CN111004318B

CN111004318B - Purification method of rhPTH (1-34) protein stock solution

Info

Publication number: CN111004318B
Application number: CN201911399951.XA
Authority: CN
Inventors: 樊欣迎; 李静; 刘月峰; 张萌萌; 郭静雅; 闻亚磊
Original assignee: Beijing Bokangjian Gene Technology Co ltd
Current assignee: Beijing Bokangjian Gene Technology Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-03-04
Anticipated expiration: 2039-12-30
Also published as: CN111004318A

Abstract

The invention discloses a purification method of rhPTH (1-34) protein stock solution. The method comprises the steps of thallus crushing, nickel ion chelating affinity chromatography, fusion protein enzyme digestion, anion exchange chromatography, reversed phase chromatography and cation exchange chromatography, wherein the thallus crushing specifically comprises the following steps: and (2) taking the thalli, resuspending the thalli by using a buffer solution A, breaking the thalli by using a high-pressure homogenizer, centrifuging the broken thalli, collecting supernate, filtering the supernate by using a 0.45-micrometer filter element, and collecting filtrate, wherein the buffer solution A comprises phosphate buffer solution, NaCl, imidazole and water, the concentration of the phosphate buffer solution is 10mmol/L, NaCl and is 500mmol/L, the concentration of the imidazole is 50-60 mmol/L, and the pH value of the buffer solution A is 8.0. By applying the technical scheme of the invention, the total yield of the rhPTH (1-34) protein stock solution is improved to more than 50 percent, and the purity is improved to more than 99.5 percent.

Description

Purification method of rhPTH (1-34) protein stock solution

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a purification method of rhPTH (1-34) protein stock solution.

Background

Parathyroid hormone (PTH) is a basic single-chain polypeptide hormone secreted by parathyroid chief cells. Its main function is to regulate the metabolism of calcium and phosphorus in vertebrates, and promote the increase of calcium level and decrease of phosphorus level in blood. PTH contributes to elevated plasma calcium concentrations and the major target organs of action are bone and kidney. It mobilizes bone calcium to enter blood, promotes reabsorption of calcium ions and excretion of phosphate by renal tubules, and increases blood calcium concentration and decreases blood phosphorus concentration. In addition, PTH indirectly promotes the absorption of calcium ions by the intestinal tract. Secretion of PTH is primarily regulated by plasma calcium ion concentration. The secretion of PTH is inhibited when the plasma calcium ion concentration is increased; plasma calcium ion concentration decreases, which stimulates secretion of PTH.

Genetic engineering studies of PTH began in the 80's of the 20 th century. The current genetic engineering methods for preparing PTH (1-34) are mainly classified into two types, one is prepared in the form of inclusion bodies and the other is prepared in the form of soluble proteins. CN100484958C fusion protein containing human parathyroid hormone 1-34 and its expression vector disclose a fusion protein, which has thioredoxin sequence and parathyroid hormone 1-34 peptide located at the downstream of thioredoxin. Preferably, the fusion protein further comprises a linker peptide comprising a recognition site for a proteolytic enzyme between the thioredoxin sequence and the peptide of parathyroid hormone 1-34. From the fusion protein, human parathyroid hormone 1-34 peptide can be prepared. However, in this embodiment, the total yield of the rhPTH (1-34) protein stock solution is only 35%, and therefore, further improvement is required.

Disclosure of Invention

The invention aims to provide a purification method of rhPTH (1-34) protein stock solution so as to provide the total yield of the rhPTH (1-34) protein stock solution.

