CN113501857A - Preparation method of high-activity recombinant protein - Google Patents

Preparation method of high-activity recombinant protein Download PDF

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CN113501857A
CN113501857A CN202110942425.4A CN202110942425A CN113501857A CN 113501857 A CN113501857 A CN 113501857A CN 202110942425 A CN202110942425 A CN 202110942425A CN 113501857 A CN113501857 A CN 113501857A
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solution
sample
dissolving
protein
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张朔
冯铃
刘辉
李雪娇
赵航
刘畅
张鹏
赵洪礼
何伟
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Liaoning He's Medical College
Shenyang Eye Industry Technology Research Institute Co ltd
Shenyang Baifa Technology Co ltd
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Liaoning He's Medical College
Shenyang Eye Industry Technology Research Institute Co ltd
Shenyang Baifa Technology Co ltd
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    • C07K1/16—Extraction; Separation; Purification by chromatography
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]

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Abstract

The invention relates to the technical field of recombinant protein purification, in particular to a preparation method of high-activity recombinant protein. The preparation method comprises the following steps: mixing an HPLC purified sample of the recombinant protein with a protein dissolving solution, and dissolving; mixing the dissolved sample with renaturation liquid for renaturation treatment; and purifying the renaturated sample by adopting an anion exchange chromatography column to obtain the high-activity recombinant protein. The invention further solves the problem that the recombinant protein purified by HPLC loses biological activity by improving the formula of the protein dissolving solution and the renaturation solution, prepares a high-activity and high-purity recombinant protein sample, has simple operation and no endotoxin and organic solvent residue, and is suitable for treating retinal neovascularization.

Description

Preparation method of high-activity recombinant protein
Technical Field
The invention relates to the technical field of recombinant protein purification, in particular to a preparation method of high-activity recombinant protein.
Background
Wet age-related macular degeneration (AMD) and Diabetic Retinopathy (DR) lack ideal treatment. The clinical treatment method mainly adopts symptomatic treatment or auxiliary treatment method to control or delay the development of the disease, and the common treatment method is as follows: hypoglycemic agents, anti-inflammatory agents, topical TNF-alpha antagonists, fenofibrate, Vascular Endothelial Growth Factor (VEGF) antagonists, RAS system inhibitors, non-enzymatic glycosylation inhibitors, combination therapies, and laser treatments. anti-VEGF therapy has a good recent effect, but involves periodic intraocular injections of anti-VEGF monoclonal antibodies (e.g., Lucentis), which are not only somewhat harmful, but also expensive and only for confirmed cases, and in addition, although the incidence is low, each injection carries serious risks of intraocular infection, retinal hemorrhage and retinal detachment, and this treatment is resistant to repeated administration.
9300 million global Diabetic Retinopathy (DR) patients in 2013, wherein 1700 million Proliferative DR (PDR) patients, 2100 million diabetic macular edema patients and 2800 million vision-threatening DR patients. The incidence rate of diabetic retinopathy is 25 percent after 5 years of diabetes attack, the incidence rate is increased to 60 percent after 10 years, and the incidence rate can reach 75 to 80 percent after 15 years. Therefore, the preparation method accelerates the development of a novel preparation for treating fundus vasculopathy, and has social benefits and huge economic benefits.
In 2006, recombinant human IL-18 (trade name: Iboktadekin) was introduced into clinical trials as an antitumor agent, developed by GlaxoSmithKline. There have been many papers reporting that IL-18 is safe in human testing and no reports have been made about ocular side effects. In addition, systemic injection of IL-18 in rodents has been reported to detect higher levels of IL-18 in the retina, which suggests bioavailability of IL-18 in the retina. IL-18 administration to patients may cause transient fever of grade 1-2, but can provide rapid remission. No significant side effects were observed on the eyes of the patients at doses up to 2000. mu.g/kg. The research result of the recombinant IL-18 in ophthalmology indicates that the IL-18 may have application value to ocular pathological neovascular diseases. Since the clinical trial of IL-18 has been proven safe, IL-18 can be used in the study of adjunctive therapy for CNV and wet AMD, and it is worth further investigation as to whether it is an effective drug.
