阅读说明：本技术 一种可溶性重组特立帕肽的非变性纯化方法及应用 (Non-denaturing purification method and application of soluble recombinant teriparatide ) 是由 余传信 杨建良 刘晓龙 丁铁林 梅丛进 董盼盼 沈孝森 何军山 于 2020-12-23 设计创作，主要内容包括：本发明公开了一种可溶性重组特立帕肽的非变性纯化方法及应用,包括以下步骤：1)菌体裂解；2)镍柱亲和纯化；3)酶切；4)镍柱穿透；5)C18-100A柱纯化；6)C18-300A反向色谱纯化；7)旋蒸与冻干。本发明提供的可溶性重组特立帕肽的非变性纯化方法及应用,采用镍亲和层析法从表达产物中分离Trx-hPTH(1-34)融合蛋白,再用酶切将重组特立帕肽从Trx-hPTH(1-34)融合蛋白中释放出来,通过三步层析方法制备纯化的重组特立帕肽,简化了纯化过程,提高了可溶性重组特立帕肽的纯化效率与纯度,对制备治疗骨质疏松症和骨折患者药物的原料药重组特立帕肽有重要帮助。(The invention discloses a non-denaturing purification method and application of soluble recombinant teriparatide, which comprises the following steps: 1) cracking thalli; 2) affinity purification by a nickel column; 3) enzyme digestion; 4) penetrating the nickel column; 5) purifying with C18-100A column; 6) C18-300A reverse phase chromatography purification; 7) rotary steaming and freeze-drying. The invention provides a non-denaturing purification method and application of soluble recombinant teriparatide, wherein a nickel affinity chromatography method is adopted to separate Trx-hPTH (1-34) fusion protein from an expression product, and then enzyme digestion is used to release the recombinant teriparatide from the Trx-hPTH (1-34) fusion protein.)

1. A method for the non-denaturing purification of soluble recombinant teriparatide, comprising the steps of:

1) fermenting, cracking and centrifuging thalli: cracking fermented bacteria expressing Trx-hPTH (1-34) fusion protein, centrifuging, and collecting supernatant of the cracked product;

2) and (3) nickel column affinity purification: loading the sample obtained in the step 1) on a nickel affinity chromatographic column, eluting Trx-hPTH (1-34) fusion protein combined on the nickel affinity chromatographic column, and obtaining affinity purified Trx-hPTH (1-34) fusion protein;

3) enzyme digestion: cleaving the Trx-hPTH (1-34) fusion protein obtained in the step 2) with enterokinase to release the recombinant teriparatide from the fusion protein; after the Trx-hPTH (1-34) fusion protein is cut, two peptide fragments are generated: trx partial peptide segment and recombinant teriparatide hPTH (1-34);

4) penetration of the nickel column: applying the enzyme digestion product obtained in the step 3) to a new nickel affinity chromatographic column, combining a Trx partial peptide segment with a 6 × His label on the nickel affinity chromatographic column, allowing the recombinant teriparatide to pass through the nickel affinity chromatographic column, and collecting a penetration peak to obtain the primarily purified recombinant teriparatide;

5) C18-100A column purification: loading the recombinant teriparatide sample obtained in the step 4) on a C18-100A column, and performing gradient elution by using acetonitrile to obtain the recombinant teriparatide hPTH (1-34) with the purity of more than 90%;

6) C18-300A reverse phase chromatography purification: putting the recombinant teriparatide protein sample purified by the C18-100A column in the step 5) on a C18-300A reverse chromatographic column, and performing gradient elution by using acetonitrile to obtain the recombinant teriparatide with the purity of more than 99 percent;

7) rotary evaporation and freeze drying: heating the sample obtained in the step 7), carrying out negative pressure rotary evaporation, removing residual acetonitrile, and then carrying out vacuum freeze drying.

2. The method for non-denaturing purification of soluble recombinant teriparatide according to claim 1, wherein the step of lysing the bacterial cells in step 1) comprises: mixing and resuspending the fermentation bacterial thallus and a column equilibrium buffer solution, then shearing and ultrasonically cracking under the ice bath condition, centrifuging, collecting the supernatant of a cracking product, and filtering; the components of the column equilibration buffer include: 25mM Tris-HCl and 1-10mM imidazole, the pH of the column equilibration buffer being 7-9.

3. The non-denaturing purification method of soluble recombinant teriparatide according to claim 1, wherein the nickel column affinity purification step in step 2) is: applying the sample obtained in the step 1) to a nickel affinity chromatography column, wherein the volume ratio of the sample obtained in the step 1) to a chromatography gel is 1:1-2, washing the column with a column balance buffer solution after applying the sample to the column, wherein the volume of the column balance buffer solution is 2-3 times of the volume of a column bed of the nickel affinity chromatography column, so as to remove the foreign proteins which are not combined with the chromatography gel, eluting Trx-hPTH (1-34) fusion protein with an elution buffer solution, collecting an elution peak, and obtaining affinity purified Trx-hPTH (1-34) fusion protein, wherein the elution buffer solution comprises the following components: 50mM sodium acetate and 0.5M NaCl, the pH of the elution buffer being 2-6.

4. The method for the non-denaturing purification of the soluble recombinant teriparatide according to claim 1, wherein the enzyme digestion step in the step 3) is as follows: mixing the Trx-hPTH (1-34) fusion protein solution obtained in the step 2) with 10 Xenzyme digestion buffer solution according to the volume ratio of 10:1, regulating the pH value of the solution to 6-8, adding enterokinase, incubating at 4-37 ℃ for 3-24h, and performing enzyme digestion, wherein 0.2-2mg of Trx-hPTH (1-34) fusion protein is added to each 1IU of enterokinase.

5. The non-denaturing purification method of soluble recombinant teriparatide according to claim 1, wherein in step 4), the mass ratio of the nickel affinity gel of the nickel affinity chromatography column to the enzyme-cleaved product obtained in step 3) is 1000: 20-60 parts of; washing the column through the column washing liquid for washing the non-specifically adsorbed teriparatide; the column washing liquid comprises the following components: 20mM Tris and 10-40mM imidazole, pH 8.0.

