阅读说明：本技术 一种纯化重组人干扰素-κ的方法和试剂盒 (Method and kit for purifying recombinant human interferon-kappa ) 是由 彭继先 李振森 晁香君 吴桂苹 韩景花 于 2020-12-25 设计创作，主要内容包括：本发明公开了一种纯化重组人干扰素-κ(rhIFN-κ)的方法和试剂盒。所述方法包括：1)将含rhIFN-κ的复性稀释液进行阴离子交换层析,收集流穿液；2)将步骤1)获得的流穿液进行阳离子交换层析,收集洗脱液；3)稀释步骤2)的洗脱液；和4)将步骤3)的稀释液进行阳离子交换层析。本发明的分离纯化方法采用三步层析进行分离,纯化步骤少,避免重组人干扰素-κ聚集,提高蛋白收率,纯度高达93％以上,且成本低、操作简单、可重复性高,适合工业化生产。(The invention discloses a method and a kit for purifying recombinant human interferon-kappa (rhIFN-kappa). The method comprises the following steps: 1) carrying out anion exchange chromatography on the renaturation diluent containing the rhIFN-kappa, and collecting flow-through liquid; 2) carrying out cation exchange chromatography on the flow-through liquid obtained in the step 1), and collecting eluent; 3) diluting the eluate of step 2); and 4) subjecting the dilution of step 3) to cation exchange chromatography. The separation and purification method provided by the invention adopts three-step chromatography for separation, has few purification steps, avoids recombinant human interferon-kappa aggregation, improves the protein yield, has the purity of over 93%, and is low in cost, simple to operate, high in repeatability and suitable for industrial production.)

1. A method of purifying recombinant human interferon- κ (rhIFN- κ), comprising the steps of:

1) carrying out anion exchange chromatography on the renaturation diluent containing the rhIFN-kappa, and collecting flow-through liquid;

2) carrying out cation exchange chromatography on the flow-through liquid obtained in the step 1), and collecting eluent;

3) diluting the eluate obtained in step 2);

4) subjecting the dilution obtained in step 3) to cation exchange chromatography.

2. The method according to claim 1, characterized in that the anion exchange chromatography in step 1) is a strong anion exchange chromatography and the cation exchange chromatography in steps 2) and 4) is a strong cation exchange chromatography.

3. The method of claim 2, wherein the anion exchange chromatography of step 1) uses an ion exchange medium with quaternary ammonium groups as ligands.

4. The method according to claim 2, wherein the cation exchange chromatography of step 2) and step 4) uses an ion exchange medium having a sulfonic acid group as a ligand.

5. The method according to claim 1, characterized in that in step 3), the eluent obtained in step 2) is mixed in a ratio of 1: (0.5-10) diluting with a buffer solution.

6. The method according to claim 1, wherein the elution is performed in step 2) using buffer B, buffer C and an aqueous NaCl solution, and the elution is performed in step 4) using buffer E, buffer F and an aqueous NaCl solution, wherein the buffer B, buffer C, buffer E and buffer F are different from each other.

7. A kit for carrying out the method of claims 1-6, comprising: strong anion exchange media for anion exchange chromatography and strong cation exchange media for cation exchange chromatography.

8. The kit of claim 7, wherein the strong anion exchange medium is an ion exchange medium having quaternary ammonium groups as ligands; and the strong cation exchange medium is an ion exchange medium with sulfonic group as ligand.

9. Use of the kit according to claim 7 or 8 for the preparation or purification of interferon.

10. The use according to claim 9, wherein the interferon is interferon- κ; preferably, the interferon is human interferon- κ; more preferably, the interferon is recombinant human interferon- κ.

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a separation and purification method of recombinant human interferon-kappa (rhIFN-kappa), in particular to a method for purifying rhIFN-kappa by using strong anion and strong cation exchange media.

Background

Interferons (IFNs) are a class of cytokines produced by monocytes and immune cells with broad-spectrum antiviral, antitumor, immunomodulatory effects that activate natural killer cells and exert antiviral activity by binding to cognate receptors on target cells. Interferons are generally classified into type I interferons, including IFN- α, IFN- β, and IFN- ω, and type II interferons, including IFN- γ. These different types of interferons are encoded by different genes and have different biological activities. Currently, much research has been devoted to further improving the isolation and purification of IFN- α, IFN- β and IFN- γ. For example, CN102219848B discloses a method for purifying recombinant human interferon beta-1 a, which adopts affinity chromatography, strong anion exchange chromatography and weak cation exchange chromatography to obtain high-purity recombinant human interferon beta-1 a; CN1259336C discloses a method for purifying consensus interferon, which comprises dialysis and cation exchange chromatography, to realize separation of renaturation intermediate and completely renatured protein and obtain high-purity protein product; CN101928341B discloses a purification process for separating recombinant human interferon alpha-1 b isomer and a detection method thereof, which adopts QHP chromatography and pH gradient elution to separate target protein from other related proteins; CN1229389C discloses the use of immunomagnetic separation technology to separate recombinant interferon, which uses immunomagnetic microspheres and magnetic separation device to separate and purify zymocyte lysate. CN103014101A provides a purification method of interferon, which adopts C18 high performance liquid column chromatography column to purify interferon, the method has higher later amplification cost, and meanwhile, when reverse phase chromatography is utilized, the environmental protection pressure of elution by organic reagent is large, and the method is not easy to be amplified to production. Therefore, a general method suitable for separating and purifying various types of interferons does not exist at present.