Recent research shows that intraocular injection of IL-18 can effectively treat the occurrence and development of the retinal neovascularisation (CNV) of artificially induced mice; moreover, the IL-18 gene knockout mice can have serious retinal neovasculopathy; in addition, studies have demonstrated that recombinant IL-18, both murine and human, is very safe for retinal pigment epithelial cells (RPE). It is reported that patients with high IL-18 content in the aqueous humor will obviously improve the visual effect when receiving Lucentis monoclonal antibody to treat macular edema and retinal vein embolism, meanwhile VEGF regulates the IL-18 content in a mutual inhibition mode, and a plurality of retina/CNV models prove that the recombinant IL-18 can regulate the fundus exudation of pathological models, so the IL-18 can become a new method for regulating the pathological neovascularization of the retina. IL-18 was reported as an IFN γ -inducing factor in 1995, and it was subsequently demonstrated that systemic injection of IL-18 (50. mu.g/kg) into mice for 6 days had an inhibitory effect on FGF-induced corneal capillary formation. It has also been reported that IL-18 has a regulatory effect on the production of pathological neovasculature of the retina in an oxygen-induced model of mouse retinopathy. IL-18 is therefore theoretically likely to be a candidate for the treatment of retinal neovascularisation.
In recent years, recombinant protein medicines are in the spotlight, and make considerable contribution to human health. Recombinant protein expression systems are classified into eukaryotic expression systems, such as CHO cells, and prokaryotic expression systems, such as E.coli. The eukaryotic expression system has the advantages that the protein expression process is similar to the protein translation process in a human body, and the expressed protein molecules can simultaneously complete the modification and the spatial configuration after expression to form active protein; its disadvantages are low expression level, high requirement for conditions and high cost. Prokaryotic expression systems have the advantages of high protein expression, low equipment requirements, low consumption cost, and the disadvantages that the expressed protein cannot complete post-translational modification and spatial conformation, and the residual of thallus components, particularly endotoxin, brings certain difficulties for subsequent protein purification. Depending on the nature of the protein molecule of interest, different expression systems may be selected. Because some protein molecules cannot be secreted and expressed in eukaryotic cells, only prokaryotic expression systems, such as IL-18, IL-2, VEGFB and the like, can be adopted. Therefore, subsequent purification of these recombinant proteins involves troublesome problems such as protein renaturation and endotoxin removal.
There are many technical difficulties in the large-scale preparation of IL-18. In 1996 (J Immunol.156,4274-4279), it was reported that "human IL-18 and its variant are expressed in E.coli and mammalian cells", IL-18 is expressed in the form of inclusion body, and its purification method adopts hydrophobic chromatography (phenyl-Sepharose), anion exchange chromatography (DEAE-Sepharose), gel chromatography (Superdex75), reverse hydrophobic chromatography (C4) and other methods once, and the purity of purified IL-18 reaches 90% by SDS-PAGE detection. In 2006 (journal of Chinese immunology, 22,141-144), "expression and anti-tumor effects of recombinant human IL-18 in Escherichia coli" was reported, human IL-18 was expressed as IL-18 in the form of inclusion bodies, the expression product was centrifuged after ice bath, the inclusion body precipitate was fully washed, dissolved with 8mol/L urea, renatured, dialyzed and subjected to G50 gel chromatography, and the purity of the purified IL-18SDS-PAGE was about 90%. 2010 chinese patent (10169340a) discloses "a method for preparing human interleukin 18; adopts the fractional ultrafiltration of the fermentation culture supernatant of the recombinant pichia pastorisConcentrated, over 50% saturation (NH)4)2SO4After precipitation, the product is purified by hydrophobic chromatography (phenyl-Sepharose) and anion exchange chromatography (Q-Sepharose HP), and the purity of the purified IL-18SDS-PAGE reaches 97%. The method has the problems of complex process, low purity, low renaturation rate, low yield and the like to a certain extent when preparing the IL-18.
The preparation of recombinant protein by using an escherichia coli expression system is concerned due to low cost and high yield, such as recombinant human IL-2, recombinant human interferon and the like, but one of the technical difficulties of purifying recombinant protein from escherichia coli is the residue of endotoxin, a High Performance Liquid Chromatography (HPLC) reverse chromatography technology is an effective method for removing endotoxin, but the recombinant protein purified by HPLC is denatured by the influence of an organic agent in a mobile phase, loses bioactivity, seriously influences the application of the purified recombinant protein, and the problem is not solved all the time.
