CN111018965A - Purification method of recombinant parathyroid hormone PTH (1-34) - Google Patents

Abstract

The application discloses a separation and purification method of recombinant parathyroid hormone PTH, which comprises the following steps: 1) crushing bacteria and processing an inclusion body, homogenizing and crushing the bacteria through high pressure, centrifuging at high speed after crushing the bacteria, collecting precipitate, and mixing the precipitate with a redissolution for redissolution after resuspension and washing; 2) affinity chromatography; the filler is selected from one of Ni-NTA, GST Sepharose and MBP Sepharose; 3) performing enzyme digestion and renaturation on the column; 4) cation exchange chromatography and/or hydrophobic chromatography; 5) and (4) desalting. The invention can efficiently extract the PTH (1-34) polypeptide with biological activity from the inclusion body, thereby enabling the large-scale industrial production of the PTH (1-34).

Description

Purification method of recombinant parathyroid hormone PTH (1-34)

Technical Field

The invention belongs to the technical field of protein separation and purification, and particularly relates to a method for separating and purifying recombinant parathyroid hormone PTH (1-34).

Background

Parathyroid hormone (PTH) is a polypeptide hormone consisting of 84 amino acids, which is synthesized, stored and secreted by Parathyroid epithelial cells. PTH entering blood circulation is quickly converted into two peptide segments of an amino terminal and a carboxyl terminal, wherein the peptide segments of amino acid residues 1-34 of the amino terminal have complete PTH bioactivity, regulate blood calcium concentration and bone metabolism and have small immunocompetence, and the peptide segment of the carboxyl terminal is PTH immunocompetent polypeptide, so the biological effect of the PTH polypeptide is not clear.

PTH exerts a major biological function against osteoporosis, and its effect on bone formation is mainly produced by acting on bone tissue cells and renal tubular epithelial cells. For renal tubular epithelial cells, PTH mainly increases the reabsorption of calcium in the distal tubule and indirectly promotes the absorption of calcium in the intestinal tract by increasing the production of active vitamin D by the kidney, so that the blood calcium concentration is increased together, and the formation of bone is facilitated. The bone tissue not only can promote bone resorption, but also can promote bone formation.

The American Eli Lilly corporation developed recombinant human PTH (1-34) as early as the nineties of the last century and was named Teraramide for the treatment of osteoporosis. Some research and development units and enterprises in China are developing recombinant human parathyroid hormone, currently, Xinlite, a Shanghai union Sertole product, has obtained production lots, some are in preclinical research or clinical research, and some enter a clinical test stage.

The purification method of the product is disclosed in patent technologies, such as CN102993293A, CN102731643A, CN1212336C, etc. However, these methods generally have the problems of requiring the use of organic solvents to affect the activity of polypeptides, being easily degraded, having low yield, and having complex process flow. In order to solve the above problems, further research on purification methods is still necessary.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a simple purification method capable of efficiently extracting biologically active PTH (1-34) polypeptide from inclusion bodies, thereby making large-scale industrial production of PTH (1-34) possible.

The invention discloses a separation and purification method of recombinant parathyroid hormone PTH, which comprises the following steps:

1) bacterial disruption and inclusion body treatment

Breaking bacteria by high-pressure homogenization, centrifuging at high speed after breaking bacteria, collecting precipitate, mixing the precipitate with a redissolving solution after resuspension and washing

2) Selecting proper affinity chromatography filler for affinity chromatography according to the label peptide segment of the fusion protein; the filler is selected from one of Ni-NTA, GST Sepharose and MBP Sepharose;

3) on-column restriction and renaturation

Wherein the enzyme is selected from TEV enzyme, EK enzyme, and Xa factor protease;

4) cation exchange chromatography and/or hydrophobic chromatography

The filler of the cation exchange chromatographic column is selected from one of SP Sepharose HP, SP Sepharose FF, Capto SP and Capto MMC;

the filler of the hydrophobic chromatographic column is one of HP or FF fillers of Phynel, Octyl or Butyl;

5) and (4) desalting.

In one embodiment according to the present invention, in step 1), step 1) comprises:

the weight of the thallus and the solution A is as follows: the volume ratio is 1: mixing at a ratio of 5-10, and pre-cooling to 1-4 deg.C.