6. The non-denaturing purification method of soluble recombinant teriparatide according to claim 1, wherein the step of purifying the C18-100A column in step 5) comprises: adjusting the primarily purified recombinant teriparatide solution obtained in the step 4) by using 2M sodium acetate, acetonitrile and deionized water to ensure that the final concentration of the sodium acetate reaches 50mM and the acetonitrile concentration reaches 5%, and adjusting the pH value of the solution to 5.5-6.5; loading into C18-100A column, wherein the mass ratio of the chromatography gel to the sample is 100: 0.5-1.0; washing the column with 10% acetonitrile, and equilibrating the column with an equilibrium solution containing 50mM NaAc and 10% acetonitrile; loading the initially purified teriparatide solution on a C18-100A column, after the loading is finished, balancing and washing the column by 5 column volumes, then carrying out gradient elution by using 100% acetonitrile, gradually increasing the elution gradient from 15% to 35%, collecting each elution peak, and carrying out SDS-PAGE analysis and related substance content analysis.

7. The non-denaturing purification method of recombinant teriparatide according to claim 1, wherein the step of reverse phase chromatography purification of C18-300A in step 6) comprises: diluting the teriparatide sample obtained in the step 5) by using a diluent according to a volume ratio of 1:3, and filling the teriparatide sample into a C18-300A column, wherein the mass ratio of glue to the sample is 100:0.5-1, washing the column with 10% acetonitrile to remove the organic solvent for sealing the column, balancing the column with an equilibrium solution, then loading, washing the column with no less than 5 column volumes of column washing solution after loading, then adding 100% acetonitrile to perform gradient elution, gradually increasing the elution gradient from 18% to 25%, collecting each elution peak, and performing SDS-PAGE analysis and related substance content analysis to obtain the recombinant teriparatide with the purity of more than 99%.

8. The method for non-denaturing purification of soluble recombinant teriparatide according to claim 1, wherein the steps of rotary evaporation and lyophilization in step 7) are as follows: the heating temperature in the rotary steaming process is 25-50 ℃; the negative pressure is more than or equal to 0.08 Pa; the freeze drying process parameters are as follows:

pre-freezing: -36 ℃ for 180 min; sublimation: the temperature is minus 20 ℃, 1200min, the vacuum degree is 0 to 60Pa, and the heating rate is 30min and 10 ℃; secondary sublimation: at the temperature of minus 5 ℃, 600min, the vacuum degree of 0-60Pa, the heating rate of 30min and 10 ℃; resolution sublimation at 25 deg.C and 1080min, vacuum degree of 0-60Pa, and temperature rise rate of 30min and 10 deg.C.

9. Use of the non-denaturing purification process of soluble recombinant teriparatide according to any one of claims 1 to 8 for the preparation of bulk drug teriparatide for use in the treatment of osteoporosis and bone fracture patients.

Technical Field

The invention belongs to the technical field of biotechnology and pharmaceutical engineering, and particularly relates to a non-denaturing purification method and application of recombinant teriparatide.

Background

Human Parathyroid Hormone (hPTH) is a major Hormone regulating calcium and phosphorus metabolism in blood, is secreted by Parathyroid chief cells, is a polypeptide consisting of 84 amino acids, can bind to Parathyroid Hormone receptors, and exerts a bidirectional effect on bone metabolism. The hPTH receptor belongs to the superfamily of G protein-coupled receptors, which are classified into type I and type II. The hPTH-I receptor is mainly distributed in the skeleton and kidney, and the hPTH-II receptor is distributed in the brain and pancreas. The N-terminal peptide chain of parathyroid hormone is combined with hPTH-I receptor to promote the growth of bone cell, and the C-terminal peptide chain is combined with hPTH-II receptor to promote the apoptosis of bone cell.

Human parathyroid hormone 1-34 peptide (hPTH1-34), also known as teriparatide, is a natural degradation product of parathyroid hormone in vivo, and is composed of amino acids 1-34 of the N-terminal of human endogenous parathyroid hormone. Compared with natural intact hPTH molecule composed of 84 amino acids, teriparatide retains intact hPTH osteoblast growth promoting effect, and eliminates bone cell apoptosis promoting effect. Therefore, teriparatide and analogues thereof are potential candidate target molecules for developing medicaments capable of effectively treating osteoporosis and fracture, and have wide clinical application prospects.

The teriparatide is a small molecular short peptide and does not contain disulfide tendons, and the preparation method by adopting a genetic engineering prokaryotic recombinant expression method has the obvious advantages of large production scale, low production cost, short production period and the like, and is one of the most common methods for preparing the teriparatide. Because parathyroid hormone is a eukaryotic cell molecule, an inclusion body is easy to form when the parathyroid hormone is expressed in escherichia coli, the inclusion body containing teriparatide is required to be denatured and dissolved firstly under the denaturation condition, and then the purification and renaturation are carried out by a chromatography method under the denaturation condition. Currently, most common methods for purifying recombinant teriparatide are inclusion body purification methods under denaturing conditions. Due to the fact that a large amount of denaturants such as urea, guanidine hydrochloride and the like are needed to be used in the inclusion body purification method under the denaturation condition, the defects of high production cost, long production period and easiness in causing environmental pollution exist, and the produced recombinant teriparatide is low in yield and poor in purity and cannot meet the requirement of large-scale commercial production. Therefore, there is an urgent need to develop engineering bacteria and fermentation and purification processes based on the soluble expression of recombinant teriparatide, so as to improve the production efficiency of recombinant teriparatide.

In order to solve the problem of difficult purification of recombinant teriparatide expressed in the form of inclusion bodies in the prior art, the applicant constructs an engineering bacterium pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3) for carrying out fusion expression on parathyroid hormone 1-34 peptide and thioredoxin, and the strain is fermented by a specific fermentation process to obtain a fermentation product of thioredoxin A-like parathyroid hormone 1-34 peptide fusion protein [ Trx-hPTH (1-34) ] with high soluble expression level. Thereby improving the production efficiency of the recombinant teriparatide and creating conditions for the production of commercial teriparatide bulk drugs.

Disclosure of Invention

In order to solve the technical problems in the method for purifying the recombinant teriparatide in the form of inclusion bodies in the prior art, the invention aims to provide a non-denaturing purification method and application of the recombinant teriparatide.

