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CN-122003444-A - Expression and purification method of human recombinant interleukin-15

CN122003444ACN 122003444 ACN122003444 ACN 122003444ACN-122003444-A

Abstract

There is provided a method for in vitro expression of purified recombinant human interleukin 15 in mammalian cells comprising the steps of a. Providing a first expression vector comprising a nucleic acid encoding a fusion protein of Maltose Binding Protein (MBP) and recombinant human IL-15 comprising from the 5 'end to the 3' end the elements 5'-MBP-Furin cleavage site-His tag-enterokinase cleavage site (EK) -rhIL-15-3', b. Providing a second expression vector comprising a nucleic acid encoding Furin protein (Furin), c. Co-transfecting the first expression vector and the second expression vector to mammalian cells, fermenting the cells for expression of recombinant human IL-15 protein, d. Centrifuging to obtain a fermentation supernatant from step c above, e. Obtaining His-EK-rhIL-15 fragment by His tag affinity chromatography, f. Cleaving the fragment with enterokinase, again purifying by His tag affinity, collecting the stream, and obtaining a purified rhg from the stream of the stream-rhg-15.

Inventors

  • ZHANG DAWEI
  • WU JIANSHENG
  • Cui Lupeng
  • XIAO LIAN

Assignees

  • 上海药明生物技术有限公司

Dates

Publication Date
20260508
Application Date
20240821
Priority Date
20230927

Claims (10)

  1. A fusion protein is characterized by comprising the following elements from the 5 'end to the 3' end, namely a 5'-MBP-Furin restriction site, a His tag, an enterokinase restriction site (EK) -rhIL-15-3'.
  2. The fusion protein of claim 1, wherein the amino acid sequence of Maltose Binding Protein (MBP) is SEQ ID NO 3, or the amino acid sequence of Furin cleavage site is SEQ ID NO 4, or the amino acid sequence of EK cleavage site is SEQ ID NO 6.
  3. The fusion protein of claim 1, wherein recombinant human IL-15 (rhIL-15) is selected from the amino acid sequence of SEQ ID No. 6, conservative variants thereof, or homologous substitutions.
  4. The fusion protein of claim 1, comprising the amino acid sequence of SEQ ID NO. 1.
  5. A method for in vitro expression of purified recombinant human interleukin 15 in mammalian cells comprising the steps of: a. Providing a first expression vector comprising a nucleic acid encoding the fusion protein of any one of claims 1-4; b. Providing a second expression vector comprising a nucleic acid encoding Furin (Furin); c. Co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing to enable the cells to express recombinant human IL-15 protein; d. Centrifuging to obtain a fermentation supernatant from step c above; e. Obtaining His-EK-rhIL-15 fragments through His tag affinity chromatography; f. Cleaving the fragment with enterokinase, again by His-tag affinity purification, collecting a flow-through comprising rhIL-15; g. Anion exchange chromatography purifying the flow-through from step f to obtain purified rhIL-15 protein.
  6. The method of claim 5, wherein the mammalian cell is a CHO-K1 cell or 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 of claim 5, wherein the fermentation culture is transient transfection and is performed in a shaker at 150rpm for 6-7 days at a temperature of 36.5 ℃ to 33 ℃ 24h after completion of transfection.
  10. The method of claim 1, wherein the rinse buffer of affinity chromatography comprises 50mM PB,200mM NaCl,10mM imidazole, ph8.0, the elution buffer comprises 50mM PB,200mM NaCl,400mM imidazole, ph8.0, or the fusion protein product is digested with 0.05% (W/W) enterokinase in 50mM Tris,100Mm NaCl,pH8.0 buffer, preferably incubated overnight at 4 ℃, or in step f the His-tagged impurity protein is affinity adsorbed with the same conditions of step e, the flow-through is collected to obtain the unlabeled rhIL-15 product, or in step g the ion exchange chromatography column is a Q-HP column, preferably the equilibration buffer of the ion exchange chromatography column is 20mm tris, ph8.0, preferably the elution buffer of the ion exchange chromatography column is 20mM Tris,1M NaCl,pH8.0, or in step g the following sub-steps are included: diluting the product from step f ten times with the equilibration buffer to a conductance of less than 8ms, then loading the product into a 5mL Q-HP column at a flow rate of 5 mL/min, performing gradient elution with the elution buffer, enabling the elution buffer ratio to be from 0 to 50% in 50 column volumes, eluting at a flow rate of 2.5 mL/min, and collecting the eluted protein.