In one embodiment according to the present invention, step 1) comprises:

homogenizing at 1-4 deg.C under 60-80Mpa for 3-5 times; centrifuging the liquid after the bacteria breaking for 15-30min at the temperature of 4 ℃ and under the conditions of 10,000 and 15,000g, and collecting the precipitate for later use.

In one embodiment according to the present invention, step 1) comprises:

resuspend inclusion bodies in resuspension, weight of inclusion bodies to resuspension: the volume ratio is 1: 5-10, stirring for 15-30min by a magnetic stirrer, centrifuging for 15-30min by 15,000g of 10,000-; the resuspension is solution a containing Triton X1000, tween20, tween80, or sacosyl at a final concentration of 0.5% to 1.5%.

In one embodiment according to the present invention, step 1) comprises:

the weight ratio of: the volume ratio is 1: adding a re-suspension solution in a proportion of 5-10 to re-suspend the inclusion body, uniformly mixing, centrifuging and collecting a supernatant for later use; the compound solution uses solution A as a solvent and contains a mixed solution of 8M urea, 6M guanidine hydrochloride or 2mol/L urea and sacosyl until the final concentration (volume percentage concentration)) is 0.3%.

In one embodiment according to the present invention, step 2) comprises:

the ratio of the wet weight of the bacteria breaking thallus to the filler is 10: 1-2, mixing the re-dissolved inclusion body with a filler uniformly, and further removing foreign protein on the filler by using a solution A containing 20-25mM imidazole and 8M urea;

preferably, the column packing is washed with several volumes of water before mixing with the reconstituted inclusion bodies and then equilibrated with liquid a containing 8M urea.

In one embodiment according to the present invention, step 3) comprises:

adding a proper amount of protease with His tag into the mixture, and performing on-column enzyme digestion, wherein the enzyme digestion solution is solution A containing 2-4M urea or guanidine hydrochloride, the amount of the enzyme digestion solution is 1-2 times of that of the filler, and the enzyme digestion condition is 4-25 ℃ for 2-5 h; wherein the weight volume ratio of the inclusion body to the enzyme is 10: 1.

in one embodiment according to the present invention, the step 4) comprises:

collecting samples after affinity chromatography or hydrophobic chromatography, diluting with solution B for 3-6 times, and adjusting pH to 6.5; then, carrying out linear gradient elution by using solution C (), setting the elution flow rate to be 5ml/min, and setting the elution gradient to be that the solution B is from 0 to 50 percent, and the elution volume is 10 column volumes; wherein the solution B is 20mM phosphate buffer solution (PB) with the pH value of 6.5; the solution C was 20mM Phosphate Buffer (PB) containing 1M NaCl, pH 6.5.

In one embodiment according to the present invention, the step 4) comprises:

mixing a sample obtained by affinity chromatography or cation exchange chromatography with 3M ammonium sulfate solution until the final concentration of ammonium sulfate is 1-1.5M;

balancing a chromatography system and a chromatography column by using a solution D, then loading, after loading, carrying out linear gradient elution by using a solution B, wherein the elution flow rate is 5ml/min, the elution gradient is that the solution B is from 0 to 100 percent, the elution volume is 10 column volumes, collecting proteins and storing at 4 ℃; the solution D is 20mM phosphate buffer solution containing 1-1.5M ammonium sulfate and pH 6.5.

In one embodiment according to the present invention, the step 5) comprises:

the desalting is carried out by one selected from ultrafiltration, diafiltration, molecular sieve or desalting column.

The invention has the characteristics and innovation points that:

by adopting the purification method, the target protein with the purity of more than 98 percent and the recovery rate of more than 40 percent can be obtained from the Escherichia coli engineering bacteria expressing PTH (1-34). The N, C terminal sequence was found to be consistent with the theoretical value by amino acid sequence determination. The mass spectrometric detection molecular weight was about 4117 daltons, consistent with the theoretical value. Cell experiments prove that the compound has good capability of stimulating cAMP secretion which is not lower than the standard product purchased by Sigma company, and the compound has good biological activity.