Interferon kappa (IFN-. kappa.) is a new member of IFN discovered in recent years that uses a receptor protein in conjunction with IFN-. alpha.and IFN-. beta.and is a type I interferon. IFN- κ is capable of activating interferon-stimulated genes and inhibiting replication of encephalomyocarditis virus (ECMV) and Human Papilloma Virus (HPV). The gene encoding IFN-kappa is selectively expressed in epithelial keratin cells, and recombinant hIFN-kappa, similar to interferons of other isotypes, can also be used to protect cells from viral infection and expression is race specific.

At present, research on IFN-kappa has few documents, and research on specific preparation technology of IFN-kappa is rare, especially the technical scheme in the aspect of industrial production. From the analysis of IFN-kappa amino acid composition sequence, it is known that IFN-kappa protein has high hydrophobicity and is easy to generate polymerization inactivation in a water phase system; the IFN-kappa amino acid sequence contains 5 sulfydryl groups, wherein C1-C3 and C2-C4 form two pairs of disulfide bonds to form a three-dimensional structure necessary for the biological activity of the IFN-kappa. The research on the separation of IFN-kappa protein by using an organic solvent-n-butyl alcohol extraction method solves the problem that IFN-kappa hydrophobic is easy to polymerize, but two pairs of disulfide bonds of IFN-kappa cannot be formed correctly, the three-dimensional structure cannot be formed by correct folding, and the biological activity of the IFN-kappa protein is influenced inevitably. Therefore, it is important to develop a suitable method for separating and purifying IFN-. kappa.s.

In order to overcome the defects of the prior art, in the invention, the use of an organic solvent is avoided by a three-step purification (strong anion exchange chromatography-strong cation exchange chromatography) method which is operated in a water phase completely, so that the reprocessing pressure at the later stage is reduced, and the effective separation and purification of the rhIFN-kappa are realized by simple operation steps.

Disclosure of Invention

The invention aims to provide a simple and feasible separation and purification method of rhIFN-kappa, which is suitable for industrial production, by adopting a three-step purification (strong anion exchange chromatography-strong cation exchange chromatography).

Another objective of the present invention is to provide a method for separating and purifying rhIFN-. kappa.in an environment-friendly manner, which avoids the cost and pressure generated in the post-treatment process of waste organic solvents by avoiding the use of a large amount of organic solvents through the rational combination of various aqueous phase-based extraction solutions.

The purpose of the invention is realized by the following technical scheme:

a method of purifying recombinant human interferon- κ (rhIFN- κ), comprising the steps of:

1) carrying out anion exchange chromatography on the renaturation diluent containing the rhIFN-kappa, and collecting flow-through liquid;

2) carrying out cation exchange chromatography on the flow-through liquid obtained in the step 1), and collecting eluent;

3) diluting the eluate obtained in step 2);

4) subjecting the dilution obtained in step 3) to cation exchange chromatography.

In a specific embodiment, in the above method, the anion exchange chromatography in step 1) is a strong anion exchange chromatography and the cation exchange chromatography in steps 2) and 4) is a strong ion exchange chromatography.

In a specific embodiment, in the above method, the rhIFN-. kappa.containing renaturation dilution may be a renaturation dilution containing rhIFN-. kappa.obtained by any conventional method.

In a specific embodiment, the rhIFN- κ -containing renaturation dilution is obtained by: culturing host cells containing the gene coding hIFN-kappa, IPTG (isopropyl-beta-D-thiogalactoside) or lactose induced expression, centrifugally collecting thalli, high-pressure homogenizing and breaking cells, centrifugally collecting inclusion bodies, denaturing, renaturing and diluting. Preferably, the host cell is an E.coli cell. More preferably, the host cell is an E.coli BL21(DE3) cell. Preferably, the denaturation is performed using a denaturing solution containing urea or guanidine hydrochloride at a high concentration. Preferably, the renaturation is carried out using a renaturation buffer containing EDTA and Trition X-100, more preferably, the renaturation buffer is 1-30mM Tris, 1-20mM sodium dihydrogen phosphate, 20-100mM NaCl, 0.2-0.6mM oxidation type reducing agent, 0-3mM EDTA, 0.05-0.2% Trition X-100, pH 7.0-8.5. Preferably, the oxidised reducing agent is selected from one or more of oxidised glutathione and oxidised cysteine.

Preferably, the gene encoding hIFN-kappa is a gene encoding hIFN-kappa which is expressed efficiently in prokaryotic cells. Preferably, the gene encoding hIFN-kappa has a codon optimized nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. Preferably, the gene coding the hIFN-kappa is the gene coding the hIFN-kappa which is efficiently expressed in Escherichia coli BL21(DE3) cells, and has a nucleotide sequence shown as SEQ ID NO. 2.