In order to achieve the above objects, according to one aspect of the present invention, there is provided a method for purifying a rhPTH (1-34) protein stock solution. The method comprises the steps of thallus crushing, nickel ion chelating affinity chromatography, fusion protein enzyme digestion, anion exchange chromatography, reversed phase chromatography and cation exchange chromatography, wherein the thallus crushing specifically comprises the following steps: taking the bacteria expressing rhPTH (1-34) protein, resuspending the bacteria by using a buffer solution A, breaking the bacteria by using a high-pressure homogenizer, centrifuging a broken bacteria solution, collecting a supernatant, filtering the supernatant by using a 0.45 mu m filter element, and collecting a filtrate, wherein the buffer solution A comprises phosphate buffer solution, NaCl, imidazole and water, the concentration of the phosphate buffer solution is 10mmol/L, NaCl and is 500mmol/L, the concentration of the imidazole is 50-60 mmol/L, and the pH value of the buffer solution A is 8.0.

Further, the nickel ion chelate affinity chromatography specifically includes: loading the nickel ion chelating affinity chromatographic column balanced by the buffer solution A with the loading amount of 1.40-1.80 g of wet bacteria/ml of filler to the nickel ion chelating affinity chromatographic column, leaching by using the buffer solution A, and eluting by using the buffer solution B to obtain a fusion protein sample; ultrafiltering, and replacing the solution with buffer C.

The buffer solution B is composed of a phosphate buffer solution, imidazole and water, wherein the concentration of the phosphate buffer solution is 10mmol/L, the concentration of the imidazole is 110-210 mmol/L, and the pH value of the buffer solution B is 8.0.

Further, buffer C was 10mmol/L Tris-HCl, pH 8.0.

Further, the enzyme digestion of the fusion protein specifically comprises: using 50mmol/L Tris-HCl and 1mmol/L CaCl₂The ultrafiltered fusion protein was diluted to 1mg/ml in a buffer pH8.0 and the ratio of 1U enterokinase: and (3) carrying out enzyme digestion for 20 hours in a constant-temperature shaking table at the temperature of 25 ℃ according to the enzymolysis ratio of 6-8 mg of fusion protein.

Further, anion exchange chromatography specifically includes: loading the enzyme-digested zymoprotein on an anion exchange chromatography column balanced by a buffer solution D, and collecting the penetration solution, wherein the buffer solution D is Tris-HCl and CaCl₂And water, wherein the concentration of Tris-HCl is 50mmol/L, CaCl₂Was 1mmol/L, and the pH of buffer D was 8.0.

Further, reverse phase chromatography specifically includes: selecting a reverse phase chromatography column, namely Source 15RPC, loading 7-10 mg of protein/ml filler, using a buffer solution F as a mobile phase A, using a buffer solution G as a mobile phase B, balancing the reverse phase chromatography column by using the mobile phase A, leaching the mobile phase A after loading, washing impurities by using 40% of the mobile phase B, continuously and gradiently eluting target protein by using 40-60% of the mobile phase B to obtain the target protein, diluting the target protein by using a buffer solution H, then carrying out cation exchange chromatography, and eluting to obtain the purified target protein.

Further, buffer F was composed of phosphate buffer, ethanol, and water, wherein the concentration of phosphate buffer was 10mmol/L, the concentration of ethanol was 10%, and the pH of buffer F was 8.0.

Further, buffer G was 80% ethanol and mobile phase B was in a gradient volume of 20 CV; buffer H was 10mmol/L phosphate buffer, pH 6.0.

Further, cation exchange chromatography specifically includes: and (2) loading the target protein diluent obtained by reversed phase chromatography to a cation exchange chromatography column balanced by a buffer solution H, washing with the buffer solution H after loading, washing impurities by using a buffer solution with the pH value of 7.0 consisting of 10mmol/L phosphate buffer solution and 50mmol/L NaCl, eluting by using a buffer solution with the pH value of 7.0 consisting of 10mmol/L phosphate buffer solution and 10-210 mmol/L NaCl, and collecting the target protein, namely the rhPTH (1-34) protein stock solution.

