CN118515782B - A fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein - Google Patents
A fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein Download PDFInfo
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- CN118515782B CN118515782B CN202410716859.6A CN202410716859A CN118515782B CN 118515782 B CN118515782 B CN 118515782B CN 202410716859 A CN202410716859 A CN 202410716859A CN 118515782 B CN118515782 B CN 118515782B
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- 108010000521 Human Growth Hormone Proteins 0.000 title claims abstract description 59
- 102000002265 Human Growth Hormone Human genes 0.000 title claims abstract description 58
- 239000000854 Human Growth Hormone Substances 0.000 title claims abstract description 57
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 46
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 28
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 112
- 238000000855 fermentation Methods 0.000 claims abstract description 46
- 230000004151 fermentation Effects 0.000 claims abstract description 46
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 26
- 238000005406 washing Methods 0.000 claims abstract description 22
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 18
- 238000001914 filtration Methods 0.000 claims abstract description 17
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 15
- 238000000746 purification Methods 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 238000004271 weak anion exchange chromatography Methods 0.000 claims abstract description 11
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 238000003860 storage Methods 0.000 claims abstract description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 5
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- 108090000623 proteins and genes Proteins 0.000 claims description 49
- 102000004169 proteins and genes Human genes 0.000 claims description 43
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- 239000007983 Tris buffer Substances 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 102000018997 Growth Hormone Human genes 0.000 claims description 20
- 108010051696 Growth Hormone Proteins 0.000 claims description 20
- 239000000122 growth hormone Substances 0.000 claims description 20
- 238000011068 loading method Methods 0.000 claims description 14
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
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- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 4
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
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- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开一种融合蛋白及基于该融合蛋白制备重组人生长激素的方法。融合蛋白包括第一肽段和第二肽段，第一肽段具有如SEQ ID NO:1所示的氨基酸序列，第二肽段具有如式(I)所示的氨基酸序列，第一肽段的N端与第二肽段的C端相连。通过基因工程菌构建、工作种子培养、发酵罐培养、菌体回收、菌体破碎、包涵体洗涤、包涵体溶解、包涵体复性步骤获取包括融合蛋白的包涵体，经过滤超滤、阴离子交换层析法粗纯、Kex2酶酶切、弱阴离子交换层析法精纯、超滤、除菌过滤、冻干保存，制得重组人生长激素。本发明能够高效制备纯度高、活性高的重组人生长激素，适合商品化生产。
The present invention discloses a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein. The fusion protein includes a first peptide segment and a second peptide segment, the first peptide segment has an amino acid sequence as shown in SEQ ID NO: 1, the second peptide segment has an amino acid sequence as shown in formula (I), and the N-terminus of the first peptide segment is connected to the C-terminus of the second peptide segment. The inclusion bodies including the fusion protein are obtained by genetic engineering bacteria construction, working seed culture, fermentation tank culture, bacterial recovery, bacterial crushing, inclusion body washing, inclusion body dissolution, and inclusion body renaturation steps, and the recombinant human growth hormone is obtained by filtration ultrafiltration, anion exchange chromatography rough purification, Kex2 enzyme digestion, weak anion exchange chromatography purification, ultrafiltration, sterilization filtration, freeze-dried storage. The present invention can efficiently prepare recombinant human growth hormone with high purity and high activity, which is suitable for commercial production.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein.
Background
Human growth hormone (human growth hormone, hGH)) is a non-glycosylated protein hormone secreted by anterior She Shi acid cells of pituitary gland, consists of 191 amino acid residues, has a molecular weight of about 22kDa, has the functions of participating in wide biological functions including protein synthesis, cell proliferation, lactation, immunoregulation and metabolism of protein, carbohydrate and lipid, is the most important hormone for promoting growth after birth of human beings, and can be widely used for treating diseases such as short stature, short bowel syndrome and aging delay of old people caused by chronic renal failure, and the like. In the past clinical application, human growth hormone can only be extracted from human pituitary glands, and has limited sources and is quite expensive. The genetic engineering technology can be used for producing recombinant human growth hormone (recombinant human growth hormone, rhGH) in a large scale, and the structure and the amino acid sequence of the recombinant human growth hormone are consistent with those of the natural growth hormone.
HGH is a non-glycosylated protein, only has 2 disulfide bonds and has no post-translational modification, so that escherichia coli becomes a first choice host for production, an escherichia coli-mediated prokaryotic expression system becomes a main expression mode of recombinant human growth hormone at home and abroad, and the escherichia coli is fast in propagation, low in cost, clear in genetic background, mature in technology and suitable for large-scale commercial production.