The applicant establishes an IL-18 Escherichia coli high-efficiency expression strain in the early period, establishes a technical route for renaturation, purification and endotoxin removal of rhIL-18 (patent No. ZL 201510455749, X, ZL 201611146887.0; patent application No. 202010771259.1), but the rhIL-18 sample is in an acidification and denaturation state after the endotoxin is removed by HPLC, the bioactivity is lost, and the application of the rhIL-18 sample is influenced by the acetonitrile residue.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a high-activity recombinant protein. The method can prepare recombinant protein with high purity and good activity, and has the advantages of simple operation and no endotoxin and organic solvent residue.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of high-activity recombinant protein, which comprises the following steps:
mixing an HPLC purified sample of the recombinant protein with a protein dissolving solution, and dissolving;
mixing the dissolved sample with renaturation liquid for renaturation treatment;
purifying the renaturated sample by adopting an anion exchange chromatography column to obtain high-activity recombinant protein;
the protein dissolving solution comprises the following components:
4-10M of dissolving agent
DTT 5～20mM
The PB buffer solution is complemented to 1L;
the renaturation liquid comprises the following components:
PB buffer 1L
5-40 mM of reducing agent.
The invention carries out the steps of redissolution, renaturation, anion column purification and the like on the sample after removing endotoxin by HPLC on the basis of earlier work, leads the inactive IL-18 to be renatured after acetonitrile treatment, obtains the rhIL-18 with high biological activity, and simultaneously further removes the impurities such as acetonitrile and the like remained in the sample, and the obtained sample can completely meet the quality requirement of the intraocular medicine for clinically treating the retinal neovasculopathy.
In the specific embodiment provided by the present invention, the recombinant protein is recombinant human interleukin 18, but is not limited to recombinant human interleukin 18.
In the invention, the HPLC purified sample is a sample obtained by purifying recombinant protein expressed by escherichia coli by HPLC reversed phase chromatography to remove endotoxin.
Preferably, the HPLC reverse phase chromatography can use C4, C8, C18 reverse phase chromatography column.
Preferably, the dissolving agent is urea and/or guanidine hydrochloride;
preferably, the dissolving agent is urea, and the concentration of the dissolving agent in the protein dissolving solution is 6-10M;
preferably, the dissolving agent is guanidine hydrochloride, and the concentration of the dissolving agent in the protein dissolving solution is 4-8M.
Preferably, the reducing agent is DTT andor 2-mercaptoethanol.
Preferably, the proteolytic solution comprises the following components:
6-8M of dissolving agent
DTT 10mM
The PB buffer solution is complemented to 1L;
the renaturation liquid comprises the following components:
PB buffer 1L
Reducing agent 20 mM.
In the specific embodiment provided by the invention, the dissolving agent is urea, and the concentration of the dissolving agent in the protein dissolving solution is 8M;
in the specific embodiment provided by the invention, the dissolving agent is guanidine hydrochloride, and the concentration of the dissolving agent in the protein dissolving solution is 6M.
Preferably, the PB buffer is 10-30 mM and has a pH of 7.0-8.0.
Preferably, the PB buffer solution in the protein dissolving solution is 15-25 mM and has pH of 7.0-7.5;
preferably, the PB buffer solution in the renaturation solution is 15-25 mM and has a pH value of 7.6-8.0.
In the specific embodiment provided by the invention, in the protein dissolving solution, the PB buffer solution is 20mM PB buffer solution with pH 7.2;
in the specific embodiment provided by the invention, in the renaturation solution, the PB buffer solution is 20mM and the pH value is 8.0.
Preferably, the volume ratio of the HPLC purified sample to the proteolytic solution is 1: (5-15); the conditions of the dissolution treatment were: dissolving at 15-30 ℃ for 1.5-2.5 h.
In the specific embodiment provided by the present invention, the volume ratio of the HPLC purified sample to the proteolytic solution is 1: 9; the conditions of the dissolution treatment were: dissolving at room temperature for 2 h.