Preferably, the gene encoding hIFN-kappa is a gene encoding a hIFN-kappa mutant that is expressed efficiently in prokaryotic cells. Preferably, the hIFN-kappa mutant is obtained by mutating the 166 th cysteine (C) of the amino acid sequence shown in SEQ ID NO.1 of hIFN-kappa into serine (S), or mutating the amino acid sequence into glycine (G) or alanine (A) with similar structure with serine. More preferably, the hIFN-kappa mutant is obtained by mutating the 166 th cysteine (C) of the amino acid sequence shown in SEQ ID NO.1 of hIFN-kappa into serine (S). Compared with a strain containing a wild type hIFN-kappa sequence, the strain containing the plasmid of the mutant sequence has higher expression yield of hIFN-kappa, higher binding affinity with an interferon-kappa receptor and better in vitro activity.

In a specific embodiment, the production of the gene encoding hIFN-. kappa.comprises the following steps:

carrying out Escherichia coli codon optimization according to the amino acid sequence of hIFN-kappa, wherein the amino acid sequence of hIFN-kappa is shown as follows:

MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK(SEQ ID NO:1)。

among them, the codon optimization principle applicable to E.coli BL21(DE3) is: (1) the region with more concentrated rare codons is optimized integrally to achieve the optimal optimization effect; (2) adjusting the AT and GC rich regions to make the GC content of the sequence appropriate; (3) the optimal codon of E.coli BL21 was not used completely in designing the gene, but the preferred codon was used only partially.

The gene sequence of hIFN-kappa expression sequence after codon optimization is as follows:

ATGCTGGATTGTAATCTGCTGAATGTGCATCTGCGTCGTGTGACCTGGCAGAATCTGCGTCATCTGAGCAGCATGAGCAATAGCTTTCCGGTTGAATGTCTGCGCGAGAATATTGCCTTTGAACTGCCGCAGGAATTTCTGCAGTATACCCAGCCGATGAAACGCGATATTAAGAAAGCCTTCTATGAAATGAGCCTGCAGGCATTTAATATCTTTAGCCAGCATACCTTTAAGTATTGGAAAGAACGTCATCTGAAACAGATTCAGATTGGCCTGGATCAGCAGGCAGAATATCTGAATCAGTGTCTGGAAGAAGATAAGAATGAGAATGAAGATATGAAGGAAATGAAGGAGAATGAAATGAAACCGAGCGAAGCACGCGTTCCGCAGCTGAGCAGTCTGGAACTGCGTCGCTATTTCCATCGTATTGATAATTTCCTGAAGGAGAAGAAATACAGCGATTGTGCCTGGGAAATTGTTCGCGTTGAAATTCGTCGCTGTCTGTATTATTTCTATAAATTTACCGCCCTGTTCCGCCGCAAATAA(SEQ ID NO:2)。

the optimized gene sequence is synthesized by Shanghai offshore technology Co., Ltd.

In a specific embodiment, the optimized hIFN-kappa gene sequence prepared and synthesized with plasmid pET30a into pET30a-hIFN-k expression vector, transformed into competent cells of Escherichia coli strain BL21(DE3), added with LB medium (without kanamycin) after the transformation at 37 ℃ for 1h, and then spread on LB solid medium containing kanamycin for overnight culture at 37 ℃. Selecting positive clone, enlarging culture, extracting plasmid, and carrying out Xho I-Apa I double enzyme digestion verification, wherein the result shows that the large fragment is about 4500bp, the small fragment is about 1500bp, and the enzyme digestion result is in line with the expectation. The correctly sequenced clone strains were stored at-80 ℃ at low temperature.

In a specific embodiment, the prepared monoclonal strain is recovered, inoculated into LB liquid culture medium (tryptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, deionized water 1L), cultured overnight at 37 ℃, transferred into a shake flask with the inoculum size of 1 per mill, cultured at 37 ℃ until OD600 is 0.6-1.2, and added with IPTG (isopropyl thiogalactoside) with the final concentration of 0.4-1.2mM for induction expression at 37 ℃ for 3 h. And (3) centrifuging the induced bacterial liquid, collecting thalli, carrying out ultrasonic disruption and centrifugation, and preserving the positive strain (BL21(DE3) -hIFNk) in a refrigerator at the temperature of-80 ℃ for later renaturation and purification.

Wherein, the anion exchange chromatography of the step 1) adopts an ion exchange medium with quaternary ammonium group as a ligand; preferably, the anion exchange chromatography of step 1) adopts an exchange medium selected from any one of Q Bestarose FF, DEAE Bestarose Fast Flow, Q sepharose FF, UNOsphere Q, Q Bestarose XL and Mono Q; more preferably, the anion exchange chromatography of step 1) adopts an exchange medium of Q BestaroseFF.

Wherein, the cation exchange chromatography in the step 2) and the step 4) adopts an ion exchange medium with sulfonic group as ligand; preferably, the cation exchange chromatography of step 2) and step 4) adopts an exchange medium selected from SP Bestarose HP, SP Bestarose FF, SP Bestarose XL, SP Sepharose XL and CM Bestarose Fast Flow; more preferably, the exchange medium adopted by the cation exchange chromatography of the step 2) and the step 4) is SP Bestarose HP.

Without being bound by theory, in the present invention, anion exchange chromatography means that the charge groups (ligands) of the anion exchanger are positively charged, while the counter-ions are negatively charged, which can exchange reactions with anions or negatively charged compounds in solution. Cation exchange means that the charge groups (ligands) of the cation exchanger are negatively charged and the counter ions are positively charged, which can exchange reactions with cations or positively charged compounds in solution. Under certain pH condition, according to the difference of the charges of the protein, exchangeable ions (namely counter ions) on the ion exchanger are adsorbed and combined with the ions separated from the solution, thereby realizing separation. Wherein proteins that do not bind or bind weakly to the ion exchange medium are eluted, and proteins with a strong affinity are bound to the column.