In addition, the method is a non-HPLC purification method, and avoids damage of organic solvents and the like to the activity of the protein. The method successfully realizes the synchronous on-column enzyme digestion, enzyme digestion and renaturation, and greatly shortens the purification process. The method is simple to operate, does not need special instruments, reagents and the like, and is favorable for industrial expanded production.

Drawings

FIG. 1 is a flow chart of a PA5055 purification process

FIG. 2 is a cation chromatogram; wherein the flow-through sample contains a small amount of contaminating proteins and the peak at elution is collected for subsequent purification.

FIG. 3 is a hydrophobic chromatogram; the former peak was hetero-protein and the latter peak was PTH (1-34) at the time of elution;

FIG. 4 shows the result of SDS-PAGE electrophoresis of a protein stock solution;

FIG. 5 shows the HPLC detection results of the protein stock solution of PTH (1-34).

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

Experimental reagents and materials

1. Bacterial strain and plasmid

Strain BL21(DE3), DH5alpha E.coli from Novagen; the strain XL-1blue escherichia coli is a product of Agilent company in the United states; the plasmid pGEX-6p-2 is a product of GE Healthcare, USA; the plasmids pET32a and pET39b (+) are products of America merck company; pMal-c4X is a product of NEB corporation; pCOLD-SUMO was purchased from Sorangbao.

2. Reagent

TEV enzyme and EK enzyme are available from Biyunnan Biotechnology Ltd, factor Xa protease Beijing Baiolai Pagaco technology Ltd.

primeSTAR HS DNA polymerase, DNA molecular weight standard, restriction enzyme, protein molecular weight standard, DNA ligase, point mutation kit (mutanBest), etc. are products of Dalibao organism (Takara); the plasmid extraction kit and the gel recovery kit are products of American Omega company; peptone and yeast extracts were purchased from Oxfoid corporation and medium from okition biotechnology, beijing.

PBS (Potassium dihydrogen phosphate (KH)₂PO₄)0.2g (domestic analytical grade), disodium hydrogen phosphate (Na)₂HPO₄·12H₂O)2.9g (domestic analytically pure), sodium chloride (NaCl)8.0g (domestic analytically pure), potassium chloride (KCl)0.2g, water is added to 1000mL, and the pH value is 7.4); 20mol/L PB buffer: potassium dihydrogen phosphate (KH)₂PO₄)0.2g of disodium hydrogen phosphate (Na)₂HPO₄·12H₂O)2.9g, potassium chloride (KCl)0.2g, water is added to 1000mL, pH is 6.0, ampicillin and kanamycin (North China pharmaceutical) are added, 5 times protein loading buffer solution is 250mmol/L Tris-HCl (pH6.8), 10% (W/V) SDS, 0.5% (W/V) bromophenol blue, 50% (V/V) glycerol, 5% (W/V) β -mercaptoethanol, glutathione sepharose 4B (GE Healthcare company, USA), Ni-NTA is purchased from Novagen company, agar powder, Tween-20 and other reagents are purchased from domestic markets.

In order to make the purposes, technical schemes and advantages of the invention more clearly understood, the invention is further described in detail with reference to the accompanying drawings and examples, in which recombinant engineering bacteria pET28a-PTH (1-34)/BL21 (the engineering bacteria introduce His tag, SUMO tag and TEV enzyme digestion site in sequence at the N-terminal of PTH (1-34), the His tag is favorable for subsequent purification, the SUMO tag is favorable for protein folding and renaturation, and no residual amino acid is left at the N-terminal of PTH (1-34) after TEV enzyme digestion) is constructed in the following chamber. The purification process flow of the present invention is shown in FIG. 1.

Example 1 construction and expression of recombinant PTH (1-34) engineering bacteria

PTH (1-34) is the amino acid sequence of SEQ ID NO. 1, and the coding DNA codon is optimized to SEQ ID NO. 2.

Three genes of TEV enzyme restriction site (ENLYFQ), enterokinase enzyme site (DDDDK) and Xa factor restriction site (IEGR) are respectively added to the N end of PTH (1-34), then (GGGGS)3, His6 and SSGSSG are sequentially added from right to left of each gene, BamHI site GGATCC is added to the 5 'end of each gene, termination codes TGATAA, HindIII site AAGCTT and NotI site GCGGCCGC are added to the 3' end, and the sequences are synthesized by Shanghai bioengineering limited company.