Preferably, the volume ratio of the HPLC purified sample to the renaturation solution is 1: (5-10); the renaturation treatment conditions are as follows: stirring for 3-5 h at 15-30 ℃, and then renaturating for more than 20h at 0-6 ℃.
In the specific embodiment provided by the invention, the volume ratio of the HPLC purified sample to the renaturation solution is 1: 8; the renaturation treatment conditions are as follows: stirring at room temperature for 4h, and renaturating at 4 ℃ for more than 20 h.
Preferably, the anion exchange chromatography column is a Capto Q chromatography column.
Preferably, the eluent used for anion exchange chromatography column purification comprises the following components:
PB buffer 1L
NaCl 0.1～0.3M。
In a particular embodiment provided by the present invention, the eluent comprises the following components:
PB buffer 1L
NaCl 0.2M。
Preferably, the PB buffer solution in the eluent is 15-25 mM and has a pH of 7.0-7.5.
In the specific embodiment provided by the present invention, the PB buffer in the eluent is 20mM PB buffer at pH 7.2.
Preferably, when the sample after renaturation treatment is purified by using an anion exchange chromatography column, the flow rate of the sample is 3-7 mL/min.
In the specific embodiment provided by the invention, when the sample after renaturation treatment is purified by adopting the anion exchange chromatography column, the flow rate of the sample is 5 mL/min.
The invention provides a preparation method of high-activity recombinant protein. The preparation method comprises the following steps: mixing an HPLC purified sample of the recombinant protein with a protein dissolving solution, and dissolving; mixing the dissolved sample with renaturation liquid for renaturation treatment; and purifying the renaturated sample by adopting an anion exchange chromatography column to obtain the high-activity recombinant protein. The invention has the following beneficial effects:
the invention further solves the problem that recombinant protein purified by HPLC loses biological activity by improving the formula of the protein dissolving solution and the renaturation solution, prepares the rhIL-18 sample with high activity and high purity and other recombinant protein samples, has simple operation and no endotoxin and organic solvent residue, and is suitable for treating retinal neovascularization.
Drawings
FIG. 1 Coomassie Brilliant blue protein assay curves;
FIG. 2 results after Capto Q purification; wherein, M: the molecular weight standard is 97.2, 66.7, 44.3 and 29.8kD from large to small; 1: samples before purification; 2, purifying the sample;
FIG. 3 shows that rhIL-18 induces KG1 cells to produce a gamma-INF standard curve;
FIG. 4A shows the microtubule formation of human endothelial cells without the addition of rhIL-18, and B shows the inhibition of the human endothelial cell microtubule formation by rhIL-18 of the present invention.
Detailed Description
The invention discloses a preparation method of high-activity recombinant protein, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the preparation method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention takes recombinant human Interleukin 18 (rhIL-18) as an example, establishes a method for purifying recombinant protein from escherichia coli, provides a preparation method for preparing high-activity recombinant protein on the basis of removing endotoxin in the recombinant protein by HPLC, and has simple operation method and can prepare the recombinant protein with high purity and good activity. The invention prepares high-activity rhIL-18, but is not limited to the preparation method of rhIL-18. The method is characterized in that on the basis of completing HPLC purification of recombinant protein rhIL-18 in the early stage, the rhIL-18 sample prepared by HPLC is subjected to renaturation and purification, the rhIL-18 with high biological activity is obtained, and simultaneously, impurities such as acetonitrile and the like remained in the sample are further removed, so that the prepared rhIL-18 sample has good activity and high purity, and can completely meet the quality requirement of clinical intraocular medicine for treating retinal neovasculopathy.
The technical scheme of the invention is as follows: through re-dissolving and renaturating the HPLC purified rhIL-18 sample and purifying by a Capto Q ion column, the problems of no activity, acetonitrile residue and the like of the HPLC purified rhIL-18 sample are solved, and the high-activity and high-purity rhIL-18 sample is obtained.