In the invention, the exchange medium adopted by anion exchange chromatography is the ion exchange medium with quaternary ammonium group as ligand. The quaternary ammonium group is a strongly basic group which dissociates OH from water^-Is strongly alkaline, OH is eluted^-Exchange with anions in solution so that they are adsorbed on the media. The strong anion exchange medium has a wide application range and can be reacted in an acidic environment. The cation exchange chromatography adopts an ion exchange medium with sulfonic group as ligand, and the sulfonic group is a strong acid group which can be easily dissociated to obtain H in solution⁺Therefore, it is strongly acidic, and in elution, H⁺Into solutionSO that cations in the solution are exchanged by SO^3－Adsorption binding to thereby realize H⁺Exchange with cations in the solution. Strong acid cation exchange resins have a very strong dissociation capability and dissociate and produce ion exchange under either acidic or basic conditions.

In the above method, in step 3), the eluent obtained in step 2) is mixed in a ratio of 1: (0.5-10) diluting with a buffer solution; preferably, the eluent obtained in step 2) is mixed in a ratio of 1: (2-8) diluting with a buffer solution in proportion; more preferably, the eluent obtained in step 2) is mixed in a ratio of 1: (4-6) diluting with a buffer solution. Through the dilution step, the contents of various ions in the eluent after the first cation exchange are slowly and properly reduced, the protein agglutination caused by too large change of the ion strength of the target protein is avoided, and therefore, the purpose that the unwanted cations in the eluent are replaced by SO as much as possible in the next cation exchange chromatography is realized^3－Adsorption is combined, and the target protein is separated out better.

In the method, the elution is performed by using a buffer solution B, a buffer solution C and an aqueous NaCl solution in the step 2), and the elution is performed by using a buffer solution E, a buffer solution F and an aqueous NaCl solution in the step 4), wherein the buffer solution B, the buffer solution C, the buffer solution E and the buffer solution F are different.

Wherein the buffer solutions B and C are solutions with phosphate buffer solution (PB), polyethylene glycol octyl phenyl ether (TritonX-100) and NaCl as main components respectively; the buffers D, E and F are solutions containing Phosphate Buffer (PB), Tween-20 and NaCl as main components.

Wherein, before the step 1), renaturation liquid obtained by denaturation and renaturation of inclusion bodies fermented and cultured by a conventional method is diluted by buffer solution G, the pH value is adjusted to 5.0-8.5, and the filtrate is collected after filtration.

Specifically, the method for purifying the recombinant human interferon-kappa (rhIFN-kappa) comprises the following steps:

(1) sample pretreatment: diluting renaturation liquid obtained by denaturation and renaturation of inclusion bodies fermented and cultured by a conventional method by using a buffer solution G, adjusting the pH value to be 5.0-8.5, filtering, and collecting filtrate;

(2) anion exchange chromatography: balancing an anion chromatographic column by using a buffer solution A, loading the filtrate obtained in the step (1), and collecting a flow-through solution;

(3) cation exchange chromatography: balancing the cation chromatographic column by using a buffer solution A, loading the flow-through solution obtained in the step (2), balancing the cation chromatographic column by using the buffer solution A and a buffer solution B again, eluting by using the buffer solution B, the buffer solution C and a NaCl aqueous solution, and collecting an eluent;

(4) diluting: diluting the eluent obtained in the step (3) with a buffer solution G, and adjusting the pH value to 5.0-8.5 to obtain a diluent;

(5) cation exchange chromatography: and (3) balancing the cation chromatographic column by using a buffer solution A, loading the diluent obtained in the step (4), balancing the cation chromatographic column by using a buffer solution D and a buffer solution E in sequence, eluting by using a buffer solution E, a buffer solution F and a NaCl aqueous solution, and collecting an eluent.

In a specific embodiment, the step (1) comprises: culturing host cell containing hIFN-kappa gene, IPTG or lactose inducing expression, centrifugal collecting thallus, high pressure homogenizing to break cell, centrifugal collecting inclusion body, denaturation, renaturation and diluting with buffer solution G. Preferably, the gene encoding hIFN-. kappa.has a sequence adapted to codon optimization of the host cell. Preferably, the host cell is an E.coli cell. More preferably, the host cell is an E.coli BL21(DE3) cell. Preferably, the denaturation is performed using a denaturing solution containing urea or guanidine hydrochloride at a high concentration. Preferably, the renaturation is carried out using a renaturation buffer containing EDTA and Trition X-100, more preferably, the renaturation buffer is 1-30mM Tris, 1-20mM sodium dihydrogen phosphate, 20-100mM NaCl, 0.2-0.6mM oxidation type reducing agent, 0-3mM EDTA, 0.05-0.2% Trition X-100, pH 7.0-8.5. Preferably, the oxidised reducing agent is selected from one or more of oxidised glutathione and oxidised cysteine.

In some preferred embodiments, buffer A, B, C contains PB, TritonX-100, and NaCl; buffer D, E, F contains PB, Tween-20 and NaCl; buffer G contained PB and TritonX-100, but not NaCl and Tween-20.