The three genes synthesized by BamHI + HindIII and BamHI + NotI double digestion and the vectors shown in Table I were used to recover the target fragment and the vectors, respectively. The construction of the recombinant expression vectors was performed in the manner shown in Table 1.

TABLE 1 construction of recombinant PTH (1-34) expression vectors

The synthetic FH8 gene (SEQ ID NO:3) was synthesized by Shanghai bioengineering, Inc., pET22b and pET28a were incorporated, the primer pair was designed to amplify the DNA sequence encoding- (GGGGGGS) n-TEVs-PTH, the primer pair pET22b and pET28a were designed to amplify separately, and the construction of the recombinant vectors pET22b-FH8- (GGGGGGS) n-TEVs-PTH and pET28a-FH8- (GGGGGGS) n-TEVs-PTH was carried out using the homologous recombination Kit Mut Express MultiS Fast Mutagenesis Kit V2 according to the manufacturer's instructions. The recombinant plasmid is transformed into Escherichia coli DH5 alpha.

The positive clones obtained in the two steps are cultured and extracted, and after the plasmids are identified correctly, the positive clones are respectively transformed into escherichia coli XL1-Blue (pGEX-6P-2) and BL21(DE3) (the rest plasmids).

Example 2: optimized construction of recombinant PTH (1-34) engineering bacteria

Preliminary results are summarized in Table 2 by summarizing the fusion proteins expressed from the recombinant vectors constructed in example 1,

TABLE 2 expression of the recombinant engineered bacteria

If the protein is a fusion protein which can be soluble expressed, the obtained target protein PTH (1-34) is in an oxidation type; if the expression is inclusion body expression, the protease enzyme digestion can be carried out only by completely renaturing the fusion protein, the old way of the existing preparation of the target protein is also obtained, a reverse phase purification step is also added in the purification process, and a large amount of organic solvent is used in the preparation process.

In order to overcome the defects of the prior art, the optimization construction strategy of the recombinant PTH (1-34) engineering bacteria is as follows: the recombinant fusion protein is expressed in the form of loose inclusion bodies, and the fusion protein can be renatured in the presence of urea with higher concentration and is cut by TEV enzyme which is easier to prepare and more economical.

The various combinations of forms were performed by optimizing the elements preceding the leader peptide by PCR, and repeated screening was performed. The characteristics that the SUMO helps the fusion protein to fold and increase the solubility are utilized; and the characteristics that the expression quantity of the pET28a vector is high and the expression quantity is further improved by expression enhancing peptide are utilized. Incompact inclusion bodies are formed by rapid expression to counteract the solubility brought by SUMO. Finally, a recombinant PTH (1-34) engineering strain, namely pET28 a-expression enhancing peptide-connecting peptide 1-His 6-connecting peptide 2-guide protein-connecting peptide 3-protease cutting site-PTH (1-34)/BL21(DE3), abbreviated as pET28a-HSTP/BL21(DE3) is screened. The amino acid sequence of the whole fusion protein is SEQ ID NO. 4.

Example 3 bacterial disruption and treatment of Inclusion bodies

And (3) resuspending the thallus: the Escherichia coli engineering bacteria pET28a-PTH (1-34)/BL21 are subjected to high-density fermentation and centrifugation to collect bacteria for later use. Taking about 50g of thalli, and weighing (g): volume (ml) to 1: 5-10, mixing with PBS (solution A) with pH6.0-pH8.5, suspending, and pre-cooling at 4 deg.C.

High-pressure bacterium breaking: and (3) washing a pipeline of the high-pressure homogenizer by using distilled water, and starting a low-temperature circulating system to precool to 1-4 ℃ for later use. Adding the precooled suspension bacteria into a high-pressure homogenizer, maintaining the pressure at 60-80Mpa for 3-5 times, taking the smear of the bacteria-breaking liquid, and performing crystal violet dyeing, wherein less than 1-2 uncrushed bacteria in each visual field of the oil lens are regarded as complete bacteria breaking.

High-speed centrifugation: the liquid after the bacteria breaking is put into a centrifugal barrel, centrifuged for 15-30min at 4 ℃ and 15,000g of 10,000-.

