阅读说明：本技术 一种重组人成纤维细胞生长因子-21包涵体的纯化工艺 (Purification process of recombinant human fibroblast growth factor-21 inclusion body ) 是由 郑诗雨 马金航 张铭浩 惠琦 何鑫 于 2020-03-09 设计创作，主要内容包括：本发明公开了一种重组人成纤维细胞生长因子-21包涵体的纯化工艺,包括以下步骤：(1)将菌体悬浮于粗提工作液中,加入溶菌酶进行搅拌和超声破碎,然后进行离心、洗涤沉淀得到包涵体；(2)将步骤(1)得到的包涵体溶解于变性溶液中得到包涵体溶液,然后将复性溶液加入包涵体溶液,搅拌均匀,得到复性后的包涵体溶液；(3)步骤(2)得到包涵体溶液采用Q柱进行分离,得到蛋白溶液；(4)步骤(3)得到的蛋白溶液采用Q柱进行浓缩,得到重组人成纤维细胞生长因子-21蛋白。该纯化工艺可以获得重组人成纤维细胞生长因子-21蛋白,并且该蛋白具有较高的纯度和活性。(The invention discloses a purification process of a recombinant human fibroblast growth factor-21 inclusion body, which comprises the following steps: (1) suspending the thallus in the crude extraction working solution, adding lysozyme, stirring and ultrasonically crushing, and then centrifuging, washing and precipitating to obtain an inclusion body; (2) dissolving the inclusion body obtained in the step (1) in a denaturing solution to obtain an inclusion body solution, then adding the renaturation solution into the inclusion body solution, and uniformly stirring to obtain the renatured inclusion body solution; (3) separating the inclusion body solution obtained in the step (2) by adopting a Q column to obtain a protein solution; (4) and (4) concentrating the protein solution obtained in the step (3) by adopting a Q column to obtain the recombinant human fibroblast growth factor-21 protein. The purification process can obtain recombinant human fibroblast growth factor-21 protein with high purity and activity.)

1. A process for purifying recombinant human fibroblast growth factor-21 inclusion bodies is characterized by comprising the following steps:

(1) suspending the thallus in the crude extraction working solution, adding lysozyme, stirring and ultrasonically crushing, and then centrifuging, washing and precipitating to obtain an inclusion body;

(2) dissolving the inclusion body obtained in the step (1) in a denaturing solution to obtain an inclusion body solution, then adding the renaturation solution into the inclusion body solution, and uniformly stirring to obtain the renatured inclusion body solution;

(3) separating the inclusion body solution obtained in the step (2) by adopting a Q Sepharose Fast Flow anion exchange column to obtain a protein solution;

during separation, 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L urea are used as buffer solution, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.05mol/L NaCl are used as impurity washing solution, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.1mol/L NaCl are used as eluent, and 25mmol/L Tris-HCl (pH7.8) and 1mol/L NaCl are used as regeneration solution;

(4) concentrating the protein solution obtained in the step (3) by adopting a Q Sepharose Fast Flow anion exchange column to obtain a recombinant human fibroblast growth factor-21;

in the concentration, 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L urea were used as a sample buffer for chromatography, and 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L NaCl were used as eluents.

2. The process for purifying inclusion body of recombinant human fibroblast growth factor-21 in claim 1, wherein in step (1), the crude extraction working solution is 25mmol/L Tris-HCl (pH8.0) buffer solution, and contains 150mmol/L NaCl, 2mmol/L EDTA-2Na and 0.5% triton.

3. The process for purifying inclusion body of recombinant human fibroblast growth factor-21 as claimed in claim 1, wherein in step (1), 1% sodium deoxycholate and 1% Triton X-100 solution are respectively used for washing.

4. The process for purifying inclusion bodies of recombinant human fibroblast growth factor-21 according to claim 1, wherein in the step (2), the denaturing solution is 8mol/L urea, and the dosage ratio of the inclusion bodies to the denaturing solution is 1:10(W: V).

5. The process for purifying recombinant human fibroblast growth factor-21 inclusion bodies according to claim 1, wherein in the step (2), the renaturation solution is 25mmol/L Tris (pH7.8), and the ratio of the inclusion bodies to the renaturation solution is 1:70(W: V).

6. The process for purifying inclusion body of recombinant human fibroblast growth factor-21 as claimed in claim 1, wherein in step (3), the inclusion body is separated by two column chromatographies, and after the first column chromatographies are completed, the second column chromatographies are performed after dialysis and desalination.

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a purification process of a recombinant human fibroblast growth factor-21 inclusion body.