The technical approach for expressing rhGH in colibacillus cells is mainly 2, one is to produce secreted rhGH, a gene of a signal peptide is added at the N end of a human growth hormone gene sequence, a soluble fusion protein of the signal peptide-rhGH is formed in colibacillus cytoplasm, the signal peptide guides the growth hormone fusion protein to enter periplasmic space, and protease is used for cutting the signal peptide sequence, so that the recombinant human growth hormone completely consistent with the primary structure of natural human growth hormone is obtained. However, the secretion efficiency of the escherichia coli is low, the yield is low, the downstream purification process technology route is complex, and the method is not a preferred method for large-scale preparation. The other is to produce the recombinant human growth hormone in intracellular form, express it in the form of inclusion body, prepare and obtain the recombinant human growth hormone with correct space conformation by inclusion body renaturation method, the expression quantity is very high, but the renaturation effect of inclusion body is often bad, the protein after renaturation is folded poorly, the stability is bad, the protein activity is low. In addition, due to the characteristic of the self expression of the escherichia coli, the N-terminal of the intracellular rhGH is one methionine more than the natural hGH in the primary structure. The presence of methionine makes such rhGH easy to induce antibody production, affecting the drug effect. There are also patents such as CN 115947864A which express recombinant human growth hormone fusion protein in the form of inclusion bodies, then enzyme-cutting the fusion protein by recombinant bovine enterokinase to form free target growth hormone and fragments thereof, the N-terminal of which does not contain free amino acid, which is identical to the amino acid sequence of natural human growth hormone, however, the downstream purification process of this patent is complicated, and the downstream purification engineering of this patent includes hydrophobic chromatography I, molecular sieve chromatography I, anion exchange chromatography I, hydrophobic chromatography II, molecular sieve chromatography II, anion exchange chromatography II and molecular sieve chromatography III, 7-step chromatography, more chromatography steps and higher cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a fusion protein and a method for preparing recombinant human growth hormone based on the fusion protein, wherein the recombinant human growth hormone protein is expressed in an inclusion body form in escherichia coli, preferably a strong induction promoter system (induction promoter system: lPL, lPR, trc, T7 and the like), growth and production stages are separated, high-density fermentation is carried out to realize high yield, the protein expression quantity is high, the defect and the defect of secretory expression can be overcome, and under the same fermentation scale, the yield is higher and the cost is lower.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a fusion protein comprises a first peptide fragment and a second peptide fragment, wherein the first peptide fragment is target growth hormone and fragments thereof, the first peptide fragment has an amino acid sequence shown as SEQ ID NO. 1, the second peptide fragment has an amino acid sequence shown as formula (I), and the N end of the first peptide fragment is connected with the C end of the second peptide fragment;
The amino acid sequence of the second peptide fragment is:
MAXB(I)
In the formula (I), M is methionine, A is at least one basic amino acid, X is at least one arbitrary amino acid, and B is an enzyme cutting site formed by one of the double basic amino acids.
Further, in formula (I), A is RR.
Further, in the formula (I), X is TG.
Further, in formula (I), B is KR or KK or PR or RR.
A method for preparing recombinant human growth hormone comprises the steps of constructing genetically engineered bacteria, culturing working seeds, culturing in a fermentation tank, recovering thalli, crushing thalli, washing inclusion bodies, dissolving the inclusion bodies, renaturating the inclusion bodies to obtain inclusion bodies containing fusion protein as defined in any one of claims 1-4, performing ultrafiltration to obtain inclusion body concentrated solution, performing crude purification on the inclusion body concentrated solution by adopting an anion exchange chromatography to obtain an anion exchange chromatography sample, performing Kex2 enzyme digestion to obtain an enzyme digestion product, performing fine purification by adopting a weak anion exchange chromatography to obtain a protein-containing eluent, and performing ultrafiltration, sterilization filtration, freeze-drying and storage to obtain the recombinant human growth hormone.
Further, the anion exchange chromatography method comprises the specific steps of washing an inclusion body concentrated solution by using a mobile phase A balance chromatographic column for 3CV, directly loading samples after balancing, wherein the loading flow rate is not more than 210cm/h, washing the mobile phase A balance chromatographic column for 2CV, washing the mobile phase B for 3CV, eluting weak binding protein, eluting the target protein by using the mobile phase A balance chromatographic column for 2CV, eluting the buffer solution, starting to collect when the UV is not less than 50mAU, stopping collecting when the UV is less than 100mAU, merging samples, and obtaining anion exchange chromatography samples, and temporarily storing at 4 ℃.