The technical scheme comprises the following specific steps:
(1) obtaining HPLC purified rhIL-18 samples: reference is made to the method of patent application No. 202010771259.1;
(2) rhIL-18 sample solubilization: adding the rhIL-18 sample into a protein dissolving solution according to the proportion of 1:9, and dissolving for more than 2 hours at room temperature;
(3) renaturation of rhIL-18 samples: slowly adding a renaturation solution into the rhIL-18 sample solution according to the proportion of 1:8, and renaturing at 4 ℃ for more than 12 hours;
(4) purification of rhIL-18 protein: enabling the rhIL-18 sample renaturation solution to pass through a Capto Q ion column at the speed of 5mL/min, fully leaching, eluting with 0.2mol/L NaCl, and collecting a target protein peak; packaging the purified protein into a hydro-acupuncture preparation or a freeze-dried preparation.
(5) Analysis of purified rhIL-18 protein: the content is measured by Coomassie brilliant blue method, the purity is measured by SDS-PAGE method, and the endotoxin is measured by limulus reagent method.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
EXAMPLE 1 purification of rhIL-18
This example rhIL-18 purification specifically comprises:
1. protein solubilization and renaturation:
adding a protein dissolving solution into an HPLC purified sample according to the proportion of 1:9, and dissolving for 2 hours at room temperature; slowly adding the renaturation solution according to the proportion of 1:8 to reduce the protein concentration to 0.2mg/mL, and magnetically stirring at room temperature for dissolving for 4 hours; renaturation is carried out for more than 20h at 4 ℃; and (5) immediately purifying.
2. Purification of rhIL-18:
balancing a Capto Q chromatographic column by using a balance liquid, and loading the renaturation liquid at the flow rate of 5 mL/min; fully eluting the hybrid protein with the equilibrium solution to a baseline, eluting with the equilibrium solution containing 0.2M NaCl, collecting the elution peak, and storing at-20 ℃ for identification.
The formula ranges and preparation of the liquids are as follows:
20mmol of PB buffer (pH7.2) was prepared in 1000mL of the following ratio:
sodium dihydrogen phosphate 2H2O 3.12g
Disodium hydrogen phosphate 12H2O 7.16g
EDTA 0.37g
The protein dissolving solution is prepared by taking 1000mL as a unit according to the following proportion:
480g of 8mol urea
10mmol DTT 1.65g
20mmol PB buffer (pH7.2) to 1000mL
The renaturation solution is prepared by taking 1000mL as a unit according to the following proportion:
20mmol of PB buffer (pH8.0) 1000mL
DTT(20mmol) 3.1g
The eluent is prepared according to the following proportion:
20mmol of PB buffer (pH7.2) 1000mL
0.2mol NaCl 11.6g
Test example 1 verification of purification Effect
1. recovery of rhIL-18 sample:
specifically, the method comprises the steps of measuring by a Coomassie brilliant blue method and measuring protein by the Coomassie brilliant blue method-enzyme mark instrument
Preparing a standard curve, respectively taking 7 EP tubes, adding bovine serum albumin mother liquor and water according to the volume of the EP tubes in the table 1, and uniformly mixing for later use. ② adding 5 mul of standard curve protein solution into 96-well plate, adding 250 mul of Coomassie brilliant blue G250 staining solution, measuring OD595 and drawing standard curve. ③ diluting the sample to proper concentration, adding 5 mul to each well, adding 250 mul Coomassie brilliant blue G250 staining solution, measuring OD595, and substituting into the standard curve to calculate the sample concentration. Bovine serum albumin mother liquor: 1 mg/mL. The results are shown in Table 2, and the standard curve is shown in FIG. 1.
TABLE 1 Standard Curve protein solution formulation protocol
Serial number Volume of mother liquor (ml) Water (ml) Concentration (mg/ml)
1 0 0.1 0
2 0.01 0.09 0.1
3 0.02 0.08 0.2
4 0.04 0.06 0.4
5 0.06 0.04 0.6
6 0.08 0.02 0.8
7 0.1 0 1.0
TABLE 2 purification recovery of rhIL-18
Before purification After purification
Concentration of 9.9mg/mL 1.37mg/mL
Volume of 7mL 30mL
Amount of protein 69.3mg 41.4mg
Recovery rate / 60％
2. Purity determination of rhIL-18 samples:
the SDS protein electrophoresis method is adopted, the concentration of the concentrated gel is 5 percent, the concentration of the separation gel is 12 percent, the constant current 25mA electrophoresis is carried out for 90 minutes, and the result shows that the purity is more than 99 percent, which is shown in figure 2.