Background

rhFGF-21 is one new member of fibroblast growth factor family, and its precursor consists of 209 amino acids, N terminal has special signal peptide sequence and mature protein 181 amino acids. The relative molecular mass is about 19.4 kD. The research also finds that the protein has an isoelectric point of 5.0-6.0 and is acidic protein. Recombinant human fibroblast growth factor-21 (rhFGF-21) is the only gene without mitogenesis found in FGFs family, and has close relation with glycolipid metabolism as an endocrine factor. The action mechanism of rhFGF-21 in lipid metabolism is mainly embodied as follows: 1) FGF-21 improves the function of pancreatic beta cells. It can improve the survival rate of islet beta cells, reduce insulin resistance and improve islet sensitivity by activating ERK1/2 and Akt signal pathways. 2) The regulation and control of FGF21 on lipid metabolism mainly include reduction of lipotoxicity, reduction of lipid neogenesis, improvement of fat beta oxidation, improvement of adiponectin content, promotion of browning of white fat, and reduction of triglyceride and phospholipid content in serum. 3) FGF21 reduces oxidative stress and inhibits the release of inflammatory factors. Therefore, FGF21 is more advantageous in treating nonalcoholic steatohepatitis.

If a large amount of FGF-21 protein is to be obtained, the expression of the protein in the engineering bacteria needs to be realized by genetic engineering technology. Since the FGF-21 expression strain constructed by the previous step is expressed in Escherichia coli in the form of inclusion bodies, the proteins in the inclusion bodies are unfolded aggregates and have no biological activity, so that the inclusion bodies are required to be dissolved to release the proteins in the inclusion bodies and perform protein renaturation to obtain the proteins with the biological activity. The inclusion bodies are mainly expressed products which can occupy 40-95% of the total body protein, and in addition, the outer membrane protein, RNA polymerase, RNA, DNA, lipids and carbohydrates of the host bacteria are also contained, so that the inclusion bodies can be prepared by primary purification by adding a surfactant, and the recombinant proteins can be purified by adopting a proper method (such as chromatography) after the inclusion bodies are separated, so that the treatment of the inclusion bodies generally comprises the following steps: the method comprises the steps of breaking thallus, washing inclusion bodies, dissolving and denaturing, renaturating and purifying, and the protein chromatography purification generally comprises three stages of capture, intermediate purification and fine purification. The aim of the capture phase is to isolate, concentrate and stabilize the protein of interest in order to maintain protein activity.

Disclosure of Invention

The invention provides a purification process of a recombinant human fibroblast growth factor-21 inclusion body, which can obtain recombinant human fibroblast growth factor-21 protein with higher purity and activity.

A process for purifying recombinant human fibroblast growth factor-21 inclusion bodies comprises the following steps:

(1) suspending the thallus in the crude extraction working solution, adding lysozyme, stirring and ultrasonically crushing, and then centrifuging, washing and precipitating to obtain an inclusion body;

(2) dissolving the inclusion body obtained in the step (1) in a denaturing solution to obtain an inclusion body solution, then adding the renaturation solution into the inclusion body solution, and uniformly stirring to obtain the renatured inclusion body solution;

(3) separating the inclusion body solution obtained in the step (2) by adopting a Q Sepharose Fast Flow anion exchange column to obtain a protein solution;

in the separation, 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L urea are used as buffer solution, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.05mol/L NaCl are used as impurity washing solution, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.1mol/L NaCl are used as eluent, and 25mmol/L Tris-HCl (pH7.8) and 1mol/L NaCl are used as regeneration solution;

(4) concentrating the protein solution obtained in the step (3) by adopting a Q Sepharose Fast Flow anion exchange column to obtain recombinant human fibroblast growth factor-21 protein;

in the concentration, 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L urea were used as a sample buffer for chromatography, and 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L NaCl were used as eluents.

Preferably, in step (1), the crude extraction working solution is 25mmol/L Tris-HCl (pH8.0) buffer solution, and contains 150mmol/L NaCl, 2mmol/L EDTA-2Na and 0.5% triton. In the crude extraction working solution, Tris-HCl buffer solution (pH8.0) can provide enough buffer capacity to ensure the pH environment of the stock solution, EDTA-2Na is used as a metal ion chelating agent to reduce the oxidation effect of metal ions on released target protein, meanwhile, EDTA can also play a role in damaging cell intima, and Triton is used as a surfactant to promote the cell disruption.

Preferably, in step (1), 1% sodium deoxycholate and 1% Triton X-100 solution are used for washing, respectively.

Preferably, in the step (2), the denaturing solution is 8mol/L urea, and the ratio of the inclusion bodies to the denaturing solution is 1:10(W: V).

Preferably, in step (2), the renaturation solution is 25mmol/L Tris (pH7.8), and the dosage ratio of the inclusion bodies to the renaturation solution is 1:70(W: V).

Preferably, in the step (3), the separation is performed by two column chromatography processes, and after the first column chromatography process is completed, the second column chromatography process is performed after dialysis and desalination.