Further, the mobile phase A is a solution containing 10-50 mM Tris and having a pH of 7-10, the mobile phase B is a solution containing 10-50 mM Tris and 30-80 mM NaCl and having a pH of 7-10, and the elution buffer is a solution containing 10-50 mM Tris and 100-200 mM NaCl and having a pH of 7-10.
The specific method for Kex2 enzyme digestion comprises the steps of slowly adjusting pH of an anion exchange chromatography sample to 8.0 by using 2M HCl, slowly adding protein diluent accounting for 1/100 of the total volume of the sample, calculating total protein of the anion exchange chromatography sample according to the results of Lowry and RP-HPLC, calculating the required Kex2 enzyme mass according to the ratio of the total protein to the Kex2 enzyme of 100:1-2500:1, adding Kex2 protease diluent buffer to the Kex2 enzyme with the calculated required amount to enable the Kex2 enzyme concentration to be 1mg/ml, adding the obtained Kex2 enzyme solution into a mixture of the anion exchange chromatography sample and the protein diluent, and carrying out enzyme digestion at room temperature.
Further, the protein diluent is a solution containing 10-100 mM Tris and 200-500 mM CaCl 2, pH 7.0-9.0, and the Kex2 protease dilution buffer is a solution containing 10-100 mM Tris and 2-5 mM CaCl 2, pH 8.0-9.0.
Further, the specific steps of the weak anion exchange chromatography for fine purification are as follows:
Pumping the enzyme-digested product into a container with proper size by adopting a peristaltic pump, pumping PB into the enzyme-digested product, diluting the enzyme-digested product to be less than 3ms/cm in electric conduction, carrying out the sample loading by using a mobile phase A equilibrium chromatographic column 3CV, carrying out the sample loading by using a sample loading flow rate not more than 130cm/h, washing 3CV by using 10% of mobile phase B, eluting 4CV by using the mobile phase A, then carrying out gradient elution, wherein the mobile phase A90%, the mobile phase B10%, the mobile phase A50% and the mobile phase B500% are started at the beginning, the collection is started when the UV is not less than 50mAU, the collection is stopped when the UV is less than 100mAU, and the sample is combined to collect protein-containing eluent;
the pH of the solution with the mobile phase A of 20-50 mM PB is 7-10;
the mobile phase B is a solution containing 20-50 mM PB and 100-500 mM NaCl, and the pH is 7-10.
The beneficial effects of the invention are as follows:
the method improves the yield of the inclusion body, has small fermentation scale under the same yield, improves the renaturation efficiency of the inclusion body, has simple purification process steps, fewer chromatography steps, shortens working hours, reduces cost, is easy to amplify and control, and has high purity and high activity of the obtained recombinant human growth hormone, thereby being suitable for commercial production.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis (reduction) of the secondary fermentation sample of step 3 of example 1 (M: marker; lane 1: 1h before induction; lane 2: 1h after induction; lane 3: 2h after induction; lane 4: 3h after induction; lane 5: 4h after induction; lane 6: 5h after induction; lane 7: 6h after induction; 22KD is recombinant human growth hormone of interest);
FIG. 2 is a SDS-PAGE electrophoresis (reduction) of recombinant human growth hormone inclusion body protein washed after hybridization in step 6 of example 1 (M: marker; lane 1: inclusion body 1 st washed pellet; lane 2: inclusion body 2 nd washed pellet; lane 3: inclusion body 3 rd washed pellet);
FIG. 3 shows anion exchange chromatography (crude purity) of step 10 of example 1, SDS-PAGE electrophoresis of each eluted fraction, SDS-PAGE electrophoresis before and after digestion of the sample in step 11 (reduction) (M: marker; lane 1: concentrated solution after ultrafiltration in step 9; lane 2: anion exchange chromatography flow-through; lane 3: elution of target protein; lane 4: protein before digestion in step 11; lane 5: protein after digestion in step 11);
FIG. 4 is a RP-HPLC test chromatogram of the sample after cleavage in step 11 of example 1;
FIG. 5 is a chromatogram of the SEC-HPLC test of the sample after cleavage in example 1;
FIG. 6 is a RP-HPLC test chromatogram of a (lean) sample after weak anion exchange chromatography of step 12 of example 1;
FIG. 7 is a SEC-HPLC test chromatogram of a (lean) sample after weak anion exchange chromatography of step 12 of example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a recombinant human growth hormone fusion protein (i.e. a fusion protein constructed in a molecular biology stage), which comprises a first peptide fragment and a second peptide fragment, wherein the first peptide fragment is target growth hormone and fragments thereof, the first peptide fragment has an amino acid sequence shown as SEQ ID NO. 1, the second peptide fragment has an amino acid sequence shown as formula (1), and the N end of the first peptide fragment is connected with the C end of the second peptide fragment.