By applying the technical scheme of the invention, on the basis of the technical scheme disclosed by CN100484958C (the fusion protein containing human parathyroid hormone 1-34 and the expression vector thereof), the parameters and the like of the chromatography step of the purification process are optimized, the yield of the protein is improved one step by one step, finally, the yield of the rhPTH (1-34) protein stock solution is improved to more than 50 percent from about 35 percent, and the purity of the rhPTH (PTH1-34) protein stock solution is improved to more than 99.5 percent from more than 99 percent.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows an artificially synthesized DNA sequence containing the coding sequence of rhPTH (1-34); and

fig. 2 shows a comparison of purity profiles of rhPTH1-34 stock solution before optimization (P20140201, reference data) and after optimization (P20190601, example 1).

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

According to an exemplary embodiment of the present invention, a method for purifying a rhPTH (1-34) protein stock is provided. The purification method comprises the steps of thallus crushing, nickel ion chelating affinity chromatography, fusion protein enzyme digestion, anion exchange chromatography, reversed phase chromatography and cation exchange chromatography, wherein the thallus crushing specifically comprises the following steps: taking the bacteria expressing rhPTH (1-34) protein, resuspending the bacteria by using a buffer solution A, breaking the bacteria by using a high-pressure homogenizer, centrifuging a broken bacteria solution, collecting a supernatant, filtering the supernatant by using a 0.45 mu m filter element, and collecting a filtrate, wherein the buffer solution A comprises phosphate buffer solution, NaCl, imidazole and water, the concentration of the phosphate buffer solution is 10mmol/L, NaCl and is 500mmol/L, the concentration of the imidazole is 50-60 mmol/L, and the pH value of the buffer solution A is 8.0.

The inventor of the invention finds that the concentration of imidazole in the equilibrium buffer solution (buffer solution A) is increased from the original 30mmol/L to 50-60 mmol/L, and the combination of the hybrid protein and the affinity column can be reduced.

In addition, in the technical scheme of the application, the inventor finds that different amounts of the above substances have influence on the elution yield and purity of the fusion protein. The saturated sample loading of the fusion protein is 54mg protein/ml filler (the fusion protein directly passes through a detection pool to obtain the highest absorption value, the highest absorption value is connected with a chromatographic column for sample loading, and the sample loading when the penetration absorption value reaches 5% of the highest absorption value is the saturated sample loading), namely 2.25g wet bacteria/ml filler (through experimental data calculation, about 24mg of fusion protein can be obtained per gram of wet bacteria, and 54mg of fusion protein is equivalent to the fusion protein content of 2.25g of wet bacteria). The inventor respectively uses 0.90g, 1.40g, 1.80g, 2.0g wet bacteria/ml filler (saturation loading 40%, 60%, 80%, 90%) samples to carry out nickel ion chelation affinity chromatography, and compares the effect of different loading on the elution yield and purity of the fusion protein. The experimental results show (Table 1), with 1.40 and 1.80g wet bacteria/ml packing obtained fusion protein yield and purity is better, therefore, preferably, with 1.40 ~ 1.80g wet bacteria/ml packing as the nickel ion chelating affinity chromatography loading range.

Table 1: influence of sample loading of nickel ion chelation affinity chromatography on yield and purity of fusion protein

Furthermore, the inventors found that eluents containing different imidazole concentrations also have a large influence on the yield and purity of the fusion protein, and directly eluted the fusion protein with buffers containing 50, 100, 150 and 200mmol/L imidazole, respectively, and the detection results showed that the yield and purity of the fusion protein eluted with 100-150 mmol/L imidazole were better than those eluted with 200mmol/L imidazole (Table 2), so buffer B (10mmol/L PB, 100-150 mmol/L imidazole, pH8.0) was preferably used as the eluent for the fusion protein in the present invention.

Table 2: influence of eluents containing different imidazole concentrations on yield and purity of fusion protein

According to an exemplary embodiment of the present invention, the inventors have also found enzyme digestion optimization conditions that can reduce the amount of enterokinase used. The results of digestion with 1U enterokinase at 25 ℃ for 20 hours for 6mg, 8mg, and 10mg of the pure fusion protein were compared (Table 3). The results showed that the enzyme digestion rate was 98.3% at the rate of 8mg of pure fusion protein added with 1U of enterokinase. The amount of the 1U enzyme digestion fusion protein is increased from 5mg to 6-8 mg, and the enzymolysis rate is basically not affected. In order to prevent excessive cutting, the enzyme cutting rate is generally not more than 99% under the condition of ensuring better enzyme cutting effect.