Further, the method comprises the following specific steps:

(1) the thallus is suspended in 25mmol/L Tris-HCl (pH8.0) buffer solution at the ratio of 1:5(W: V), the content of NaCl is 150mmol/L, EDTA-2Na is 2mmol/L, 0.8mg lysozyme is added into per gram of thallus and stirred, the thallus is kept at 37 ℃ for 30min while being continuously stirred, and the thallus is broken by ultrasound after being kept at 37 ℃ until the solution is not viscous. And (3) centrifuging the liquid after the bacteria breaking to obtain precipitates, washing the precipitates respectively by using 1% of sodium deoxycholate and 1% of Triton X-100, and finally obtaining the precipitates, namely the inclusion bodies.

(2) The inclusion body is dissolved in 8mol/L urea, renaturation liquid is pumped in at a low flow rate until the final concentration of the urea is 1mol/L, and the obtained liquid is the inclusion body solution after renaturation.

(3) The first step of chromatography adopts Q Sepharose Fast Flow anion exchange column chromatography: using 25mmol/L Tris-HCl (pH7.8) and 1.0mol/L urea as the sample loading buffer solution of the first step of chromatography, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.05mol/L NaCl as the eluent, and 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea and 0.1mol/L NaCl as the eluent, and collecting protein peaks. 25mmol/L Tris-HCl (pH7.8), 1mol/L NaCl as regeneration liquid.

(4) And (3) after the obtained eluent is dialyzed and desalted, repeating the Q column again, wherein the obtained eluent is a purer target protein solution, but the target protein concentration is lower, so that the Q column needs to be repeated again for sample concentration.

(5) The concentration chromatography adopts Q Sepharose Fast Flow anion exchange column chromatography: the protein peak was collected by eluting with 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea as the sample buffer for chromatography, 25mmol/L Tris-HCl (pH7.8), 1.0mol/L NaCl as the eluent.

Drawings

FIG. 1 is the electrophoresis chart of the adsorption test of rhFGF-21 protein on a Q column in example 1;

FIG. 2 is the electrophoresis chart of the adsorption test of rhFGF-21 protein on a DEAE column in example 1;

FIG. 3 is an electrophoretogram of rhFGF-21 inclusion body 1% Triton washed 2 times in example 2;

FIG. 4 is the rhFGF-21 inclusion body 1% Triton sodium deoxycholate washing electrophoresis chart in example 2;

FIG. 5 is the Triton washing electrophoretogram of rhFGF-21 inclusion body urea sodium deoxycholate in example 2;

FIG. 6 is the electrophoresis chart of the inclusion body renaturation of rhFGF-21 in example 3 through DEAE column adsorption test;

FIG. 7 is the electrophoresis chart of the rhFGF-21 inclusion body renaturation Q-column adsorption test in example 3.

Detailed Description

The instruments and reagents used in the present invention were as follows:

required instrumentation: centrifuge, high pressure homogenizer, and chromatographic column.

Required reagents: purifying working solution

Solution A: 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea

And B, liquid B: 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea 50mmol/L NaCl solution C: 25mmol/L of LTris-HCl (pH7.8), 1.0mol/L of urea 100mmol/L of NaCl solution D: 25mmol/L Tris-HCl (pH7.8), 1.0mol/L urea 300mmol/L NaCl E solution: 25mmol/L Tris-HCl (pH7.8), 1.0mol/L NaCl

Chromatography conditions are as follows:

anion exchange column chromatography: and selecting a Q column as a chromatographic packing on the basis of the adsorption test, and carrying out the chromatographic test.

Regeneration: and washing the regenerated chromatographic column by using the E solution.

Washing with water: the column was washed clean of salt with purified water.

Balancing: and (4) balancing the column by using the solution A, and balancing 1.5-3 column volumes until the baseline of the detector is stable.

Loading: pumping the sample into a chromatographic column, collecting the emergent peak, and detecting the emergent peak.

Balancing: the column was equilibrated with solution A until the baseline of the detector was stable.

Impurity washing: and (4) pumping the solution into a chromatographic column by using a B liquid, collecting the washing impurity peak, and reserving the sample for detection.

And (3) elution: and (4) pumping the solution into a chromatographic column by using a C liquid, collecting an elution peak, and reserving a sample for detection.

Impurity washing: and (4) pumping the solution into a chromatographic column by using a D liquid, collecting an elution peak, and reserving a sample for detection.

Regeneration: eluting 1-2 column volumes by using E solution, collecting the emergent peaks, and detecting the residual sample.

The collected eluent is dialyzed and then is repeated for one time to obtain the eluent which is the purer target protein stock solution, and the concentration is required to be concentrated once because the concentration of the sample is thinner.

Concentration chromatography conditions (Q column):

regeneration: and washing the regenerated chromatographic column by using the E solution.

Washing with water: the column was washed clean of salt with purified water.

Balancing: and (4) balancing the column by using the solution A, and balancing 1.5-3 column volumes until the baseline of the detector is stable.

Loading: pumping the sample into a chromatographic column, collecting the emergent peak, and detecting the emergent peak.

Balancing: the column was equilibrated with solution A until the baseline of the detector was stable.

And (3) elution: pumping the solution into a chromatographic column by using an E liquid, and collecting an elution peak to obtain the required target protein stock solution.