Washing of inclusion bodies: adopting solution A containing 0.5-1.5% of Triton X100 according to the weight percentage: the volume ratio is 1: resuspending the inclusion body at a ratio of 5-10, stirring with a magnetic stirrer for 15-30min, centrifuging at 10,000-15,000g for 15-30min, and collecting the precipitate for later use.

Inclusion body redissolving: adopting a solution A containing 8M urea according to the weight: the volume ratio is 1: resuspending the inclusion body at a ratio of 5-10, stirring with a magnetic stirrer for 15-30min, centrifuging at 10,000-15,000g for 15-30min, and collecting the supernatant for later use.

Example 4 Ni-NTA affinity chromatography, on TEV enzyme column cleavage and renaturation

Ni-NTA affinity chromatography packing is selected for preliminary purification, GST affinity packing products of different models are obtained by different companies, Ni-NTA affinity chromatography packing of invitrogen is adopted in the embodiment, and the consumption of the wet weight of the bacteria breaking thallus per 100g of packing is 10-20 ml. Cleaning the filler by pure water for 2-5 volumes, balancing the filler by using solution A containing 8M urea, mixing the dissolved inclusion body with the filler, gently mixing for 10-20min, removing unbound protein, and further removing foreign protein on the filler by using solution A containing 20-25mM imidazole and 8M urea. And then adding a proper amount of TEV enzyme with a His tag (1g of inclusion body added with about 0.1ml of enzyme) for on-column enzyme digestion, wherein the enzyme digestion solution is solution A containing 2M urea, the dosage of the enzyme digestion solution is 1-2 times of that of the filler, the enzyme digestion condition is 4-25 ℃ for 2-5h, and the enzyme digestion process is vertically suspended to ensure that the enzyme is fully contacted with the fusion protein, so that the enzyme digestion is full, and the on-column renaturation is completed while the enzyme digestion is carried out. And collecting the supernatant for subsequent purification after the enzyme digestion is finished.

Example 5 cation exchange chromatography

The samples for affinity chromatography were collected, diluted 3-6 times with solution B (20mM PB, pH6.5) and adjusted to pH6.5 or so for further use. And (3) adopting a liquid B equilibrium chromatography system and a 5ml SP HP chromatographic column, then carrying out loading, and eluting unbound heteroproteins by using a liquid A after the loading is finished. Eluting with C solution (20mM PB, 1M NaCl, pH6.5) with linear gradient, setting elution flow rate to 5ml/min, eluting gradient from 0 to 50% of B solution, eluting 10 column volumes, eluting impurities such as nucleic acid with 100% of B solution, collecting eluted protein, and storing at 4 deg.C. The chromatogram is shown in FIG. 2, where a small amount of contaminating proteins is present in the flow-through sample, the two peaks merge during elution, and the peak at elution is collected for subsequent purification.

Example 6: hydrophobic chromatography and displacement buffer

Adding 3M ammonium sulfate solution into the sample obtained by the cation exchange chromatography until the final concentration of ammonium sulfate is 1-1.5M, balancing the chromatography system by adopting D liquid (20mM PB, 1-1.5M ammonium sulfate, pH6.5) and 5ml Phynel HP chromatography column, then loading, and eluting the unbound hetero protein by using A liquid after loading. Performing linear gradient elution with liquid B, setting the elution flow rate to be 5ml/min, the elution gradient is that the liquid B is from 0 to 100 percent, the elution volume is 10 column volumes, collecting protein and storing at 4 ℃ for later use. As shown in FIG. 3, two peaks appeared in the elution, the former peak was a hetero-protein and the latter peak was the PTH (1-34) protein of interest, and the two peaks could be separated efficiently.

Desalting by directly replacing buffer solution with 50ml G25 desalting column, using 200mM sodium acetate and 150mM NaCl, sampling after desalting, performing SDS-PAGE identification, subpackaging samples, and freezing at-80. The electrophoresis result of the desalted sample is shown in FIG. 4, and the sample has extremely high purity, and other foreign proteins are basically not seen.

