the linker in the present invention is preferably a His tag; other tags can also be used in combination with a specific affinity chromatography column.
[0066] The plasmid used in this article is preferably a shuttle expression plasmid that can be expressed in Escherichia coli and mammalian cells, including restriction enzyme sites (SalI / NotI), a replication origin site, such as OriP, pUC ori, a marker gene, such as ampicillin resistance or tetracycline resistance, etc., a size of 3948 bp, and a signal peptide sequence, such as HSV thymidine kinase polyadenylation signal (TK pA), etc.
[0067] As used herein, "affinity chromatography" is a chromatographic method that utilizes the binding properties of a stationary phase to separate molecules. Affinity chromatography involves attaching molecules with a specific binding capacity to the substance to be separated to a gel filtration column. A preferred affinity chromatography method for the present invention is an affinity chromatography ligand for the His tag, nickel ions, chelated to a macroporous affinity solid support, IMAC resin. Alternatively, commercially available affinity columns such as Ni-Excel (Cytiva, Catalog No. 17371201) for the tag peptide can be used.
[0068] As used herein, the term "reverse purification" refers to the removal of impurities by adsorbing impurity proteins via affinity chromatography, allowing the protein to flow through. Chromatographic columns commonly used in the art can be easily determined based on the impurities and target protein observed during chromatography. For example, the Ni-Excel chromatography column used herein can adsorb impurity proteins with a His tag.
[0069] Enterokinase as used herein is an enzyme that specifically recognizes its binding motif (enzyme cleavage site) to cleave the fusion protein domain, and is available from Shanghai WuXi Biologics Co., Ltd.
[0070] The ion exchange chromatography column used herein is used for further fine purification of IL-15 protein and can be selected from ion exchange chromatography columns Poros 50 HQ or Q-HP. In this study, the Q-HP column is preferred.
[0071] The molecular weight of the rhIL-15 of the present invention can be determined by LC-MS, etc., and the proliferation stimulating ability of IL-15 can be verified using, for example, the Mo7e cell proliferation assay (Avanzi GC, Brizzi MF, Giannotti J, Ciarletta A, Yang YC, Pegoraro L, Clark SC. M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J Cell Physiol. 1990 Dec; 145(3): 458-64. doi: 10.1002 / jcp.1041450310. PMID: 2273055.).
[0072] Example 1 Expression and purification of recombinant human IL-15
[0073] 1. Materials and Methods
[0074] 1) The equipment and materials used in this study are shown in Tables 1 and 2
[0075] Table 1 Equipment
[0076] Table 2: Reagents and Equipment
[0077] 2) Methods:
[0078] Recombinant vector construction: The full gene synthesis expression component of the present invention, the MBP-Furin restriction site-His-EK-IL15 gene (the coding sequence of the fusion protein shown in SEQ ID NO: 1) and the Furin gene (Uniprot accession number P09958, https: / / www.uniprot.org / uniprotkb / P09958 / entry), was constructed and respectively constructed into the expression vectors pWX155 and pWX4.1 (provided by Shanghai WuXi Biotech Co., Ltd., the backbone of the vector is the commercial pTT5 vector backbone), to obtain the recombinant expression vectors pWX155 / IL-15 and pWX4.1 / Furin, which were then transformed into the E. coli BL21 / DH5α host bacteria. The recombinant plasmid culture containing the specified sequence was streaked onto a solid LB plate containing ampicillin sodium and cultured in an inverted manner at 37°C overnight. A single colony was picked from the plate and expanded into 200 mL of LB medium (Sigma, Catalog # L7658) containing ampicillin sodium. Plasmid extraction was then performed according to the NucleoBond Xtra Midi EF Kit (MACHEREY-NAGEL, Catalog # 740420.50) instructions. Sufficient amounts of recombinant plasmids pWX155 / IL-15 and pWX4.1 / Furin were obtained. The pWX155 / IL-15 and pWX4.1 / Furin plasmids were diluted, and their concentrations were determined using a Nano-Drop (Thermo Scientific, Model # ND2000). Gene sequencing was then performed to facilitate subsequent transfection and expression.
[0079] ii) Transient transfection expression: Using the WuXi Biologics Protein Science platform protein expression platform process, CHO-K1 host cells were diluted to the specified density using the manufacturer's platform process medium. The pWX155 / IL-15 and pWX4.1 / Furin plasmids were mixed at a mass ratio of 5:1. The premixed plasmids and transfection reagent, polyethyleneimine (PEI), were then added to CHO-K1 and HEK293 cells, respectively, to complete transfection (using the platform-specified amounts). A 5-L volume was used for each host cell. The cells were then cultured in a shaker at 36.5°C, 120 RPM, 6% CO₂, and 85% humidity. The day of transfection was designated Day 0. On Day 1, the shaker settings were adjusted to 31°C, 120 RPM, 6% CO₂, and 85% humidity. Culture continued under these shaker settings until Day 7, when the cells were harvested. To ensure nutrient supply during cell culture, feed medium and glucose were added to the cells at a certain ratio on Day 0 and Day 4 after transfection according to the platform process. Cells were harvested on Day 7 for downstream purification.