In a specific embodiment, buffer A is a solution comprising 10-30mM PB, 0.05-0.25% (by volume) Triton X-100, and 40-60mM NaCl, pH 4.0-6.8. Preferably, the buffer A is a solution comprising 15-25mM PB, 0.1-0.2% TritonX-100 and 45-55mM NaCl, pH 4.5-6.5.

In a specific embodiment, the buffer B is a solution comprising 10-30mM PB, 0.05-0.25% (by volume) Triton X-100 and 800-1200mM NaCl, pH 7-9. Preferably, the buffer B is a solution containing 15-25mM PB, 0.1-0.2% (by volume) TritonX-100 and 900-1100mM NaCl, pH 7.5-8.5.

In a specific embodiment, the buffer C is a solution comprising 10-30mM PB, 0.05-0.25% (by volume) Triton X-100, and 40-60mM NaCl, pH 7-9. Preferably, the buffer C is a solution comprising 15-25mM PB, 0.1-0.2% TritonX-100 and 45-55mM NaCl, pH 7.5-8.5.

In a specific embodiment, the buffer D is a solution comprising 10-30mM PB, 0.05-0.25% Tween-20, and 40-60mM NaCl, pH 4.0-6.8. Preferably, the buffer D is a solution comprising 15-25mM PB, 0.1-0.2% Tween-20 and 45-55mM NaCl, pH 4.5-6.5.

In a specific embodiment, the buffer E is a solution comprising 10-30mM PB, 0.05-0.25% Tween-20 and 40-60mM NaCl, pH 7-9. Preferably, the buffer E is a solution comprising 15-25mM PB, 0.1-0.2% Tween-20 and 45-55mM NaCl, pH 7.5-8.5.

In a specific embodiment, the buffer F is a solution comprising 10-30mM PB, 0.05-0.25% Tween-20 and 800-1200mM NaCl, pH 7-9. Preferably, the buffer F is a solution comprising 15-25mMPB, 0.1-0.2% Tween-20 and 900-1100mM NaCl, pH 7.5-8.5.

In a specific embodiment, the buffer G is a solution comprising 10-30mM PB and 0.05-0.25% Triton X-100 at pH 6-7.5. Preferably, the buffer G is a solution comprising 15-25mM PB and 0.1-0.2% TritonX-100, pH 6.5-7.0.

In the present invention, several buffers having different compositions and different pH ranges are used, and the dissociation degree of ions in the solution is decreased by changing the pH, and the charge is decreased, so that the affinity to the exchanger is weakened and the buffer is eluted. By using a specific combination of buffer concentration and pH, the elution capacity of the ion exchanger is continuously enhanced, thereby achieving efficient separation of the protein of interest.

Wherein, the exchange medium adopted by the anion exchange chromatography of the step (2) is Q BestaroseFF.

Wherein, the cation exchange chromatography of the step (3) and the step (5) adopts SP Bestarose HP as an exchange medium.

Wherein, in the step (1), the volume ratio of the renaturation solution to the buffer solution G is 1: (0.5-5).

Wherein, in the step (2), the sample is loaded at the flow speed of 150-250 cm/h.

Wherein, in the step (2), the rhIFN-kappa of the target protein is not hung on the column.

Wherein, in the step (3), sampling is carried out at a flow speed of 150-250 cm/h; and eluting with buffer solution B, buffer solution C and 50-500mM NaCl aqueous solution at a flow rate of 40-80 cm/h.

Wherein, in the step (3), the eluent collection conditions are set as follows: 5% buffer solution B-20% buffer solution B for 5 min; 280nm is selected as a detection wavelength, the eluent starts to be collected when the ultraviolet absorption value is increased to 180mAU, and the collection is stopped when the ultraviolet absorption value is reduced to 230 mAU.

Wherein, in the step (4), the volume ratio of the eluent to the buffer G is 1: (0.5 to 10).

Wherein, in step (5), the sample collection conditions are set as: 0% buffer solution F-47% buffer solution F for 3-10 min; selecting 280nm as detection wavelength, starting to collect eluent when the ultraviolet absorption value is increased to 180mAU, and stopping collecting when the ultraviolet absorption value is reduced to 200mAU, wherein the obtained product is purified interferon-kappa.

Specifically, the rhIFN-kappa purifying method comprises the following steps:

(1) sample pretreatment:

culturing host cells containing a gene for coding hIFN-kappa, inducing expression by IPTG or lactose, centrifugally collecting thalli, crushing cells by high-pressure homogenization, centrifugally collecting inclusion bodies, performing denaturation and renaturation to obtain renaturation solution, wherein the volume ratio of the renaturation solution to buffer solution G is 1: (0.5-5), diluting with a buffer solution G, and adjusting the pH value: 5.0-8.5, conductivity: 2-10 ms/cm, filtering with a filter membrane, and collecting filtrate;

(2) strong anion exchange chromatography:

preprocessing a Q Bestarose FF column: balancing the chromatographic column by using a buffer solution A for 5-10 CV, and stopping balancing when the conductivity of the effluent is 7.0-8.5 ms/cm;

sample loading: sampling the filtrate obtained in the step (1) at a flow speed of 150-250 cm/h;

column balancing: washing the chromatographic column with 3-8CV buffer solution A to make the target protein show flow-through without hanging the column under the condition, and collecting the flow-through solution;