Example 7: HPLC detection

The purity of the above purified PTH (1-34) protein was checked using a C18 column (available from Agilent Co.), the column was equilibrated with 0.1% aqueous TFA, and 5ul of a sample was applied and eluted with 0.1% TFA acetonitrile at a column temperature of 60 ℃ and a flow rate of 0.5 ml/min. The elution procedure was: 10-100% B, 30 min. The mass spectrogram is shown in FIG. 5, and the result shows that the purity of PTH (1-34) is about 99%, which meets the purity requirement of biological products on protein drugs. Preparation of 0.1% TFA aqueous solution: 1LI grade water was mixed with 1ml TFA and filtered through a 0.22 μm filter. Preparation of 0.1% TFA acetonitrile solution: 1L acetonitrile was mixed with 1ml TFA.

Example 8: cellular animals

1) Cell preparation: 2 bottles of Saos-2 cells were taken, the medium was discarded, and 3ml of PBS was added to each bottle, and the medium was washed and discarded. Digestion was carried out with 1ml of each pancreatin, and digestion was terminated by adding 5ml of incomplete medium. And (3) blowing and beating by using an elbow dropper, transferring the liquid into a 15ml centrifuge tube after the cells are completely exfoliated, centrifuging for 1500r by 5min, removing supernatant, adding 1ml of complete culture medium, and counting the cells after uniformly mixing. After counting, the whole culture medium is used for preparing 2 multiplied by 10⁵200 ul/ml in 96-well plates at 37 ℃ with 5% CO₂Overnight incubation for about 28 h.

(2) Preparation of PTH samples and media: PTH samples were adjusted to 250ug/ml with sodium acetate buffer (pH 4.0). Complete medium (McCoy's medium + 10% fetal bovine serum + 1% HEPES) and viability medium (0.1% bovine serum albumin plus complete medium to 10ml +0.5M IBMX + corresponding protein samples) were prepared.

(3) Stimulating cells and sample processing: 8 EP tubes were used, numbered 1-7. 180ul of viability assay medium was added to each of the 2-7 tubes. 312ul of viability assay medium was added to 1 tube and the volume of viability assay medium added to 1 tube was calculated after protein concentration was determined so that the protein concentration in 1 tube was 4000 ng/ml. 60ul of the mixture is taken from the 1 tube and transferred to the 2 tube to be mixed uniformly, the concentration is 1000ng/ml, and 60ul of the mixture is taken from the 2 tube and transferred to the 3 tube to be mixed uniformly, and then the mixture is added to the 7 th tube. The cell plate is provided with 7 holes corresponding to numbers 1-7, each hole is added with 150ul of protein sample for stimulation, another hole is used as a cell blank, and stimulation is carried out at 37 ℃ for 15 min. Duplicate wells were set up in the experiment and PTH standard purchased from Sigma was used as a control.

After the stimulation was completed, the supernatant was aspirated off, 200ul PBS was added to each well, and after washing twice, 80ul 0.1M HCl was added to each well and left at room temperature for 20 Min. After the completion, the cells were blown up several times with a pipette tip, 70ul of the liquid was taken out from each well and put into an EP tube, and 70ul (0.5M PB solution +0.1M NaOH solution) was added to each well to neutralize the pH to about 7.24. Centrifugation at 1500rpm 10 min. This is the sample.

(4) ELISA operation: the cAMP content in each stimulated sample was determined by competitive inhibition, following strictly the kit instructions from cayman.

The results show that: PTH (1-34) purified by this protocol has a biological activity of stimulating the production of cAMP by Saos-2 cells and its activity is not lower than that of the standard product of Sigma.

Although the present application has been described in detail with respect to the general description and the specific examples, it will be apparent to those skilled in the art that certain changes and modifications may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.

Sequence listing

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Claims (10)

1. A method for separating and purifying recombinant parathyroid hormone PTH, which comprises the following steps:

1) bacterial disruption and inclusion body treatment

Performing high-pressure homogenate to break bacteria, performing high-speed centrifugation after the bacteria breaking, collecting precipitates, and mixing and redissolving the precipitates with a redissolution after the precipitates are subjected to heavy suspension and washing;

2) affinity chromatography; the filler is selected from one of Ni-NTA, GST Sepharose and MBP Sepharose;

3) performing enzyme digestion and renaturation on a column:

wherein the enzyme is selected from TEV enzyme, EK enzyme, and Xa factor protease;

4) cation exchange chromatography and/or hydrophobic chromatography:

the filler of the cation exchange chromatographic column is selected from one of SP Sepharose HP, SP Sepharose FF, Capto SP and Capto MMC;

the filler of the hydrophobic chromatographic column is one of HP or FF fillers of Phynel, Octyl or Butyl;

5) and (4) desalting.