Table 3: optimization of enzyme digestion conditions

Further, the inventors have also studied reverse phase chromatography (Source 15RPC) and found that the amount of the sample has an influence on the yield of this purification. When the following experiments are carried out according to the conditions that the loading amount of the filler is 4mg, 7 mg, 10mg, 13 mg and 16mg of protein/ml, the effects of the loading amount on the yield of the target protein and the purification efficiency are compared, and the results show that (shown in table 4), the loading amount of the filler of 7 mg to 10mg of protein/ml is used for loading, the yield of the eluted target protein is improved to about 88 percent, and the purity is over 99 percent.

Table 4: comparing the influence of different sample loading on the yield of the target protein in reverse phase chromatography

Furthermore, in an exemplary embodiment of the present invention, the inventors also found the effect of the elution desalination concentration on the final protein yield, and the experimental data is as follows, using buffer I (10mmol/L PB, pH7.0) as mobile phase A and buffer J (10mmol/L PB, 500mmol/L NaCl, pH7.0) as mobile phase B, performing a continuous gradient elution of 0-40% B of 30CV, and the results show that impurities are eluted with 50mmol/L NaCl first, and the yields of the target protein eluted with 50-110, 110-210, 210-310, 310-400 mmol/L NaCl are compared, and the results show (Table 5) that the target protein mainly exists in the elution peak with a concentration of 110-210 mmol/L NaCl, and the yield of the target protein is 94.93%. Therefore, the target protein is eluted by using a buffer solution of 10mmol/L PB, 150mmol/L NaCl and pH7.0 to obtain the rhPTH (1-34) protein with the purity of more than 99.5 percent.

Table 5: elution results of cation exchange chromatography with different salt concentrations

According to an exemplary embodiment of the present invention, a method for purifying a rhPTH (1-34) protein stock is provided. The purification method comprises the following steps:

1. crushing of thallus

Taking the thalli expressing rhPTH (1-34) protein, resuspending the thalli by using a buffer solution A (10mmol/L PB (phosphate buffer), 500mmol/L NaCl, 50-60 mmol/L imidazole and pH8.0), breaking bacteria by using a high-pressure homogenizer, centrifuging broken bacteria, collecting supernate, filtering by using a 0.45 mu m filter element, and collecting filtrate.

2. Nickel ion chelate affinity chromatography

The sample loading range of the nickel ion chelating affinity chromatography is 1.40-1.80 g of wet bacteria/ml filler, the nickel ion chelating affinity chromatography column is fully leached by buffer solution A (10mmol/L PB, 500mmol/L NaCl, 50-60 mmol/L imidazole, pH8.0) after being balanced by the buffer solution A, and the buffer solution B (10mmol/L PB, 110-210 mmol/L imidazole, pH8.0) is used for elution, so that a fusion protein sample is obtained. The resulting mixture was ultrafiltered, and the replacement solution was buffer C (10mmol/L Tris-HCl, pH 8.0).

3. Cleavage of fusion proteins

Using 50mmol/L Tris-HCl and 1mmol/L CaCl₂And (3) diluting the ultrafiltered fusion protein to 1mg/ml by using a buffer solution with the pH of 8.0, and adding 1U of enterokinase: and (3) carrying out enzyme digestion for 20 hours in a constant-temperature shaking table at the temperature of 25 ℃ according to the enzymolysis ratio of 6-8 mg of fusion protein.

4. Anion exchange chromatography

Loading the enzyme-cleaved zymoprotein into buffer solution D (50mmol/L Tris-HCl, 1mmol/L CaCl)₂Ph8.0) and an anion exchange chromatography column (Q Sepharose High Performance) to collect the permeate.