(3) strong cation exchange chromatography:

preprocessing an SP Bestarose HP column: balancing the chromatographic column by using a buffer solution A for 5-10 CV, and stopping balancing when the conductivity of the effluent is 7.0-8.5 ms/cm;

sample loading: sampling the flow-through liquid obtained in the step (2) at a flow speed of 150-250 cm/h;

column balancing: washing with buffer solution A for 3-8CV, and washing with buffer solution B for 2-8 CV;

and fourthly, elution: eluting with buffer solution B, buffer solution C and 50-200mM NaCl aqueous solution at flow rate of 40-80 cm/h;

collecting the eluent: the system is set as follows: 5% buffer solution B-20% buffer solution B for 5 min; selecting 280nm as detection wavelength, starting to collect eluent when the ultraviolet absorption value is increased to 180mAU, and stopping collecting when the ultraviolet absorption value is reduced to 230 mAU;

(4) diluting: and (3) mixing the eluent obtained in the step (3) with a buffer G in a volume ratio of the eluent to the buffer G of 1: (0.5-10) diluting with a buffer solution G, adjusting the pH to 5.0-8.5 and the conductivity: 2-10 ms/cm to obtain a diluent;

(5) strong cation exchange chromatography:

preprocessing an SP Bestarose HP column: balancing the chromatographic column by using a buffer solution A for 5-10 CV, and stopping balancing when the conductivity of the effluent is 7.0-8.5 ms/cm;

sample loading: sampling the diluent in the step (4) at a flow speed of 150-250 cm/h;

column balancing: washing 3-8CV with buffer solution D, and washing 2-8CV with buffer solution E;

and fourthly, elution: eluting with buffer solution E, buffer solution F and 50-500mM NaCl water solution at flow rate of 40-80 cm/h;

collecting the eluent: the system is set as follows: 0% buffer solution F-47% buffer solution F for 3-10 min; selecting 280nm as detection wavelength, starting to collect eluent when the ultraviolet absorption value is increased to 180mAU, and stopping collecting when the ultraviolet absorption value is reduced to 200mAU, wherein the obtained product is purified interferon-kappa.

A recombinant human interferon-kappa (rhIFN-kappa) prepared by the method according to the separation and purification method of the invention.

A kit for carrying out the above method comprising: strong anion exchange media for anion exchange chromatography and strong cation exchange media for cation exchange chromatography.

Wherein, the strong anion exchange medium is an ion exchange medium with quaternary ammonium group as ligand; and the strong cation exchange medium is an ion exchange medium with sulfonic group as ligand.

The kit of the invention is used for preparing or purifying interferon.

In the use, the interferon is interferon- κ; preferably, the interferon is human interferon- κ; more preferably, the interferon is recombinant human interferon- κ.

The invention has the beneficial effects that:

(1) the invention adopts a purification and separation method of full water phase, overcomes the defect that rhIFN-kappa is easy to polymerize into polymer, and does not use organic solvent in the whole separation process, thereby greatly reducing the environmental protection pressure and cost in the later stage of the whole purification process, and obviously reducing the total cost of the separation and purification of the hIFN-kappa.

(2) Compared with the prior art, the separation and purification process adopts two chromatographic column fillers of strong anions and strong cations for separation, has less purification steps, improves the yield of rhIFN-kappa, and has the purity of more than 93 percent.

(3) The invention adopts three-step purification of anion exchange resin and two-step cation exchange resin, well avoids the formation of interferon-kappa polymer, and the separation and purification method has low cost, simple operation and high repeatability, and is suitable for industrial production.

Drawings

FIG. 1 is a schematic flow chart of the method for separating and purifying recombinant human interferon-. kappa.of the present invention.

FIG. 2 is an HPLC chromatogram of recombinant human interferon-. kappa.isolated by the method of the present invention.

FIG. 3 is a SDS-PAGE gel of the recombinant human interferon-. kappa.separated by the method of the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments, and those skilled in the art will understand that the described embodiments are for illustrative purposes only and are not all the present invention. Based on the embodiments of the present invention, those skilled in the art will better understand and appreciate the technical solutions claimed in the present invention and the technical effects achieved thereby.

In the following examples, reagents other than the specifically prepared reagents were commercially available.

Separation and purification of rhIFN-kappa protein

Example 1

The steps of the separation and purification of the present invention were carried out according to the separation and purification scheme shown in FIG. 1.

Experimental materials:

q Bestarose FF (Q-FF) column: boglon (shanghai) biotechnology limited; SP Bestarose HP (SP-HP) column: boglon (shanghai) biotechnology limited; butyl Sepharose HP (GE Healthcare) column: shanghai Yubo Biotech Co., Ltd.

The instrument comprises the following steps: protein purification apparatus AKTAPure.

The various buffers used in the following procedures are specified below:

and (3) buffer solution A: 20mM PB, 0.1% TritonX-100 and 50mM NaCl, pH 6.5;

and (3) buffer solution B: 20mM PB, 0.1% TritonX-100 and 1000mM NaCl, pH 7.5;

and (3) buffer C: 20mM PB, 0.1% TritonX-100 and 50mM NaCl, pH 7.5;

and (3) buffer solution D: 20mM PB, 0.1% Tween-20 and 50mM NaCl, pH 6.5;

and (3) buffer solution E: 20mM PB, 0.1% Tween-20 and 50mM NaCl, pH 7.5;

and (3) buffer solution F: 20mM PB, 0.1% Tween-20 and 1000mM NaCl, pH 7.5;

buffer G: 20mM PB, 0.1% TritonX-100, pH 6.5.