[0080] iii) Affinity chromatography: The cell suspension expressed on day 7 was centrifuged at 10,000 × g for 30 minutes using a floor-standing centrifuge. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain a clarified supernatant containing the target protein. First, 25mL Ni-Excel was used to capture the target protein. Specifically, an AKTA chromatography system was used. The sample was first loaded at a flow rate of 15ml / min. 50mM PB, 200mM NaCl, 10mM imidazole, pH8.0 was used as the EQ balance solution to balance five column volumes to flush the baseline. 50mM PB, 200mM NaCl, 10mM imidazole, pH8.0, 0.1% TX114 (V / V) was used as Wash2 (washing buffer) to wash 12 column volumes for no less than 60 minutes to remove endotoxins. EQ balance solution was then used to wash for 6 column volumes to balance. 50mM PB, 200mM NaCl, 40mM imidazole, pH8.0 was used as the eluent to elute the target protein at a flow rate of 2.5ml / min for 7 column volumes. 0.5M NaOH was then used to regenerate the column at a flow rate of 5ml / min for 15 minutes. Finally, EQ was used to regenerate the column. The AKTA baseline was flushed with 10 column volumes of equilibration buffer. The eluate was collected as the target protein from the first affinity chromatography step and analyzed by SDS-PAGE. Figure 2a shows the supernatant, flow-through, and eluate from CHO-K1-expressed IL-15 in non-reducing and reducing gels; Figure 2b shows the supernatant, flow-through, and eluate from HEK293-expressed IL-15 in non-reducing and reducing gels. SDS-PAGE revealed that the target protein was essentially captured by the Ni-Excel in the first step, yielding a relatively pure His-tagged IL-15 protein.
[0081] iv) Enterokinase digestion: To remove the His tag, the protein eluted from the affinity chromatography was digested with enterokinase. The eluate was first dialyzed into a 50 mM Tris, 100 mM NaCl, pH 8.0 buffer. 0.05% w / w enterokinase solution was then added to the protein solution, and digestion was carried out overnight at 4°C. The SDS-PAGE before and after digestion is shown in Figure 3. Before digestion, i.e., column B, there was a distinct band at approximately 15 kDa. After digestion, i.e., column A, the target band at approximately 15 kDa was lower than the pre-digestion band, indicating that digestion was initially completed.
[0082] v) Ni-Excel Reverse Purification: After enzymatic digestion, the His-tag and IL-15 protein remain in the protein solution. Ni-Excel is used to affinity capture the His-tagged impurities again, using the same purification method and conditions as the affinity chromatography step above. After purification, the flow-through is collected to obtain the untagged IL-15 protein. As shown in Figure 4, the protein band in the FT lane is primarily concentrated around 15 kDa, and the purity is significantly improved.
[0083] vi) Q-HP Fine Purification: To further purify the IL-15 protein, this study used a Q-HP ion exchange chromatography column for purification. 20 mM Tris, pH 8.0, was used as the equilibration buffer (Buffer A), and 20 mM Tris, 1 M NaCl, pH 8.0, was used as the elution buffer (Buffer B). The digested IL-15 protein solution was first diluted tenfold with Buffer A equilibration buffer to a conductivity below 8 ms. The protein was then loaded onto a 5 mL Q-HP column at a flow rate of 5 mL / min. Following loading, the column was eluted using a gradient of Buffer B, increasing the Buffer B ratio from 0 to 50% over 50 column volumes at a flow rate of 2.5 mL / min. The eluted protein was collected to obtain pure IL-15. SDS-PAGE gel analysis is shown in Figure 5, showing that the purity of IL-15 from both sources was significantly improved after purification. The target bands in the SDS-PAGE images in Figures 5a and 5b appeared fuzzy, due to the numerous glycosylation sites in IL-15. The proteins were deglycosylated and re-run on the gel. As shown in Figure 5c, the target protein appeared as a single band on the SDS-PAGE, confirming that the fuzzy bands in the gel images were due to glycosylation. This study ultimately yielded 52.56 mg of IL-15 protein from 5L of CHO K1 cells and 47.52 mg of IL-15 protein from 5L of HEK293 cells.
[0084] LC-MS verification: The molecular weight of the purified IL-15 was confirmed by LC-MS. As shown in Figure 6, the theoretical molecular weight of the protein is 12773.50. The molecular weight measured by LC-MS analysis is 12770, which is consistent with the theoretical molecular weight of the protein, confirming that it is IL-15 protein.
[0085] Example 2 IL-15 activity detection
[0086] The activity of the two IL15 proteins obtained from different sources in Example 1 was verified using the Mo7e cell proliferation assay. The two proteins were numbered 20221109-HEK and 20221109-CHO, respectively. A commercial IL15 protein was used as a positive control, numbered GMP-10360-HNAE.
[0087] 1. Materials and Methods
[0088] The instruments and reagents used in this experiment are shown in Table 1 and Table 2 of Example 1 above.