5. Reverse phase chromatography

Selecting a reverse phase chromatography column Source 15RPC, wherein the loading amount is 7-10 mg protein/ml filler. Buffer F (10mmol/L PB, 10% ethanol, pH8.0) was used as mobile phase A, and buffer G (80% ethanol) was used as mobile phase B. And (3) fully balancing the reversed-phase chromatographic column by using the mobile phase A, fully leaching by using the mobile phase A after sampling, washing impurities by using 40% B, and eluting the target protein by using 40% -60% B continuous gradient (gradient volume of 20 CV). The obtained target protein is diluted by buffer solution H (10mmol/L PB, pH6.0), and then cation exchange chromatography is carried out, and the purified target protein is obtained after elution.

6. Cation exchange chromatography

And (3) loading a target protein diluent obtained by reversed phase chromatography to a cation exchange chromatography column balanced by a buffer solution H, fully eluting with the buffer solution H after loading, washing impurities with 10mmol/L PB, 50mmol/L NaCl and pH7.0 buffer solution, eluting with 10mmol/L PB, 110-210 mmol/L NaCl and pH7.0 buffer solution, and collecting the target protein, namely the rhPTH (1-34) protein stock solution.

The following examples are provided to further illustrate the advantageous effects of the present invention.

Control and reference data

CN100484958C (fusion protein containing human parathyroid hormone 1-34 and its expression vector) discloses the following relevant process steps (examples 1-5):

design and artificial synthesis of DNA sequence for expressing hPTH (1-34)

Based on the hPTH (1-34) amino acid sequence (see Table 1), optimized DNA sequences suitable for E.coli expression were synthesized according to E.coli codon preference. When synthesizing hPTH (1-34), Kpn I cleavage site GGTACC was introduced at the 5 'end of the gene, termination codes TAA and Sal I cleavage site GT' CGAC were introduced at the 3 'end, and coding sequence GACGACGACGACAAG of enterokinase cleavage recognition site was added after the Kpn I cleavage site introduced at the 5' end to obtain sequence l (see FIG. 1). To facilitate subsequent subcloning, the synthetic gene was cloned into pUC18 for the purpose of preserving the synthetic DNA sequence. Plasmid pUC18 contained the same Kpn I and Sal I cleavage sites as plasmid pET32a (+).

Second, construction process of recombinant plasmid

The following procedures of molecular cloning techniques, unless otherwise specified, are referred to in the literature: molecular cloning experiments refer to (Huang Petang et al, eds. [ Sam Brooks et al, science Press, 2002). DNA extraction kit (UNIQ-10), DNA gel recovery kit (UNIQ-10), ligation kit and the like used for DNA manipulation were purchased from Shanghai Biotechnology engineering services, Inc. Cloning vector pUC18, restriction enzyme was purchased from Fermentas Life Science. The expression vectors pET32a (+), E.coli TOP 10 and BL21(DE3) were purchased from Novagen. The E.coli host for cloning was TOP 10 and the E.coli host for expression was BL21(DE 3). The BL21(DE3) genotype is: hsdS gal (λ cIts857 ind1 Sam7 nin5 lacUV5-T7 gene I). BL21(DE3) carries T7RNA polymerase gene, and T7RNA polymerase is produced in large quantity under the induction of IPTG, thus opening the expression of exogenous gene and enabling the exogenous gene to be expressed efficiently.

1. Preparing a target gene fragment

Extracting pUC18 plasmid DNA containing hPTH (1-34) coding sequence with DNA extraction kit, double cutting with Kpn I/Sal I, separating small fragment in 1% agarose gel electrophoresis, cutting gel containing about 130bp fragment, recovering about 130bp fragment with gel DNA recovering kit, and electrophoretic checking.

2. Preparation of expression vector fragments

Extracting pET32a (+) plasmid DNA by using a DNA extraction kit, carrying out double digestion by using Kpn I/Sal I, carrying out electrophoresis separation on a large fragment by using 1% agarose gel, cutting off gel containing the large fragment, recycling the large fragment by using a gel DNA recovery kit, and carrying out electrophoresis verification for later use.