In order to achieve the purpose and achieve the technical effect, the invention adopts the technical scheme that:

a method for the non-denaturing purification of soluble recombinant teriparatide comprising the steps of:

1) fermenting, cracking and centrifuging thalli: cracking fermented zymocyte bacteria expressing Trx-hPTH (1-34) fusion protein, centrifuging, and collecting supernatant of cracked products;

2) and (3) nickel column affinity purification: loading the sample obtained in the step 1) on a nickel affinity chromatographic column, eluting the Trx-hPTH (1-34) fusion protein combined on the nickel affinity chromatographic column, and obtaining affinity purified Trx-hPTH (1-34) fusion protein;

3) enzyme digestion: cleaving the Trx-hPTH (1-34) fusion protein obtained in the step 2) with enterokinase to release the recombinant teriparatide from the fusion protein; after the Trx-hPTH (1-34) fusion protein is cut, two peptide fragments are generated: trx partial peptide segment and recombinant teriparatide hPTH (1-34);

4) penetration of the nickel column: loading the enzyme digestion product obtained in the step 3) on a new nickel affinity chromatographic column, combining a Trx partial peptide segment with a 6 × His label on the nickel affinity chromatographic column, allowing the recombinant teriparatide to pass through the nickel affinity chromatographic column, and collecting a penetration peak to obtain the primarily purified recombinant teriparatide;

5) C18-100A column purification: loading the recombinant teriparatide sample obtained in the step 4) on a C18-100A column, and performing gradient elution by using acetonitrile to obtain the recombinant teriparatide hPTH (1-34) with the purity of more than 90%;

6) C18-300A reverse phase chromatography purification: putting the recombinant teriparatide protein sample purified by the C18-100A column in the step 5) on a C18-300A reverse chromatographic column, and performing gradient elution by using acetonitrile to obtain the recombinant teriparatide with the purity of more than 99 percent;

7) rotary evaporation and freeze drying: heating the sample obtained in the step 7), carrying out negative pressure rotary evaporation, removing residual acetonitrile, and then carrying out vacuum freeze drying.

Further, the step of lysing the thallus in the step 1) comprises the following steps: mixing the fermentation bacterial thallus with a column equilibrium buffer solution, re-suspending, shearing and cracking under an ice bath condition, centrifuging, collecting a supernatant of a cracking product, and filtering; the components of the column equilibrium buffer solution comprise: 25mM Tris-HCl and 1-10mM imidazole, the pH of the column equilibration buffer being 7-9.

Further, the nickel column affinity purification in step 2) comprises the following steps: applying the sample obtained in the step 1) to a nickel affinity chromatography column, wherein the volume ratio of the sample obtained in the step 1) to a chromatography gel is 1:1-2, washing the column with a column balance buffer solution after applying the sample to the column, wherein the volume of the column balance buffer solution is 2-3 times of the volume of a column bed of the nickel affinity chromatography column, so as to remove the foreign proteins which are not combined with the chromatography gel, eluting Trx-hPTH (1-34) fusion protein with an elution buffer solution, collecting an elution peak, and obtaining the affinity purified Trx-hPTH (1-34) fusion protein, wherein the elution buffer solution comprises the following components: 50mM sodium acetate and 0.5M NaCl, the pH of the elution buffer being 2-6.

Further, the enzyme digestion step in the step 3) is as follows: mixing the Trx-hPTH (1-34) fusion protein solution obtained in the step 2) with 10 Xenzyme digestion buffer solution according to the volume ratio of 10:1, regulating the pH value of the solution to 6-8, adding enterokinase, incubating at 4-37 ℃ for 3-24h, and performing enzyme digestion, wherein 0.2-2mg of Trx-hPTH (1-34) fusion protein is added to each 1IU of enterokinase.

Further, in the step 4), the mass ratio of the nickel affinity glue of the nickel affinity chromatographic column to the enzyme digestion product obtained in the step 3) is 1000: 20-60 parts of; washing the column through the column washing liquid for washing the non-specifically adsorbed teriparatide; the column washing liquid comprises the following components: 20mM Tris and 10-40mM imidazole, pH 8.0.

Further, the step of purifying the C18-100A column in the step 5) comprises the following steps: adjusting the primarily purified recombinant teriparatide solution obtained in the step 4) by using 2M sodium acetate, acetonitrile and deionized water, so that the final concentration of the sodium acetate reaches 50mM, the concentration of the acetonitrile reaches 5%, and the pH value of the solution is adjusted to 5.5-6.5; loading into C18-100A column, wherein the mass ratio of the chromatography gel to the sample is 100: 0.5-1.0; washing the column with 10% acetonitrile, and equilibrating the column with an equilibrium solution containing 50mM NaAc and 10% acetonitrile; loading the initially purified teriparatide solution on a C18-100A column, after the loading is finished, balancing and washing the column by 5 column volumes, then carrying out gradient elution by using 100% acetonitrile, gradually increasing the elution gradient from 15% to 35%, collecting each elution peak, and carrying out SDS-PAGE analysis and related substance content analysis.

Further, the step of C18-300A reverse phase chromatography purification in step 6) comprises the following steps: diluting the teriparatide sample obtained in the step 5) by using a diluent according to a volume ratio of 1:3, and filling the teriparatide sample into a C18-300A column, wherein the mass ratio of glue to the sample is 100:0.5-1, washing the column with 10% acetonitrile to remove the organic solvent for sealing the column, balancing the column with an equilibrium solution, then loading, washing the column with no less than 5 column volumes of column washing solution after loading, then adding 100% acetonitrile to perform gradient elution, gradually increasing the elution gradient from 18% to 25%, collecting each elution peak, and performing SDS-PAGE analysis and related substance content analysis to obtain the recombinant teriparatide with the purity of more than 99%.

Further, the steps of rotary evaporation and freeze-drying in the step 7) are as follows: the heating temperature in the rotary steaming process is 25-50 ℃; the negative pressure is more than or equal to 0.08 Pa; the freeze drying process parameters are as follows:

pre-freezing: -36 ℃ for 180 min; sublimation: the temperature is minus 20 ℃, 1200min, the vacuum degree is 0 to 60Pa, and the heating rate is 30min and 10 ℃; secondary sublimation: at the temperature of minus 5 ℃, 600min, the vacuum degree of 0-60Pa, the heating rate of 30min and 10 ℃; resolution sublimation at 25 deg.C and 1080min, vacuum degree of 0-60Pa, and temperature rise rate of 30min and 10 deg.C.