3. Endotoxin assay for rhIL-18 samples:
the method comprises the following steps: the limulus reagent method was performed according to "the three-part appendix of the pharmacopoeia of the people's republic of China" 2010 edition, XII E "bacterial endotoxin test method (gel limit test)", and the results are shown in table 3.
TABLE 3 determination of endotoxin in rhIL-18 purified samples
Figure BDA0003215383290000091
Figure BDA0003215383290000101
The result shows that the rhIL-18 endotoxin after being purified by the purification method meets the relevant standard.
4. Determination of the biological Activity of rhIL-18:
(1) and human leukemia cell KG1 cell subculture: using 10% FCS in RPMI1640 medium, 5% CO2Culturing KG1 cells at 37 ℃; when the cell density reaches 2X 106Passage at individual cells/mL; the density of the passage cells is 4 multiplied by 105cells/mL.
(2) Preparing rhIL-18 into 50nmol by using RPMI1640 culture medium without serum for standby; the cell density of 96-well cell culture plates prepared by adding 100ul of 20% FCS 1640 medium to each well of the first 5 columns was 2X 106cells/mL, A, B, C, D plus ConA, final concentration 0.5 mg/L. Adding 100ul of 10-fold diluted rhIL-18 into each hole of the 1 st row to the 5 th row, and uniformly mixing, wherein the final concentration of the first row is 25 nmol; (4 wells per dilution). After mixing, the cell culture plate was placed at 37 ℃ in 5% CO2And culturing in an incubator for 24 h.
(3) The culture supernatants were collected by ELISA and the standard curve is shown in FIG. 3, and the results are shown in Table 4.
TABLE 4 IFN-. gamma.expression level
Final rhIL-18 concentration (nmol) IFN-gamma expression level (pg/mL)
25 7821.09
12.5 5422.16
6.25 1729.61
3.125 903.779
The results show that rhIL-18 can stimulate KG1 to produce IFN-gamma and has biological activity.
5. Inhibition assay of rhIL-18 on human endothelial cell microtubule formation:
EA.hy926 cells were treated with different concentrations (0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL) of IL-18 for 48h in advance. Mixing Matrigel artificial Matrigel with a serum-free 1640 medium pre-cooled at 4 ℃ according to the ratio of 1: mixing at a ratio of 1, spreading on the bottom of a 24-well plate at a volume of 300 μ L per well, and adding 5% CO2Curing the glue in a constant temperature incubator at 37 ℃ for 30 min. IL-18 treated EA.hy926 cells were taken and adjusted to 1.2X 10 concentration with serum-free 1640 medium5After the concentration of the micro-tube, the micro-tube is inoculated into the 24-well plate paved with the glue, 1mL of the micro-tube is cultured for 16h in a conventional way, the formation of the micro-tube-like structure is observed every 4h, and 5 visual fields are randomly photographed under an optical microscope (100 times). Images were analyzed using Microvision Saisam software to count microtubular structures at 5 fields per well.
TABLE 5 number of microtubular structures
rhIL-18 concentration (ng/mL) Number of micro-tube-like structures
0 83
0.1 78
1 59
10 17
100 5
The result shows that the rhIL-18 provided by the invention has an effect on inhibiting the generation of microtubule-like structures.
Comparative example 1
Obtaining the HPLC purified rhIL-18 sample was performed as described in patent application No. 202010771259.1, in particular example 1:
1. preparative liquid phase purification of recombinant IL-18
Purifying the recombinant IL-18 by using a preparation liquid to remove endotoxin, wherein the sample is a sample disclosed as a patent number: 201510455749.X liquid sample collected on gel chromatography column of recombinant IL-18 prepared.
The chromatographic column medium is C18, the specification is 21.2mm multiplied by 250mm, and the particle size is 7 mu m.
Mobile phase A: a mixed solution of pyrogen-free water and 0.1% trifluoroacetic acid, for example: 1L pyrogen-free water was added with 1ml trifluoroacetic acid.
Mobile phase B: to a mixed solution of acetonitrile and 0.1% trifluoroacetic acid, for example, 1L of acetonitrile, 1ml of trifluoroacetic acid was added.