[0089] 2. Experimental Procedures: (The experimental procedures used in this example can be found in the reference: Avanzi GC, Brizzi MF, Giannotti J, Ciarletta A, Yang YC, Pegoraro L, Clark SC. M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J Cell Physiol. 1990 Dec; 145(3): 458-64. doi: 10.1002 / jcp.1041450310. PMID: 2273055)
[0090] 1) Cell culture: Cultivate cells until the flask is 80%-90% full.
[0091] 2) Cell seeding: Collect Mo7e cells, count them, and seed them into 96-well plates at a density of 20k / well. Place them in a CO2 incubator and incubate them (incubator conditions: 37°C, 5% CO2);
[0092] 3) Sample Addition: Dilute the IL-15 and positive control from Example 1 from an initial concentration of 1.08 mg / ml to 1000 ng / ml (detected using a spectrophotometer), then sequentially dilute the sample three-fold in 10 steps. Add the protein sample to the cell culture using a pipette and incubate in a 37°C, 5% CO2 incubator for 72 h.
[0093] 4) Detection: After the incubation, WST8 (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazole monosodium salt) was added to detect the OD450 value.
[0094] Analysis of the samples' effects on Mo7e cell proliferation, as shown in Figure 7, shows that both IL-15 proteins from different sources produced in Example 1 promoted Mo7e cell proliferation. Table 3 below lists the ED values for stimulation. As shown in Table 3, the ED50 values for both IL-15 proteins obtained in Example 1 were lower than those of the positive control, demonstrating excellent cell proliferation-promoting activity.
[0095] Table 3: Summary of sample activity results
[0096] Although the above disclosure has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it is obvious to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit and scope of the disclosure described in the appended claims. Therefore, the above description should not be construed as limiting the scope of the disclosure.
Claims
1. A fusion protein, characterized in that The fusion protein includes the following elements from the 5' end to the 3' end: 5'-MBP-Furin enzyme cleavage site-His tag-enterokinase enzyme cleavage site (EK)-rhIL-15-3'.
2. The fusion protein as claimed in claim 1, wherein the amino acid sequence of maltose binding protein (MBP) is SEQ ID NO: 3; or the amino acid sequence of the Furin cleavage site is SEQ ID NO: 4; or the amino acid sequence of the EK cleavage site is SEQ ID NO:
6.
3. The fusion protein of claim 1, wherein the recombinant human IL-15 (rhIL-15) is selected from the amino acid sequence of SEQ ID NO: 6, conservative variants thereof, or homologous substitutions. The fusion protein according to claim 1 , comprising the amino acid sequence of SEQ ID NO:
1.
5. A method for expressing and purifying recombinant human interleukin-15 in vitro in mammalian cells, comprising the steps of: a. Providing a first expression vector, the first expression vector comprising a nucleic acid encoding the fusion protein of any one of claims 1-4; b. providing a second expression vector, the second expression vector comprising a nucleic acid encoding Furin; c. co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing the cells to allow the cells to express recombinant human IL-15 protein; d. Centrifuge the fermentation supernatant obtained from step c above; e. Obtain His-EK-rhIL-15 fragment by His tag affinity chromatography; f. digesting the fragment with enterokinase, affinity purifying the fragment again by His tag, and collecting the flow-through, which contains rhIL-15; g. Purify the flow-through from step f by anion exchange chromatography to obtain purified rhIL-15 protein. The method of claim 5 , wherein the mammalian cell is a CHO-K1 cell or a HEK293 cell.
7. The method of claim 5, wherein the nucleic acid sequence encoding the fusion protein is cloned into the SalI / NotI site of the first expression vector.
8. The method of claim 5, wherein the furin encoding nucleic acid sequence is cloned into the SalI / NotI site of the second expression vector.
9. The method according to claim 5, wherein the fermentation culture is transient transfection, cultured in a shaking incubator at 150 rpm for 6-7 days, and the culture temperature is lowered from 36.5°C to 33°C 24 hours after the completion of the transfection.
10. The method of claim 1, wherein the wash buffer of the affinity chromatography comprises 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0; the elution buffer comprises: 50 mM PB, 200 mM NaCl, 400 mM imidazole, pH 8.0; or the fusion protein product is digested with 0.05% (W / W) enterokinase in 50 mM Tris, 100 Mm NaCl, pH 8.0 buffer, preferably incubated overnight at 4°C; or in step f, the impurity protein with a His tag is affinity adsorbed under the same conditions as in step e, and the flow-through is collected to obtain an untagged rhIL-15 product; or in step g, the ion exchange chromatography column is a Q-HP column, and preferably the equilibration buffer of the ion exchange chromatography column is 20 mM Tris, pH 8.0; preferably the elution buffer of the ion exchange chromatography column is 20 mM Tris, 1 M NaCl, pH 8.0; or in step g, the following sub-steps are included: The product from step f was diluted ten-fold using the equilibration buffer to make its conductivity lower than 8 ms, and then loaded onto a 5 mL Q-HP column at a flow rate of 5 mL / min. After loading, gradient elution was performed using the elution buffer, with the elution buffer ratio ranging from 0 to 50% within 50 column volumes, and the elution flow rate was 2.5 mL / min, and the eluted protein was collected.