The invention discloses an application of a non-denaturing purification method of soluble recombinant teriparatide in preparing bulk drug teriparatide for treating osteoporosis and fracture patients.

Compared with the prior art, the invention has the beneficial effects that:

1) the invention establishes a purification method of the high-purity recombinant teriparatide under the non-denaturing condition, simplifies the purification process and improves the purification efficiency and purity of the recombinant teriparatide;

2) the invention establishes a method for releasing and preliminarily purifying the recombinant teriparatide from the Trx-hPTH (1-34) fusion protein by nickel column affinity chromatography-enzyme digestion-nickel column penetration, and a denaturant is not needed in the process;

3) the invention provides a method for removing impurities in recombinant teriparatide by sequentially adsorbing and eluting C18-100A and C18-300A and then carrying out rotary evaporation.

Drawings

FIG. 1 is a block diagram of the workflow of the present invention;

FIG. 2 is an SDS-PAGE analysis of a lysate of the fermentation bacteria according to the present invention; FIG. 1a shows the expression product lysis supernatant; FIG. 2a is a representation of the expression product lysis precipitate; FIG. 3a is the ultrafiltration residue of the expression product lysis supernatant; FIG. 4a is an ultrafiltration filtrate of expression product lysis supernatant; FIG. 5a is a protein standard molecular weight marker;

FIG. 3 is an SDS-page electrophoresis of a primary purified sample of a nickel affinity layer according to the present invention; FIG. 1b is a sample of the column supernatant from the lysis of the expression product; 2b, 3b and 4b are respectively penetration peaks; 5b is the elution peak; 6b is a human serum albumin control; m is a protein standard molecular weight marker;

FIG. 4 is an SDS-page electrophoresis of the Trx-hPTH (1-34) fusion protein enzyme cleavage product of the present invention; FIG. 1c shows the rx-hPTH (1-34) fusion protein before cleavage; 2c is an enzyme digestion product; 3c is HSA; 4c is a protein standard molecular weight marker;

FIG. 5 is SDS-PAGE analysis electrophoresis chart of a penetrating sample of a nickle affinity chromatographic column of the enzyme digestion product of the present invention; FIG. 1d shows the restriction enzyme digestion product of Trx-hPTH (1-34) fusion protein; FIG. 2d is the pellet after centrifugation of the enzyme product; FIG. 3d is a column penetration peak; FIG. 4d is a column wash peak; FIG. 5d is a protein standard molecular weight marker;

FIG. 6 is an analysis chromatogram of related substances in the column C18-100A for purifying teriparatide according to the present invention;

FIG. 7 is a table of peaks of related substances in the purified teriparatide using C18-100A column according to the present invention;

FIG. 8 is an analysis chromatogram of related substances in the column C18-300A for purifying teriparatide according to the invention;

FIG. 9 is a chart showing the peak analysis of related substances in the purification of teriparatide using C18-300A column according to the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention is provided to enable those skilled in the art to more easily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.

As shown in FIGS. 1 to 9, a non-denaturing purification method of soluble recombinant teriparatide, the recombinant teriparatide expression product used in the present invention is derived from engineering bacteria: a fermentation product of pET28b-Trx-hPTH (1-34)/e.coli BL21(DE3), the fusion protein consisting of thioredoxin (Trx), linker peptide and teriparatide (hPTH (1-34)), the linker peptide between thioredoxin and teriparatide carrying a 6 xhis tag and an enterokinase cleavage site (DDDDK) located just upstream of the N-terminus of teriparatide. Therefore, it is possible to isolate Trx-hPTH (1-34) fusion protein from the expression product by nickel affinity chromatography, release the recombinant teriparatide from the Trx-hPTH (1-34) fusion protein by enterokinase cleavage, and then prepare the purified recombinant teriparatide by further chromatography.

The preparation method of the engineering bacterium pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3) comprises the following steps: constructing a gene sequence of a fusion protein (Trx-hPTH (1-34)) of encoding escherichia coli thioredoxin (Trx) and human parathyroid hormone 1-34 peptide (hPTH (1-34)), carrying out enzyme digestion connection, constructing a recombinant expression plasmid for expressing the Trx-hPTH (1-34) fusion protein, and carrying out inducible expression.

The invention designs a gene sequence of a fusion protein (Trx-hPTH (1-34)) for coding escherichia coli thioredoxin (Trx) and human parathyroid hormone 1-34 peptide (hPTH (1-34)), the full length is 599 bases, the 5 'end of the gene sequence is a gene sequence for coding Trx, the 3' end is a gene sequence for coding hPTH (1-34), a gene sequence for coding connecting peptide is arranged between the gene sequence for coding Trx peptide segment and the gene sequence for coding hPTH (1-34), the gene segment for coding Trx-hPTH (1-34) fusion protein is completely synthesized by manpower, the full length is 599 bases, and the nucleotide sequence is SEQ IN NO: 3.

In order to facilitate the purification of the recombinant human parathyroid hormone 1-34 peptide, a DNA sequence coding for a connecting peptide is introduced between Trx and hPTH (1-34), the sequence comprises a sequence coding for 6 histidines (6 XHis) and a sequence coding for enterokinase enzyme cutting site (DDDDK), wherein, the C end of the enterokinase sequence is directly connected with the N end of the hPTH (1-34), and the DNA coding for the gene sequence of the Trx-hPTH (1-34) fusion protein is subjected to Escherichia coli codon optimization.

For the convenience of the next cloning operation, a Nco I cleavage site was introduced at the 5 'end and an Xho I cleavage site was introduced at the 3' end of the Trx-hPTH (1-34) fusion protein gene sequence. The Trx-hPTH (1-34) fusion protein consists of 194 amino acids and has theoretical molecular weight of 21332.18Da, the nucleotide sequence of the Trx-hPTH (1-34) fusion protein is SEQ IN NO:1, the amino acid sequence of the Trx-hPTH (1-34) fusion protein is SEQ IN NO:2, and the encoding nucleotide sequence of the Trx-hPTH (1-34) fusion protein is inserted into an expression vector and a cell-host system.