3. Construction of recombinant plasmid pET-PTH (1-34)

The prepared DNA fragments of I and 2 were mixed uniformly at different volume ratios (2/8, 3/7, 4/6 and 5/5), ligated with T4 DNA ligase at 16 ℃ for 30min, the ligation product was transformed into E.coli TOP 10 competent cells, spread on LB agarose plates (1% peptone, 0.5% yeast extract, 1% NaCI, 2% agar) containing 100. mu.g/ml Amp, and cultured overnight at 37 ℃.

4. Recombinant screening

10 colonies were picked, cultured overnight at 37 ℃ in 5ml of LB medium containing 100. mu.g/ml Amp, and plasmid DNA was extracted using a DNA extraction kit. The extracted DNAs were digested with EcoR and Pst I, respectively, and then subjected to 1% agarose gel electrophoresis to identify recombinants. Since EcoR I is a single enzyme cutting site in pET32a (+), and is removed by Kpn I/Sal I double enzyme cutting when constructing the recombinant, the recombinant cannot be cut by EcoR; while the Pst I single site in pET32a (+) is not in the multiple cloning region and rhPTH (1-34) gene contains one Pst I site, digestion of the pET32a (+) vector plasmid with Pst I results in a single DNA fragment of 5.9kb molecular weight, while digestion of the recombinant plasmid with Pst I should yield two DNA fragments of about one of l.2kb and 4.7 kb. The correct recombinant plasmid was designated pET-PTH (1-34).

5. Sequencing verification of plasmid pET-PTH (1-34)

Third, induced expression of engineering bacteria

The recombinant plasmid pET-PTH (1-34) DNA is used for transforming escherichia coli BL21(DE3), and the obtained product is the fusion protein for expressing rhPTH (1-34)The genetically engineered bacterium of (1). 8 single colonies were picked, cultured overnight at 37 ℃ in 5ml LB medium (1% peptone, 0.5% yeast extract, 1% NaCI) containing 100. mu.g/ml Amp, transferred to 50ml LB medium containing 100. mu.g/ml Amp at 37 ℃ in a volume of 1/100, and the remaining bacterial solution was frozen with 15% glycerol. OD₆₀₀When the concentration reached 0.5, IPTG was added to a final concentration of 0.5mM for induction of expression, and a sample was taken after 4 hours for SDS-PAGE electrophoresis. Compared with the uninduced control, the recombinants after induction all have an expression band with the expected molecular weight of about 20 kDa.

Fourthly, fermentation of engineering bacteria BL21(DE3) -PTH (I-34)

Taking 1 glycerol strain (1mL) of the engineering bacteria BL21(DE3) -PTH (1-34) for expressing Trx-hPTH, inoculating the glycerol strain into 400mL LB culture medium (1% peptone, 0.5% yeast extract, 1% NaCI), and carrying out shake-flask culture at 37 ℃ and 200rpm for 14-16h to obtain activated seeds. Inoculating 10% of activated seed into 3.5L TB medium (1.2% protein, 2.4% yeast extract, 0.4% glycerol, 17mM KH)₂PO₄，72mM K₂HPO₄) (5L B.Braun fermenter), incubated at 37 ℃ for 3-4 h. Inoculating the culture solution into 70L TB medium (100L B. Braun fermenter) at 5% inoculum concentration, fermenting at 37 deg.C, pH7.0, and DO not less than 30%, and culturing to OD₆₀₀When the expression is started to be induced, the final concentration of IPTG for induction is 0.5mM, and the induction time is 3-4 h. Under the condition, the concentration of the fermentation liquor can reach more than 30g of bacterial wet weight// L fermentation liquor, and the expression quantity of the target fusion protein is more than 25%.