The method comprises the following specific operation steps:

(1) sample pretreatment:

culturing Escherichia coli cells containing a gene for coding hIFN-kappa, carrying out IPTG induced expression, centrifugally collecting thalli, carrying out high-pressure homogenization and cell disruption, centrifugally collecting inclusion bodies, carrying out 8M urea denaturation and renaturation of a renaturation buffer solution to obtain a renaturation solution, wherein the volume ratio of the renaturation solution to the buffer solution G is 1: 2, diluting with a buffer solution G, adjusting the pH to 6.5, adjusting the conductivity to 5ms/cm, filtering with a 0.45-micron filter membrane, and collecting the filtrate; wherein the renaturation buffer solution is 20mM Tris, 10mM sodium dihydrogen phosphate, 50mM NaCl, 0.4mM oxidized glutathione, 2mM EDTA, 0.1% TritionX-100 and pH 7.0.

(2) Strong anion exchange chromatography:

preprocessing a Q Bestarose FF (Q-FF) column: balancing the chromatographic column with buffer solution A for 8CV, stopping balancing when the conductivity of the effluent is 7.5ms/cm, and finishing the pretreatment of the chromatographic column;

sample loading: loading the filtrate obtained in the step (1) at a flow rate of 200 cm/h;

column balancing: washing the chromatographic column 5CV with a buffer solution A to ensure that the target protein does not hang the column under the condition and shows flow-through, and collecting flow-through liquid;

(3) strong cation exchange chromatography:

preprocessing an SP Bestarose HP (SP-HP) column: balancing the chromatographic column with buffer solution A for 8CV, stopping balancing when the conductivity of the effluent is 7.5ms/cm, and finishing the pretreatment of the chromatographic column;

sample loading: sampling the flow-through liquid obtained in the step (2) at a flow speed of 200 cm/h;

column balancing: rinsing with buffer solution A for 5CV, and rinsing with buffer solution B for 3 CV;

and fourthly, elution: eluting with buffer solution B, buffer solution C and 100mM NaCl aqueous solution at a flow rate of 60 cm/h;

collecting the eluent: the system is set as follows: 5% buffer solution B-20% buffer solution B for 5 min; selecting 280nm as detection wavelength, starting to collect eluent when the ultraviolet absorption value is increased to 180mAU, and stopping collecting when the ultraviolet absorption value is reduced to 230 mAU;

(4) diluting: and (3) mixing the eluent collected in the step (3) with a buffer G in a volume ratio of 1: 5, diluting the mixture by using a buffer solution G to obtain a diluent, and adjusting the pH to 7.0 and the conductivity to 5ms/cm for later use;

(5) strong cation exchange chromatography:

preprocessing an SP Bestarose HP column: balancing the chromatographic column with buffer solution A for 8CV, stopping balancing when the effluent conductivity is 8ms/cm, and finishing the pretreatment of the chromatographic column;

sample loading: loading the diluent in the step (4) at a flow speed of 200 cm/h;

column balancing: rinsing with buffer solution D for 5CV, and rinsing with buffer solution E for 3 CV;

and fourthly, elution: eluting with buffer solution E, buffer solution F and 300mM NaCl aqueous solution at the flow rate of 60 cm/h;

collecting the eluent: the system is set as follows: 0% buffer solution F-47% buffer solution F for 5 min; selecting 280nm as detection wavelength, starting to collect eluent when the ultraviolet absorption value is increased to 180mAU, and stopping collecting when the ultraviolet absorption value is reduced to 200mAU, wherein the obtained product is purified interferon-kappa.

Example 2

Buffers a to G in example 1 were replaced with the following buffers, respectively:

and (3) buffer solution A: 10mM PB, 0.05% TritonX-100 and 50mM NaCl, pH 4.0;

and (3) buffer solution B: 10mM PB, 0.1% TritonX-100 and 800mM NaCl, pH 7.0;

and (3) buffer C: 10mM PB, 0.25% TritonX-100 and 40mM NaCl, pH 8.0;

and (3) buffer solution D: 10mM PB, 0.05% Tween-20 and 40mM NaCl, pH4.0;

and (3) buffer solution E: 10mM PB, 0.1% Tween-20 and 40mM NaCl, pH 7.0;

and (3) buffer solution F: 10mM PB, 0.25% Tween-20 and 800mM NaCl, pH8.0;

buffer G: 10mM PB, 0.05% TritonX-100, pH6.0.

The remaining reaction materials and the operating procedures and conditions were the same as in example 1.