The expression vector of the invention can be eukaryotic cell expression vector, yeast expression vector, and can also be Escherichia coli expression vector, the preferred expression vector of the invention is Escherichia coli expression vector, wherein, the Escherichia coli expression vector can be pGEX series, pET series, pMAL series, pQE series, pTrc99a series, pTrcHis series, pBV220 series, pTXB series, pLLP-ompA series, pIN-III-ompA series, etc., preferably pET expression plasmid series, more preferably pET28b (+).

The invention provides a construction method of a recombinant expression plasmid for expressing Trx-hPTH (1-34) fusion protein, which comprises the following steps:

1) firstly, a polynucleotide sequence fragment (Trx-hPTH (1-34)) is prepared by a chemical synthesis method, namely a gene sequence for coding the Trx-hPTH (1-34) fusion protein. The sequence contains a nucleotide sequence for coding human parathyroid hormone 1-34 peptide [ hPTH (1-34) ], a nucleotide sequence for coding thioredoxin (Trx) and a nucleotide sequence for coding intermediate connecting peptide, wherein the 5 'end of the polynucleotide sequence is provided with a restriction endonuclease Nco I site, and the 3' end of the polynucleotide sequence is provided with a restriction endonuclease Xho I site;

2) carrying out restriction enzyme digestion on the Trx-hPTH (1-34) gene fragment synthesized in the step 1) and an expression vector pET28b (+) DNA by using Nco I and Xho I, carrying out agarose DNA electrophoresis on the enzyme digestion product, purifying the enzyme digested Trx-hPTH (1-34) DNA fragment and an expression vector pET28b (+) DNA fragment by using a gel recovery kit, and using T₄Connecting the two purified DNA fragments by using DNA ligase, electrically transforming the connection product into a recipient bacterium E.coli DH5 alpha, coating the receptor bacterium E.coli DH5 alpha on an LB plate containing kanamycin (35 mu g/ml), and growing the product at 37 ℃ to obtain a strain containing a recombinant expression plasmid pET28b-Trx-hPTH (1-34); several single colonies were picked from LB plates and inoculated into LB liquid medium containing kanamycin (35. mu.g/ml), grown overnight at 37 ℃, centrifuged to collect the cells, and a plasmid DNA purification kit was used to prepare recombinant expression plasmid pET28b-Trx-hPTH (1-34) DNA.

The invention provides a construction method of engineering bacteria for expressing Trx-hPTH (1-34) fusion protein, which comprises the following steps:

the purified recombinant expression plasmid pET28b-Trx-hPTH (1-34) for expressing Trx-hPTH (1-34) fusion protein is transformed into an Escherichia coli host bacterium for induction expression, and the preferred Escherichia coli host bacterium is E.coli BL21(DE 3). Specifically, the recombinant expression plasmid pET28b-Trx-hPTH (1-34) is transformed into a competent cell of Escherichia coli BL21(DE3) obtained in a commercial way, and an engineering bacterium pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3) capable of expressing Trx-hPTH (1-34) fusion protein is obtained through kanamycin screening and protein induction expression, wherein the engineering bacterium for expressing the Trx-hPTH (1-34) fusion protein is Escherichia coli, which is deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, having the address of China, Beijing City, institute of microbiology, Beijing, Yangxing, Xilu No. 1 of the Yangxi district of Beijing, the deposit number is CGMCC21219, and the deposit time is 23 days 11 years and 23 days.

The invention provides a fermentation method for improving the soluble expression of Trx-hPTH (1-34) fusion protein, which is a fermentation method for expressing the soluble Trx-hPTH (1-34) fusion protein at a high level and comprises the following process steps:

(1) seed activation: the working seeds of the engineered bacteria stored at-70 ℃ were removed, thawed at 4 ℃ and then streaked onto LB solid plates containing 35. mu.g/ml kanamycin, and inverted in a 37 ℃ incubator overnight.

(2) Preparing first-level seeds: 100 single colonies were picked from LB solid plates, inoculated into 100ml LB liquid medium containing 35. mu.g/ml kanamycin, cultured at 37 ℃ for 3-5 hours at 200rpm, and OD was measured₆₀₀In the range of 0.5-2, primary seeds are obtained.

(3) Preparing secondary seeds: mixing the first-stage seed liquid according to the proportion of 1: 10-100 of the culture medium was inoculated into fresh liquid LB medium containing 35. mu.g/ml kanamycin, cultured at 30-37 ℃ for 4-8 hours at 200rpm to OD₆₀₀Secondary seeds were obtained at values of 2.5-3.0.

(4) Fermentation culture: preparing a fermentation culture medium, inoculating a secondary seed liquid according to the proportion of 1:100 for fermentation culture;

the fermentation medium comprises the following components in percentage by mass: 2% of tryptone, 1% of yeast extract powder, 1% of glycerol, 0.4% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.4% of dipotassium hydrogen phosphate, 0.24% of potassium sulfate, 0.113% of calcium chloride and 0.1% of ammonium chloride, and adjusting the pH to 7.0 by using ammonia water; the temperature is controlled at 37 ℃ in the fermentation process, DO is controlled to be more than 30% through stirring speed, ventilation flow and tank pressure, and the pH is controlled to be 6.9-7.1 through adding ammonia water.

(5) Feeding: feeding when the DO in the tank is greatly increased to 50-70%, wherein the total volume of a feeding liquid is one tenth of that of a fermentation medium, the feeding liquid comprises a feeding material I and a feeding material II, the volume of the feeding material I is 2 times that of the feeding material II, and feeding of the feeding material II is carried out after feeding of the feeding material I is finished;

the components of the feed medium include:

and (3) feed liquid I: 7% of yeast powder, 7% of peptone and 7% of glycerol

And (3) supplement liquid II: 7% of yeast powder and 7% of glycerol.

(6) Induction: when OD is reached₆₀₀When 10-15 mM of the total concentration is reached, adding sterile IPTG through a feeding tube to a final concentration of 0.3-0.6mM for induction, and inducing again after 3hAdding sterile IPTG to a final concentration of 0.4-0.7mM for second induction, and finishing fermentation after 2-5 h;

(7) and (3) collecting thalli: after the fermentation is finished, the fermentation liquor is centrifuged by a continuous centrifuge, and thalli are collected and stored at the temperature of minus 20 ℃.