Fifthly, purification of rhPTH (1-34)

The fermentation biomass in step four was collected by a continuous flow centrifuge (CEPA Z41, B.Braun Co., Germany), suspended in buffer A (10mM PB (phosphate buffer), 500mM NaCl, 30mM imidazole, pH8.0), disrupted by an APV-1000 high pressure homogenizer (APV Co. Denmark), the endocrinic fusion protein was dissolved in buffer A, and centrifuged at 9000rpm for 30 min. And taking supernatant, and purifying rhPTH (1-34) by the following methods in sequence:

1. nickel ion chelate affinity chromatography (Chelating Sepharose Fast Flow)

And (3) separating the supernatant obtained by high-pressure homogenate and bacterium breaking through a nickel ion chelating affinity chromatography column (10mmol/L PB, 500mmol/L NaCl, 30mmol/L imidazole and pH8.0 of an equilibrium buffer solution), fully leaching the supernatant with the equilibrium buffer solution after the sample loading is finished, and eluting the supernatant with the buffer solution (10mmol/L PB, 200mmol/L imidazole and pH8.0) to obtain the fusion protein sample.

2. Conditions of enzyme digestion

Using buffer (50mmol/L Tris-HCl, 1mmol/L CaCl)₂) Diluting the ultrafiltered fusion protein to 1mg/ml, adding 1U enterokinase into 5mg fusion protein, and performing shake enzymolysis at 25 deg.C for 20 hr.

3. Anion exchange column chromatography

The cleaved protein solution was applied to a Q Sepharose High Performance column equilibrated with buffer C (50mM Tris-HC1, pH 8.0). rhPTH (1-34) has a theoretical isoelectric point of 8.29, is positively charged at pH8.0 and does not bind to the anion exchange column, while the hetero-protein binds to the anion exchange column due to its negative charge. The transudate liquid is collected, and rhPTH (1-34) protein samples with the purity of more than 95 percent can be obtained.

4. Reverse phase chromatography (Source 15RPC)

Mobile phase a (deionized water), mobile phase B (80% ethanol). The buffer solution (10mmol/L PB, pH8.0) balances the chromatographic column, the chromatographic column is leached by the mobile phase A after the sample is loaded, the mobile phase B with 24 percent to 64 percent is used for continuous gradient elution (the gradient volume is 20CV), the target protein elution peak appears when the content of the mobile phase B is 40 percent to 60 percent, and the purity reaches more than 98 percent.

5. Cation exchange chromatography (SP Sepharose Fast Flow)

And (3) loading the eluent of the previous step on a cation chromatographic column, fully eluting with an equilibrium buffer solution (10mmol/L PB, pH6.0), and eluting the target protein with the buffer solution (10mmol/L PB, 400mmol/L NaCl, pH7.0) to obtain the rhPTH (1-34) protein with the purity of more than 99%.

Example 1

The preparation of rhPTH (1-34) in the present example was identical to the steps one to four in the above control and reference data, except for the purification step, which was optimized as follows:

the purification method comprises the following steps:

1. crushing of thallus

2. Nickel ion chelate affinity chromatography

3. Cleavage of fusion proteins

4. Anion exchange chromatography

5. Reverse phase chromatography

6. Cation exchange chromatography

According to the optimized purification process, a plurality of batches of target proteins are purified, the intermediate products obtained in each process step have stable performance, the purity of the stock solution is more than 99.5 percent, the total yield is improved to more than 50 percent from about 35 percent (figure 2, tables 6, 7, 8 and 9), the biological activity of the stock solution is not obviously different, the content of the stock solution is improved, the detection indexes for the stock solution are not obviously different, and the quality of the stock solution is not influenced.

Table 6: optimization parameters and results of purification process

Table 7: statistics of purification results before optimization

Table 8: post-optimization purification results statistics

Note: affinity chromatography yield ═ affinity chromatography elution peak protein (mg)/wet weight of cells (g);

the total yield is the stock solution protein content/(affinity chromatography elution peak protein content × 0.20), and the coefficient 0.20 is the ratio of rhPTH1-34 to the fusion protein molecular weight.