And (3) taking the mobile phase A and the mobile phase B as eluents, wherein the flow rate is 10mL/min, and eluting by using a gradient elution mode, wherein the column temperature is 25 ℃, and the detection wavelength is 280 nm.
Gradient elution procedure:
time (min) Flow rate (ml/min) A％ B％
0 10 100 0
55 10 30 70
The sample peak with retention time of 15.308min was collected.
2. Removing acetonitrile and renaturation after liquid phase collection
1) Removing acetonitrile by a rotary evaporation method: a rotary evaporator is adopted, the temperature is 32 ℃, the pressure is-100 KPa, the rotating speed is 35-45rpm, and the time is 20-30 minutes. The volume after evaporation was about half that collected for the liquid phase.
2) Renaturation: and slowly adding the renaturation solution into the evaporated sample solution until the pH value of the sample solution is 7.0-7.2. The renaturation solution is 20mmol of PB buffer solution (pH8.0) and 20mmol of DTT.
3) And (3) dialysis: the sample was dialyzed against 100-fold volume of 20mmol PB buffer (pH7.0-7.2) by changing the solution every 12 hours for 24 hours to remove the residual acetonitrile.
3. results of rhIL-18 bioactivity assay:
TABLE 6 IFN-. gamma.expression level
Final rhIL-18 concentration (nmol) IFN-gamma expression level (pg/mL)
25 565.69
12.5 324.64
6.25 127.17
3.125 35.56
4. results of inhibition assay of rhIL-18 on human endothelial cell microtubule formation:
TABLE 7 number of microtubular structures
rhIL-18 concentration (ng/mL) Number of micro-tube-like structures
0 85
0.1 82
1 74
10 66
100 53
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for preparing high-activity recombinant protein is characterized by comprising the following steps:
mixing an HPLC purified sample of the recombinant protein with a protein dissolving solution, and dissolving;
mixing the dissolved sample with renaturation liquid for renaturation treatment;
purifying the renaturated sample by adopting an anion exchange chromatography column to obtain high-activity recombinant protein;
the protein dissolving solution comprises the following components:
4-10M of dissolving agent
DTT 5～20mM
The PB buffer solution is complemented to 1L;
the renaturation liquid comprises the following components:
PB buffer 1L
5-40 mM of reducing agent.
2. The method of claim 1, wherein the dissolving agent is urea and/or guanidine hydrochloride;
the dissolving agent is urea, and the concentration of the dissolving agent in the protein dissolving solution is 6-10M;
the dissolving agent is guanidine hydrochloride, and the concentration of the dissolving agent in the protein dissolving solution is 4-8M.
3. The method according to claim 1, wherein the reducing agent is DTT andor 2-mercaptoethanol.
4. The method according to claim 1, wherein the proteolytic solution comprises the following components:
6-8M of dissolving agent
DTT 10mM
The PB buffer solution is complemented to 1L;
the renaturation liquid comprises the following components:
PB buffer 1L
Reducing agent 20 mM.
5. The method according to claim 1, wherein the PB buffer is 10-30 mM and has a pH of 7.0-8.0.
6. The method according to claim 1, wherein the PB buffer solution is 15-25 mM and pH 7.0-7.5;
in the renaturation solution, the PB buffer solution is 15-25 mM and has pH of 7.6-8.0.
7. The preparation method of claim 1, wherein the volume ratio of the HPLC purified sample to the proteolytic solution is 1 (5-15); the conditions of the dissolution treatment are as follows: dissolving at 15-30 ℃ for 1.5-2.5 h.
8. The method of claim 1, wherein the volume ratio of the HPLC purified sample to the renaturation solution is 1: (5-10); the renaturation treatment conditions are as follows: stirring for 3-5 h at 15-30 ℃, and then renaturating for more than 20h at 0-6 ℃.
9. The method of claim 1, wherein the eluent used for the anion exchange chromatography column purification comprises the following components:
PB buffer 1L
NaCl 0.1～0.3M。
10. The method according to any one of claims 1 to 9, wherein when the sample after the renaturation treatment is purified by using an anion exchange chromatography column, the flow rate of the sample is 3 to 7 mL/min.
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