The invention discloses a gene sequence for coding Trx-hPTH (1-34) fusion protein, a recombinant expression plasmid pET28b-Trx-hPTH (1-34) for expressing the Trx-hPTH (1-34) fusion protein, an engineering bacterium pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3) for expressing the Trx-hPTH (1-34) fusion protein, and a fermentation method for improving the soluble expression of the Trx-hPTH (1-34) fusion protein, which can be used for producing a raw material drug human parathyroid hormone 1-34 (also called teriparatide) for treating osteoporosis and fracture patients. The recombinant expression plasmid for expressing the Trx-hPTH (1-34) fusion protein and the engineering bacteria do not contain ampicillin genes, beta-lactam antibiotics are not needed in the fermentation process of the engineering bacteria, and the environmental pollution can be reduced.

The invention adopts the following steps to prepare a preliminary purification product of the recombinant teriparatide:

1) cell lysis

Adopting a low-temperature mechanical shearing, ultrasonic cracking or homogenizer method to crack the fermented fermentation bacteria thallus expressing Trx-hPTH (1-34) fusion protein, centrifuging and collecting the supernatant of the cracked product;

2) nickel column affinity purification

Putting the supernatant of the lysate into a nickel affinity chromatography column, and eluting Trx-hPTH (1-34) fusion protein specifically bound on the nickel affinity chromatography column by using an elution buffer solution;

3) enzyme digestion

Cutting the Trx-hPTH (1-34) fusion protein by using bovine enterokinase to release the recombinant teriparatide from the fusion protein;

4) penetration

And (3) putting the enzyme digestion product in the step 3) on a new nickel affinity chromatographic column, and after the Trx-hPTH (1-34) fusion protein is subjected to enzyme digestion by enterokinase, generating two peptide fragments, wherein one is a Trx partial peptide fragment, and the other is recombinant teriparatide. The Trx part is provided with a 6 XHis tag, the protein of the Trx part can be combined on a nickel affinity chromatographic column when an enzyme digestion product is put on the nickel affinity chromatographic column, the recombinant teriparatide of the other part passes through the nickel affinity chromatographic column because the recombinant teriparatide of the Trx part is not provided with the 6 XHis tag, and the collected liquid passing through is the primarily purified recombinant teriparatide;

5) C18-100A column purification

Taking a nickel affinity purified teriparatide sample, loading the teriparatide sample on a C18-100A column, and performing gradient elution by using acetonitrile to obtain rhPTH (1-34) with purity of more than 90%;

6) C18-300A reverse phase chromatography purification

Loading rhPTH (1-34) protein sample purified by C18-100A column on C18-300A reverse chromatographic column, and gradient eluting with acetonitrile to obtain teriparatide with purity of more than 99%;

7) rotary steaming and freeze-drying

And (3) putting the purified teriparatide sample into a rotary evaporation bottle, heating for negative pressure rotary evaporation, removing residual acetonitrile, and then carrying out vacuum freeze drying.

In step 1), the weight (g) of the mixture of the fermentation bacterial cells and the column equilibrium buffer: volume (ml) ratio 1: 5, and precooling at 2-4 ℃; the column equilibration buffer comprises 25mM Tris-HCl and 1-10mM imidazole, the concentration of imidazole is preferably 5 mM; the pH of the column equilibration buffer is 7-9, preferably 8.0.

In the step 1), shearing thallus, grade C and shearing for 30min by using a shearing machine in ice bath, and then carrying out continuous ultrasonic treatment for 25min in ice bath under the conditions that the ultrasonic power is 1200W and the suspension time is 2s per ultrasonic treatment for 2s, so as to crack the thallus. Centrifuging at 14400 Xg and 4 deg.C for 30min, collecting supernatant of lysate, and discarding precipitate containing inclusion body; the lysate supernatant was filtered through a 0.45 μm filter.

In the step 2), a supernatant sample of the filtered lysate is applied to a nickel affinity chromatographic column, the ratio of the weight of the sample to the volume of chromatographic gel of the nickel affinity chromatographic column is 1:1-2, the column is washed by column balance buffer solution after the application of the column is finished so as to remove foreign proteins which are not combined with the chromatographic gel, and the volume of the column balance buffer solution for washing the column is 2-3 times of the volume of a column bed; eluting Trx-rhPTH (1-34) fusion protein by using an elution buffer solution, and collecting an elution peak to obtain the affinity purified Trx-rhPTH (1-34) fusion protein. The elution buffer comprises 50mM sodium acetate and 0.5M NaCl, and the pH value of the elution buffer is 2-6, and the preferred pH value is 4.0.

In the step 3), the Trx-rhPTH (1-34) fusion protein obtained in the step 2) is mixed with 10 Xenzyme digestion buffer solution according to the volume ratio of 10:1, and the pH value of an enzyme digestion system is adjusted by using 1M Tris solution. Adding enterokinase, incubating for a period of time at a proper temperature under magnetic stirring, and performing enzyme digestion; wherein the 10 Xenzyme digestion buffer solution comprises 200mM Tris and 20mM CaCl₂(ii) a The pH value of the enzyme digestion system is 6-8, and the preferable pH value is 7.0-7.4; the temperature of enzyme digestion incubation is 4-37 ℃, and the preferable temperature is 37 ℃; the enzyme digestion incubation time is determined according to enzyme digestion conditions, and the optimized enzyme digestion incubation time is 3-24h, preferably 10 h; the enzyme cutting time is 16 h; the enterokinase is bovine enterokinase, the dosage of the enterokinase is calculated by 1IU enterokinase, the dosage of the Trx-rhPTH (1-34) fusion protein is 0.2-2mg, and 1IU is preferred: 0.5 mg.

In the step 4), the enzyme digestion product in the step 3) is applied to a new nickel affinity chromatography gel column, and the mass ratio of the nickel affinity chromatography gel column nickel affinity gel to the sample is 1000: 20-60.

In the step 4), after the Trx-hPTH (1-34) fusion protein is digested by enterokinase, a penetration peak is collected, and then column washing liquid (20mM Tris and 10-40mM imidazole; pH 8.0), washing the non-specifically adsorbed teriparatide, preferably with imidazole at a concentration of 20mM, collecting the washing peak, and combining the penetration peak and the washing peak to obtain the primarily purified recombinant teriparatide.