Description

Expression and purification method of human recombinant interleukin-15 (1) Technical field The invention relates to the field of recombinant protein production by genetic engineering, in particular to a method for constructing a vector for expressing recombinant human interleukin 15 (rhIL-15), expressing the recombinant human interleukin 15 in cells and purifying rhIL-15 from cell fermentation liquor in a large scale. (2) Background art Interleukin (IL) -15 is a pleiotropic cytokine that plays a key role in innate and adaptive immunity. It mainly regulates activation and proliferation of T cells and Natural Killer (NK) cells, similar in structure to interleukin 2 (IL 2), IL-15 binds and signals through a complex consisting of the IL-2/IL-15 receptor beta chain (CD 122) and the common gamma chain (γ -C, CD 132). After being infected by viruses, IL-15 is secreted in the body mainly by mononuclear phagocytes, inducing proliferation of natural killer cells, thereby killing virus-infected host cells. In addition, the cell can also maintain the response of memory T cells to invading pathogens, inhibit apoptosis, activate dendritic cells and other functions. IL15 has a four-helix structure of short-chain helical cytokines, i.e., a "top-bottom" four-helix structure. IL15 is most similar in structure to IL2, with two protein sequences up to 39.5% similar, and IL15 and IL-2 are most similar in the A, C, D helical region, which form a complex with the gc subunit of IL-2Rb and IL-2R to signal. At present, recombinant human interleukin 15 (rhIL-15) has remarkable biological functions in promoting NK cells and T cells to activate proliferation and enhancing anti-tumor immunity of CD8+ T cells. However, most researches mainly obtain IL-15 through escherichia coli expression, and the expressed protein structure is different from the natural structure due to the fact that a escherichia coli system of a prokaryote lacks a corresponding translation and modification system. The IL-15 expressed by mammalian cells may represent a natural form of cytokine, is more stable in structure, has a longer half-life, is less immunogenic in the in vivo system, and is more easily maintained in vivo. IL-15 is about 14kDa in size, and because of small molecular weight and poor structural stability of protein, the expression level of IL-15 in mammalian cells is extremely low, and it is reported in literature that IL-15 is degraded (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). immediately after expression in HEK293 cells, so there is still a need in the art to develop a method for efficiently and stably expressing and purifying rh-IL 15. (3) Summary of the invention Thus, the present invention provides a method for highly expressing recombinant human interleukin 15 (rhIL-15) and its purification. In one aspect of the invention, there is provided a fusion protein comprising a maltose binding protein and recombinant human IL-15, 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'. 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 Furin cleavage site is SEQ ID NO 4, or the amino acid sequence of EK cleavage site is SEQ ID NO 6. In another embodiment of this aspect, recombinant human IL-15 (rhIL-15) is selected from the amino acid sequence of SEQ ID NO. 6, a conservative variant thereof, or a homologous substitution. In a preferred embodiment of this aspect, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO. 1. In another aspect of the invention, a method for expressing purified recombinant human interleukin 15 in mammalian cells in vitro is disclosed, comprising the steps of: a. providing a first expression vector comprising a nucleic acid encoding a fusion protein of a Maltose Binding Protein (MBP) of the invention with recombinant human IL-15, the fusion protein comprising the following elements from 5 'to 3': 5'-MBP-Furin cleavage site-His tag-enterokinase cleavage site (EK) -rhIL-15-3'; b. Providing a second expression vector comprising a nucleic acid encoding Furin (Furin); c. Co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing to enable the cells to express recombinant human IL-15 protein; d. Centrifuging to obtain a fermentation supernatant from step c above; e. Obtaining His-EK-rhIL-15 fragments through His tag affinity chromatography; f. Cleaving the fragment with enterokinase, again by His-tag affinity purification, collecting a flow-through comprising rhIL-15; g. Anion exchange chromatography purifying the flow-through from step