Example 3

Buffers a to G in example 1 were replaced with the following buffers, respectively:

and (3) buffer solution A: 30mM PB, 0.25% TritonX-100 and 60mM NaCl, pH 6.8;

and (3) buffer solution B: 30mM PB, 0.25% TritonX-100 and 1200mM NaCl, pH 9.0;

and (3) buffer C: 30mM PB, 0.25% TritonX-100 and 60mM NaCl, pH 9.0;

and (3) buffer solution D: 30mM PB, 0.25% Tween-20 and 60mM NaCl, pH 6.8;

and (3) buffer solution E: 30mM PB, 0.25% Tween-20 and 60mM NaCl, pH 9.0;

and (3) buffer solution F: 30mM PB, 0.25% Tween-20 and 1200mM NaCl, pH9.0;

buffer G: 30mM PB, 0.25% TritonX-100, pH 7.5.

The remaining reaction materials and the operating procedures and conditions were the same as in example 1.

Example 4

The strong anion exchange chromatography column Q bestrol FF of operation (2) in example 1 was replaced with a weak anion exchange chromatography column DEAE bestrol Fast Flow (i.e., DEAE bestrol FF), and the strong cation exchange chromatography column SP bestrol HP of operations (3) and (5) in example 1 was replaced with a weak cation exchange chromatography column CM bestrol Fast Flow (i.e., cmbestrol FF), and the rest of the reaction materials and operation steps and conditions were the same as in example 1.

Example 5

The strong anion exchange chromatography column Q bestrol FF of procedure (2) in example 1 was replaced with a weak anion exchange chromatography column DEAE bestrol Fast Flow (i.e., DEAE bestrol FF), and the strong cation exchange chromatography column SP bestrol HP of procedure (3) in example 1 was replaced with a weak cation exchange chromatography column CM bestrol Fast Flow (i.e., CM bestrol FF), and the rest of the reaction materials and procedures and conditions were the same as in example 1.

Example 6

The strong cation exchange chromatography column SP Bestarose HP in operation (3) in example 1 was replaced with a weak cation exchange chromatography column CM Bestarose Fast Flow (i.e. CM Bestarose FF), and the rest of the reaction materials and operation steps and conditions were the same as in example 1.

Comparative example 1

The strong anion exchange chromatography column Q bestrol FF of operation (2) of example 1 was replaced with a weak anion exchange chromatography column DEAE bestrol Fast Flow (i.e., DEAE bestrol FF), and the strong cation exchange chromatography column SP bestrol HP of step (3) was replaced with a weak cation exchange chromatography column CM bestrol Fast Flow (i.e., cmbestrol FF), while omitting step (4) and step (5), and the rest of the reaction materials and operation steps and conditions were the same as in example 1.

Comparative example 2

In the operation steps of example 1, steps (4) and (5) were omitted, and the remaining reaction materials and operation steps and conditions were the same as those of example 1.

Comparative example 3

In the procedure of example 1, step (5) was replaced with hydrophobic chromatography, and the remaining reaction materials and procedures and conditions were the same as in example 1. The specific steps of hydrophobic chromatography are as follows: and (3) eluting and separating the diluent in the step (4) by using a Butyl Sepharose HP (GE Healthcare) hydrophobic chromatographic column under the conditions that the conductivity is 50ms/cm and the flow rate is 60cm/h, and collecting eluent, wherein the obtained product is purified interferon-kappa.

(II) measurement of results

The purity and yield results of the recombinant human interferon-. kappa.proteins obtained in examples 1-6 and comparative examples 1-3 were determined as shown in Table 1 below. The recombinant human interferon-kappa protein prepared in the detection example 1 is detected by the conventional SEC-HPLC method and SDS-PAGE method in the prior art, and the detection results are shown in FIGS. 2 and 3.

SEC-HPLC chromatography conditions: using SEC-300 analytical column, size of chromatographic column 4.6 x 300mm, pore diameterThe particle size is 5 mu m, external water is 10000-1000000 Da, the flow rate is 0.26ml/min, the buffer solution system is 20mMPB, 500mMNaCl and 0.1% Tween 20, the pH value is 7.2, and the detection is carried out at 280 nm.

TABLE 1

As can be seen from table 1 above, the chromatographically purified samples of examples 1-6 of the invention all achieved yields of at least 60% and protein purities of at least 93%. The combination of weak anion and weak cation exchange chromatography and the combination of strong anion and strong cation resulted in a reduction in protein yield of about 31% and about 8%, respectively, and a reduction in recovered protein purity of about 18% and about 12%, respectively, as compared to example 1 of the present invention. The strong cation chromatography step of the third step is replaced by a hydrophobic chromatography step, and the yield and the purity of the recombinant human interferon-kappa protein are also obviously reduced. In addition, when the purified interferon-kappa protein obtained in example 1 of the present invention is analyzed, as shown in the SEC-HPLC result shown in fig. 2, the separation and purification method of the present invention realizes the good separation of the interferon-kappa protein, and a single main peak appears; as shown in FIG. 3, SDS-PAGE results of the target protein human interferon-kappa show that the purity of the separated and purified protein is very high, and the molecular weight of the purified target protein is close to that of the natural human interferon-kappa. The above results show that the three-step chromatography and two cation exchange chromatography steps of the present invention significantly improve the separation and purification of interferon-kappa protein.

The present invention is not limited to the embodiments listed above, and those skilled in the art will appreciate that various substitutions, modifications and combinations of the technical features of the present invention can be made by reading the specification of the present invention without departing from the spirit and the spirit of the present invention, and the technical aspects after the substitutions, modifications and combinations are all covered by the scope of the claims of the present invention.

Sequence listing

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