In the step 5), 2M sodium acetate, acetonitrile and water are used for adjusting the primarily purified recombinant teriparatide solution obtained in the step 4) to ensure that the final concentration of the sodium acetate reaches 50mM and the acetonitrile concentration reaches 5 percent, the pH value is adjusted to 5.5-6.5 by using acetic acid, and the optimized pH value is 6.0; then loading on a C18-100A column, wherein the ratio of the mass (g) of the chromatographic gel to the amount (g) of the sample is 100:0.5-1.0, washing the column with 10% acetonitrile, and balancing the column with a balance solution of pH 6.0 and containing 50mM NaAc and 10% acetonitrile; loading the initially purified teriparatide solution on a column, washing the column by 5 column volumes of an equilibrium solution containing 50mM NaAc and 10% acetonitrile at pH 6.0 after loading, then performing gradient elution by using 100% acetonitrile, gradually increasing the elution gradient from 15% to 35%, collecting each elution peak, and performing SDS-PAGE analysis and related substance content analysis.

In step 6), the mixture was washed with 50mM Na at pH 2.3₂SO₄And 10% acetonitrile in a ratio of 1:3 to dilute the teriparatide sample obtained in step 5). Loading into a C18-300A column, wherein the ratio of the mass (g) of the chromatographic gel to the amount (g) of the sample is 100:0.5-1. The column was washed with 10% acetonitrile, and then with 50mM Na at pH 2.3₂SO₄And a 10% acetonitrile solution equilibration column; the diluted teriparatide sample was applied to the column and the end of the application was repeated at 5 column volumes of pH 2.3 containing 50mM Na₂SO₄Washing the column with 10% acetonitrile solution, performing gradient elution with 100% acetonitrile from 18% to 25%, collecting each elution peak, performing SDS-PAGE analysis and related substance content analysis to obtain purity>99% teriparatide.

In the step 7), the heating temperature in the rotary steaming process is 25-50 ℃, and preferably 35 ℃; the negative pressure is more than or equal to 0.08 Pa; the freeze-drying process parameters are as follows:

pre-freezing: -36 ℃ for 180 min; sublimation: the temperature is minus 20 ℃, 1200min, the vacuum degree is 0 to 60Pa, and the heating rate is 30min and 10 ℃; secondary sublimation: at the temperature of minus 5 ℃, 600min, the vacuum degree of 0-60Pa, the heating rate of 30min and 10 ℃; resolution sublimation at 25 deg.C and 1080min, vacuum degree of 0-60Pa, and temperature rise rate of 30min and 10 deg.C.

Example 1

The fermented fermentation bacterial thallus expressing Trx-hPTH (1-34) fusion protein is cracked and centrifuged, and the supernatant of the cracked product is collected:

taking 1000g of fermentation bacterial thallus of engineering bacteria pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3), and suspending the thallus by using 5L of column equilibrium buffer solution (containing 25mM Tris-HCl and 5mM imidazole; pH 8.0); shearing the thallus in ice bath with shearing machine, hanging C gear, and shearing for 30 min; then continuously carrying out ultrasonic treatment for 25min in an ice bath under the conditions that the ultrasonic power is 1200W and the ultrasonic treatment is suspended for 2s every 2s, and cracking thalli; centrifuging at 14400 Xg and 4 deg.C for 30min, collecting lysate supernatant, filtering with 0.45 μm filter membrane, and performing SDS-PAGE analysis on the lysate supernatant, precipitate, filtrate and ultrafiltration residue respectively to obtain lysate supernatant, 1 as lysate supernatant shown in FIG. 2; 2 is a cracking precipitate; 3 is ultrafiltration residue; 4 is a filtrate; and 5 is a protein standard molecular weight marker. As can be seen from FIG. 2, the supernatant of the lysate contained a large amount of the Trx-hPTH (1-34) fusion protein of interest, and the precipitate contained less of the protein of interest. Thus, the protein of interest in the fermentation product is predominantly soluble and is present in the lysate supernatant and is amenable to nickel affinity chromatography purification under non-denaturing conditions.

Example 2

And (3) purifying the Trx-hPTH (1-34) fusion protein by nickel column affinity chromatography:

equilibrating the column with column equilibration buffer (containing 25mM Tris-HCl, 5mM imidazole; pH 8.0) at a flow rate of 100ml/min, loading the ultrafiltrate at a flow rate of 100ml/min, and starting to collect breakthrough peaks when UV280 rises to above 1500 (the three stages of breakthrough collection start, middle and end, sample retention are 2, 3 and 4); after the sample loading is finished, washing the column by using a 3L column equilibrium buffer solution, and stopping washing the column when the UV280 is lower than 100; eluting with elution buffer (50mM sodium acetate, 0.5M NaCl; pH 4.0) at a flow rate of 50ml/min, and collecting eluate peaks when UV280 is greater than 200 (if collecting peaks have inflection points, collecting by changing bottle); determining OD280 and OD260 of the collected peak, and sending the sample to perform SDS-PAGE analysis, wherein 1 is the upper column sample, 2, 3 and 4 are the penetration peak, 5 is the elution peak and 6 is HSA, as shown in FIG. 3; m is a protein standard molecular weight marker. From fig. 3, it can be seen that none of the 3 breakthrough peaks contains the target protein, and the target protein is only present in the elution peak, so that the purity is high.

Example 3

And (3) carrying out enzyme digestion on the Trx-hPTH fusion protein:

25g of Trx-hPTH fusion protein sample was added with 10 Xenzyme digestion buffer (0.5M Tris-HCl solution and 20mM CaCl₂) In the method, the final concentration of Tris is 50mM, and the final concentration of calcium chloride is 2 mM; adjusting the pH of the sample to 7.4 with 1M Tris solution; adding into50000U enterokinase (corresponding amount of 0.5 mg/U), enzyme-cutting under magnetic stirring at 37 deg.C for 10 hr, and performing SDS-PAGE analysis on the sample after enzyme-cutting to obtain a result as shown in FIG. 4, wherein 1 is fusion protein before enzyme-cutting, 2 is enzyme-cutting product, and 3 is HSA; and 4 is a protein standard molecular weight marker. As can be seen from FIG. 4, after the enzyme digestion, the fusion protein is completely cut, the target protein yield is high, and the cutting effect is good.