TABLE 9 comparative analysis of quality of stock solutions of rhPTH1-34 before optimization (method in reference data) and after optimization (example 1)

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A purification method of rhPTH (1-34) protein stock solution comprises the steps of thallus crushing, nickel ion chelation affinity chromatography, fusion protein enzyme digestion, anion exchange chromatography, reversed phase chromatography and cation exchange chromatography, and is characterized in that the thallus crushing specifically comprises the following steps: taking thalli expressing rhPTH (1-34) protein, resuspending the thalli with a buffer solution A, breaking the thalli with a high-pressure homogenizer, centrifuging a broken bacteria solution, collecting a supernatant, filtering the supernatant with a 0.45-micrometer filter element, and collecting a filtrate, wherein the buffer solution A consists of a phosphate buffer solution, NaCl, imidazole and water, the concentration of the phosphate buffer solution is 10mmol/L, NaCl and is 500mmol/L, the concentration of the imidazole is 50-60 mmol/L, and the pH value of the buffer solution A is 8.0;

the nickel ion chelating affinity chromatography specifically comprises: loading the nickel ion chelating affinity chromatographic column balanced by the buffer solution A with the loading amount of 1.40-1.80 g of wet bacteria/ml of filler to the nickel ion chelating affinity chromatographic column, leaching by using the buffer solution A, and eluting by using a buffer solution B to obtain a fusion protein sample; performing ultrafiltration, wherein the replacement solution is buffer solution C;

the buffer solution B consists of a phosphate buffer solution, imidazole and water, wherein the concentration of the phosphate buffer solution is 10mmol/L, the concentration of the imidazole is 100-150 mmol/L, and the pH value of the buffer solution B is 8.0;

the enzyme digestion of the fusion protein specifically comprises the following steps: using 50mmol/L Tris-HCl and 1mmol/L CaCl₂The ultrafiltered fusion protein was diluted to 1mg/ml in a buffer pH8.0 and the ratio of 1U enterokinase: performing enzyme digestion on the fusion protein of 6-8 mg in a constant-temperature shaking table at 25 ℃ for 20 hours according to the enzymolysis ratio;

the reverse phase chromatography specifically comprises: selecting a reverse phase chromatography column (Source 15RPC), loading 7-10 mg of protein/ml filler, using a buffer solution F as a mobile phase A and a buffer solution G as a mobile phase B, balancing the reverse phase chromatography column by using the mobile phase A, leaching by using the mobile phase A after loading, washing impurities by using 40% of the mobile phase B, eluting the target protein by using 40-60% of the mobile phase B in a continuous gradient manner to obtain the target protein, diluting the target protein by using a buffer solution H, performing cation exchange chromatography, and eluting to obtain the purified target protein;

the cation exchange chromatography specifically comprises: loading a target protein diluent obtained by the reverse phase chromatography to a cation exchange chromatography column balanced by the buffer solution H, washing with the buffer solution H after loading, washing impurities with a buffer solution with the pH value of 7.0 consisting of 10mmol/L phosphate buffer solution and 50mmol/L NaCl, eluting with a buffer solution with the pH value of 7.0 consisting of 10mmol/L phosphate buffer solution and 10-210 mmol/L NaCl, and collecting a target protein, namely the rhPTH (1-34) protein stock solution;

the buffer C is 10mmol/L Tris-HCl, and the pH value is 8.0;

the anion exchange chromatography specifically comprises: loading the enzyme-digested zymolytic protein on an anion exchange chromatography column balanced by a buffer solution D, and collecting the penetration solution, wherein the buffer solution D is Tris-HCl and CaCl₂And water, wherein the concentration of Tris-HCl is 50mmol/L, CaCl₂At a concentration of 1mmol/L, pH of buffer D is 8.0;

the buffer solution F consists of a phosphate buffer solution, ethanol and water, wherein the concentration of the phosphate buffer solution is 10mmol/L, the concentration of the ethanol is 10%, and the pH value of the buffer solution F is 8.0;

the buffer solution G is 80% ethanol, and the gradient volume of the mobile phase B is 20 CV; the buffer H is 10mmol/L phosphate buffer, and the pH value of the buffer H is 6.0.