The amino acid sequence of the first peptide fragment is:
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:1)
The amino acid sequence of the second peptide fragment is:
MAXB(I)
In the formula (I), M is methionine, A is at least one of basic amino acids, generally comprises 1-2 amino acids, X is at least one arbitrary amino acid, generally comprises 1-4 amino acids, and B is an enzyme cutting site formed by one of double basic amino acids (such as KR or KK or PR or RR, etc.). The cleavage site of the double basic amino acid can be specifically recognized and cleaved by recombinant double basic amino acid endopeptidase (Kex 2).
The amino acid sequence of the recombinant human growth hormone fusion protein is as follows:
MAXBFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(II)
The present invention is based on the preparation of high purity recombinant human growth hormone from fusion proteins in the form of intracellular inclusion bodies expressed by E.coli, comprising the steps of:
Step 1, construction of genetically engineered bacteria
The N end of the first peptide segment (the amino acid sequence is shown as SEQ ID NO: 1) is connected with the C end of the second peptide segment (the amino acid sequence is shown as formula (I)), recombinant human growth hormone fusion protein (fusion protein for short, MAXB+hGH) is obtained, the amino acid sequence is shown as formula (II), the nucleotide sequence for coding the recombinant human growth hormone fusion protein is designed, the initiation codon is ATG, the termination codon is TAA, the sequence between the initiation codon ATG and the termination codon TAA is the gene sequence of the fusion protein, the CATATG of the front end sequence of the fusion protein is Nde I enzyme cutting site, the CTCGAG is Xho I enzyme cutting site, the Nde I enzyme cutting site and the Xho I enzyme cutting site of the MCS region corresponding to the cloning vector pET-30a (+) of the target gene are respectively obtained, the target gene is synthesized to the cloning vector pET-30a (+) by the Shanghai biological engineering limited company, and the plasmid fusion protein expression vector of the plasmid fusion protein is obtained through E.coli21 (DE 3), cloning, screening, enzyme cutting hormone, enzyme identification and the plasmid fusion protein is transformed.
The specific steps of transforming host cell E.coli BL21 (DE 3) are 1, taking BL21 competent cells out of a refrigerator at-80 ℃ and putting them on ice for thawing, adding 5 mu L plasmid into competent cells for mixing, and putting them on ice for 30min. 2. The EP tube containing the plasmid-cell mixture was placed in a water bath, and after heat shock at 42℃for 90s, it was quickly removed and placed on ice for 2min. 3. 1mL of LB liquid medium (without antibiotics) was added, and shaking culture was performed at 37℃for 2 hours to resuscitate the cells. 4. mu.L of the transformation solution was spread on a solid LB medium containing 50mg/L kanamycin sulfate, and cultured overnight at 37℃for 16 hours.
The specific step of clone screening is to select single colonies of genetic engineering bacteria growing on a solid LB medium of 50mg/L kanamycin sulfate, and then to put the single colonies into the LB medium for culture at 37 ℃ for 16h.
The plasmid extraction comprises extracting plasmid vector of growth hormone fusion protein in genetically engineered bacteria according to SanPrep column type plasmid DNA small extraction kit instruction of Shanghai Co.
Enzyme digestion identification, namely carrying out double enzyme digestion on a plasmid vector for extracting the growth hormone fusion protein according to the restriction enzyme Nde I and Xho I enzyme specifications produced by TaKaRa company, and carrying out agarose gel electrophoresis identification, wherein the size of the double enzyme digestion section is compositely expected, and the construction of the genetically engineered bacterium for expressing the growth hormone fusion protein is successful.
Step 2, working seed culture
1ML of working seeds of the genetically engineered bacteria are taken and all inoculated into LB shake flask culture medium (180 mL) containing kanamycin monosulfate (50 mug/mL) for shake flask culture, wherein the culture temperature is 37.0 ℃ and the rotation speed is 220rpm. When the concentration OD600 of the thalli is more than or equal to 2 and the form of the thalli detected by an optical microscope is the typical form of escherichia coli, the culture of the working seeds is ended without other microbial contamination.