Example 4

Penetration of the nickel column:

and (4) centrifuging the enzyme digestion product, and collecting the supernatant. Applying a new nickel affinity chromatographic column at the flow rate of 50ml/min, starting to collect breakthrough peaks when UV280 rises to about 10, washing 3 column volumes by using column washing liquid (20mM Tris-HCl, 20mM imidazole; pH 8.0) after the sample application is finished, collecting washing peaks, combining the breakthrough peaks, and determining OD260 and OD 280; samples were submitted for SDS-PAGE analysis. The results are shown in FIG. 5, where 1 is the cleavage product, 2 is the enzyme product precipitate, 3 is the penetration peak, 4 is the washing peak, and 5 is the protein standard molecular weight marker. FIG. 5 shows that the target protein is mainly present in the breakthrough peak, and the wash peak is free from the target protein.

Example 5

C18-100A column purification:

penetrating a Trx-rhPTH (1-34) fusion protein preliminarily separated and purified by a nickel affinity chromatographic column into a sample, wherein the total amount is 6.3L, adjusting by using a mixed solution of 2M sodium acetate, acetonitrile and water to ensure that the final concentration of the sodium acetate reaches 50mM, the concentration of the acetonitrile reaches 5%, and adjusting the pH value to 6.0 by using acetic acid; washing the C18-100A column with 10% acetonitrile at flow rate of 15mL/min for 30 min; the column was equilibrated with 10 column volumes of mobile phase A (10% acetonitrile, 50mM sodium acetate; pH 6.0) at a flow rate of 15 mL/min. Loading a Trx-rhPTH (1-34) fusion protein penetration sample which is primarily separated and purified by a nickel affinity chromatography column at the flow rate of 15 mL/min; after the loading was complete, the column was washed with 5 column volumes of mobile phase A (pH 6.0, containing 50mM NaAc and 10% acetonitrile) at a flow rate of 15 mL/min; followed by a gradient elution with mobile phase B (100% acetonitrile) from 15% to 35% for a total time of 40min at a flow rate of 15 mL/min. The elution peaks were collected from the UV220 monitoring profile, the OD280 and OD260 values of each sample were determined, and the samples were sent for substance analysis, and the results are shown in FIGS. 6 and 7. As can be seen from FIG. 7, the purity of the teriparatide sample purified by the C18-100A column reaches 98%, the highest impurity content is 0.56%, and the purification effect is good.

Example 6

C18-300A column purification:

taking a sample of rhPTH (1-34) protein purified by C18-100A reverse chromatography column, adding 50mM Na at pH 2.3₂SO₄And 10% acetonitrile in 1: 3. Loading into a C18-300A column, wherein the ratio of the mass (g) of the chromatographic gel to the amount (g) of the sample is 100:0.5-1. Washing the C18-300A column with 10% acetonitrile at a flow rate of 40mL/min to remove the organic solvent for sealing the column, wherein the washing time is 30 min; the C18-300A column was equilibrated with a solution containing 50mM NaAC, 10% acetonitrile (pH 2.3) at a flow rate of 40mL/min, not less than 10 column volumes. Loading the treated loading solution at the flow rate of 40mL/min, and washing the column with a solution (pH 2.3) containing 50mM NaAC and 10% acetonitrile at the flow rate of 40mL/min for not less than 5 column volumes after loading; then, a gradient elution was performed with 100% acetonitrile, the gradient increasing stepwise from 18% to 25% for a total time of 50min and a flow rate of 40 mL/min. Each elution peak was collected according to the UV280 monitoring profile. Samples were taken for relevant substance analysis and the results are shown in FIGS. 8-9. As can be seen from FIG. 9, the purity of teriparatide purified by C18-300A reaches 99.89%, and both impurities are less than 0.5%, so that the result meets the requirement.

Example 7

Rotary evaporation and freeze drying:

setting the water temperature of a water bath kettle of a rotary evaporator, putting the mixed sample into a 1000ml rotary evaporation bottle when the water temperature reaches 35 ℃, installing the rotary evaporator on the bottle, setting the rotating speed at 50rpm, starting a circulating water type multipurpose vacuum pump, and starting rotary evaporation. Timing is started when the vacuum degree reaches 0.09MPa, and the rotary evaporation is finished after the timing is carried out for 15 min. The OD280 value of the sample was measured with a spectrophotometer, and the total protein amount (the absorption coefficient of rhPTH (1-34) was 1.34, and the value of the protein concentration was OD280/1.34) was calculated. Transferring the rotary-distilled concentrated solution into a beaker subjected to dry heat sterilization, transferring the rotary-distilled concentrated solution into a clean bench, subpackaging the rotary-distilled concentrated solution into sterilized medium borosilicate glass tube injection bottles according to 5 ml/bottle, and then carrying out vacuum freeze drying, wherein the freeze drying conditions are set as shown in table 1. After the freeze-drying is finished, sampling and carrying out quality inspection, wherein the detection result shows that the product quality meets the requirements of WHO, USP and imported registration quality standards.

TABLE 1

Phases	Temperature (. degree.C.)	Time (min)	Degree of vacuum (Pa)	Temperature raising strategy
					Prefreezing	-36	180	--	--
Sublimation	-20	1200	0-60	10℃/30min
					Second sublimation	-5	600	0-60	10℃/30min
Resolution sublimation	25	1080	0-60	10℃/30min

The invention constructs an engineering bacterium pET28b-Trx-hPTH (1-34)/E.coli BL21(DE3) for expressing the parathyroid hormone 1-34 peptide and thioredoxin fusion protein, and initially provides a fermentation method for improving the soluble expression of the Trx-hPTH (1-34) fusion protein, and the strain can obtain a fermentation product of the thioredoxin parathyroid hormone 1-34 peptide fusion protein (Trx-hPTH (1-34)) with high soluble expression level through fermentation by a specific fermentation process. On the basis, the invention provides a non-denaturing purification method of soluble teriparatide, which can completely release the recombinant teriparatide from Trx-hPTH (1-34) fusion protein under the non-denaturing condition, effectively remove impurities in the recombinant expressed teriparatide, and ensure that the purity of the obtained recombinant teriparatide can reach more than 99 percent, thereby improving the production efficiency of the recombinant teriparatide and creating conditions for the production of commercial teriparatide bulk drugs.

The parts of the invention not specifically described can be made of existing products or existing technologies, and are not described herein.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.