2. The method of claim 1, wherein in step 1), step 1) comprises:

the weight of the thallus and the solution A is as follows: the volume ratio is 1: mixing at a ratio of 5-10, and pre-cooling to 1-4 deg.C.

3. The method of claim 1 or 2, wherein step 1) comprises:

homogenizing at 1-4 deg.C under 60-80Mpa for 3-5 times; centrifuging the liquid after the bacteria breaking for 15-30min at the temperature of 4 ℃ and under the conditions of 10,000 and 15,000g, and collecting the precipitate for later use.

4. The method of any one of claims 1-3, wherein step 1) comprises:

resuspend inclusion bodies in resuspension, weight of inclusion bodies to resuspension: the volume ratio is 1: 5-10, stirring for 15-30min by a magnetic stirrer, centrifuging for 15-30min by 15,000g of 10,000-; the resuspension is solution a containing Triton X1000, tween20, tween80, or sacosyl at a final concentration of 0.5% to 1.5%.

5. The method of any one of claims 1-4, wherein step 1) comprises:

the weight ratio of: the volume ratio is 1: adding a re-suspension solution in a proportion of 5-10 to re-suspend the inclusion body, uniformly mixing, centrifuging and collecting a supernatant for later use; the compound solution takes solution A as a solvent and contains mixed solution of 8M urea, 6M guanidine hydrochloride or 2mol/L urea and sacosyl to the final concentration of 0.3%.

6. The method of any one of claims 1-5, wherein step 2) comprises:

the ratio of the wet weight of the bacteria breaking thallus to the filler is 10: 1-2, mixing the re-dissolved inclusion body with a filler uniformly, and further removing foreign protein on the filler by using a solution A containing 20-25mM imidazole and 8M urea;

preferably, the column packing is washed with several volumes of water before mixing with the reconstituted inclusion bodies and then equilibrated with liquid a containing 8M urea.

7. The method of any one of claims 1-6, wherein step 3) comprises:

adding a proper amount of protease with His tag into the mixture, and performing on-column enzyme digestion, wherein the enzyme digestion solution is solution A containing 2-4M urea or guanidine hydrochloride, the amount of the enzyme digestion solution is 1-2 times of that of the filler, and the enzyme digestion condition is 4-25 ℃ for 2-5 h; wherein the weight volume ratio of the inclusion body to the enzyme is 10: 1.

8. the method according to any one of claims 1-6, wherein the step 4) comprises:

collecting samples after affinity chromatography or hydrophobic chromatography, diluting with solution B for 3-6 times, and adjusting pH to 6.5; then, carrying out linear gradient elution by using solution C (), setting the elution flow rate to be 5ml/min, and setting the elution gradient to be that the solution B is from 0 to 50 percent, and the elution volume is 10 column volumes; wherein the solution B is 20mM phosphate buffer solution (PB) with the pH value of 6.5; the solution C was 20mM Phosphate Buffer (PB) containing 1M NaCl, pH 6.5.

9. The method according to any one of claims 1-8, wherein the step 4) comprises:

mixing a sample obtained by affinity chromatography or cation exchange chromatography with 3M ammonium sulfate solution until the final concentration of ammonium sulfate is 1-1.5M;

balancing a chromatography system and a chromatography column by using a solution D, then loading, after loading, carrying out linear gradient elution by using a solution B, wherein the elution flow rate is 5ml/min, the elution gradient is that the solution B is from 0 to 100 percent, the elution volume is 10 column volumes, collecting proteins and storing at 4 ℃; the solution D is 20mM phosphate buffer solution containing 1-1.5M ammonium sulfate and pH 6.5.

10. The method according to any one of claims 1-9, wherein said step 5) comprises:

the desalting is carried out by one selected from ultrafiltration, diafiltration, molecular sieve or desalting column.

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