Step 3, culturing in a fermentation tank
The culture scale is further enlarged by a two-stage stepwise amplified thallus fermentation system. The primary fermentation culture step comprises the steps of inoculating working seeds cultured in a shaking bottle into a primary fermentation tank (5L) filled with 1.8L of sterile culture medium according to an inoculation amount of 1 percent, and carrying out primary fermentation culture until the concentration OD600 of the thalli is more than or equal to 20, the thalli form detected by an optical microscope is typical of the escherichia coli form, and no other microorganism is polluted, wherein the primary fermentation culture is finished, the culture temperature is 30.0 ℃, the rotating speed is 120-600 rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation amount is 10-100L/min, the dissolved oxygen is more than or equal to 20.0%, and the culture time is 6-12 h. The secondary fermentation culture step comprises the steps of transferring all fermentation liquor in a primary fermentation tank into a secondary fermentation tank (50L) filled with 18L of sterile culture medium for secondary fermentation culture, wherein the culture temperature is 30.0 ℃, the rotation speed is 120-800 rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation amount is 200.0-1000.0L/min, the dissolved oxygen is 20.0-80.0%, when the carbon source (glycerin) is consumed, the pH value is rising and the dissolved oxygen rises in the culture process, feeding is carried out, the feeding rate is regulated, the carbon source, the pH value and the dissolved oxygen in the secondary fermentation tank meet the secondary fermentation culture condition, the culture is carried out for 7-9 h, the thallus concentration OD600 is more than or equal to 100.0, 100-400 g of lactose inducer or IPTG inducer (controlling the concentration of the IPTG inducer in the secondary fermentation system to be 0.1-1 mM), the temperature reduction induction is 26-30 ℃, the induction time is 4-15 h, and the sample is carried out under the temperature reduction to 15 ℃ when the carbon source (glycerin) is consumed, and the pH value is rising and the dissolved oxygen rises, so that the SDS-PAGE test is carried out to detect the conditions of the recombinant human growth hormone, and the recombinant protein is detected, and the growth conditions are fused.
The aseptic culture medium and the feed supplement culture medium adopted in the primary fermentation culture and the secondary fermentation culture comprise the following components:
the primary fermentation tank culture medium comprises 5g/L of peptone, 5g/L of yeast powder, 4g/L of disodium hydrogen phosphate, 3.4g/L of sodium dihydrogen phosphate, 1g/L of magnesium sulfate and 21g/L of glycerin.
The secondary fermentation tank culture medium comprises 5.8g/L of yeast powder, 4g/L of dipotassium hydrogen phosphate, 4g/L of monopotassium phosphate, 7g/L of disodium hydrogen phosphate, 1.2g/L of magnesium sulfate, 0.2g/L of ammonium chloride, 1.2g/L of ammonium sulfate and 0.02g/L of calcium chloride.
The feed medium comprises 30g/L peptone, 15g/L yeast powder, 7g/L dipotassium hydrogen phosphate, 7g/L potassium dihydrogen phosphate, 10g/L disodium hydrogen phosphate, 2.5g/L magnesium sulfate and 10g/L ammonium chloride.
Step4, recovering the thalli
And (3) centrifuging the secondary fermentation culture solution to obtain thalli, wherein the centrifugation parameter is 15000rpm/min and 1 hour.
Step 5, crushing thalli to obtain inclusion bodies
Adding the thalli into a lysis buffer solution (containing 10-50 mM Tris (tromethamine), 0.1-5 mM metal ion chelating agent EDTA or EGTA, 0.5-2M denaturant urea or 0.1-0.5M denaturant guanidine hydrochloride, 1-5% nonionic surfactant (Triton X-100 or Tween-20) according to the mass volume ratio of the thalli to the lysis buffer solution of 1:10, dispersing the thalli by a high-speed dispersing machine at the speed of 700-1000 bar, homogenizing and breaking the walls for 1-5 times, centrifuging for 30min at the condition of 4 ℃ and 9000rpm, and collecting inclusion body precipitates.
Step 6, washing inclusion bodies
Adding an inclusion body washing buffer solution I (containing 10-50 mM Tris, 0.1-5 mM metal ion chelating agent EDTA or EGTA, a nonionic surfactant (Triton X-100 or Tween-20) with a pH value of 7.0-8.5) into the inclusion body precipitate, adding the inclusion body washing buffer solution I according to the mass-volume ratio of the thalli added in the step 5 to the inclusion body washing buffer solution I of 1:3, dispersing the inclusion body precipitate by using a high-speed dispersing machine for 1 st washing, centrifuging at 4 ℃ and 9000rpm for 30min, and collecting the inclusion body precipitate. And then adding the inclusion body washing buffer solution II (10-50 mM Tris, 0.1-5 mM metal ion chelating agent EDTA or EGTA, pH value of 7.0-8.5) according to the mass volume ratio of the thalli to the inclusion body washing buffer solution II of 1:3 in the step 5, dispersing the inclusion body precipitate by using a high-speed dispersing machine to carry out 2 nd washing on the inclusion body precipitate, and centrifuging for 30min at 4 ℃ and 9000rpm, and then collecting the inclusion body precipitate. And then adding pure water according to the mass-volume ratio of the thalli to the pure water of 1:3 in the step 5, dispersing the inclusion body sediment by using a high-speed dispersing machine, washing the inclusion body sediment for the 3 rd time, centrifuging for 30min at 4 ℃ and 9000rpm, collecting the inclusion body sediment, and weighing the inclusion body.
Step 7, inclusion body is dissolved
Crushing the inclusion body precipitate obtained in the step 6 into small blocks, calculating the volume of inclusion body dissolution buffer (containing 10-50 mM glycine, 7-8M urea or 5-6M guanidine hydrochloride, 0.1-5 mM metal ion chelating agent EDTA or EGTA and having a pH value of 9-11) according to the mass-volume ratio of the inclusion body precipitate to the inclusion body dissolution buffer of 1:15, adding beta-mercaptoethanol into the inclusion body dissolution buffer to make the final concentration of the beta-mercaptoethanol be 30-60 mM, adding inclusion body fragments, and accelerating the dissolution of the inclusion body by using a high-speed dispersing machine, wherein the dissolution temperature is controlled at 4 ℃. And after the inclusion body is completely dissolved, continuously adding an inclusion body dissolving buffer solution, and slowly diluting, wherein the inclusion body dissolving buffer solution is added according to the volume ratio of a dissolving system obtained after the inclusion body is completely dissolved to the inclusion body dissolving buffer solution of 1:3, so as to obtain an inclusion body mixture I.
Step 8, inclusion body renaturation
Adding the inclusion body mixture I into a newly prepared inclusion body dilution buffer solution with the volume ratio of the inclusion body mixture I to the inclusion body dilution buffer solution with the volume ratio of 1:3 (containing 10-50 mM glycine, 0.1-5 mM metal ion chelating agent EDTA or EGTA and the pH value of 9-11) by adopting a peristaltic pump, controlling the flow rate of the inclusion body mixture I to be 5ml/min by the peristaltic pump, stirring (50-200 rpm) the mixture at a low speed by using a magnetic stirrer in the adding process, adding the mixture into the newly prepared inclusion body renaturation buffer solution with the volume ratio of 1:1 (containing 10-50 mM glycine, 0.1-5 mM metal ion chelating agent or EGTA and the pH value of 9-11) with the ice bath (with the temperature of 0-4 ℃), and renaturating the mixture at a low speed of 100rpm for 8-16 h to obtain the inclusion body mixture II.
Step 9, filtering and ultra-filtering
Filtering the inclusion body mixture II by using a 0.45 mu m capsule filter, and then performing ultrafiltration by using a10 KD ultrafiltration membrane bag to obtain concentrated solution, wherein ice bath is needed before and after filtration.
Step 10, performing crude purity by anion exchange chromatography
Mobile phase A10~50mM Tris,pH 7~10.
Mobile phase B10~50mM Tris+30~80mM NaCl,pH 7~10.
Elution buffer 10~50mM Tris+100~200mM NaCl,pH 7~10.
Washing 3CV with mobile phase A balance chromatographic column, directly loading sample with flow rate not greater than 210cm/h, washing 3CV with mobile phase A balance chromatographic column 2CV with mobile phase B, eluting weak binding protein, eluting target protein with mobile phase A balance chromatographic column 2CV, collecting when UV is not less than 50mAU, stopping collecting when UV is less than 100mAU, mixing samples, and collecting anion exchange chromatographic sample at 4deg.C for temporary storage. RP-HPLC was performed and Lowry was performed.
Step 11, kex2 enzyme digestion
And (3) slowly adjusting the pH of the anion exchange chromatography sample in the step (10) to 8.0 by using 2M HCl, and slowly adding protein diluent (containing 10-100 mM Tris, 200-500 mM CaCl 2 and pH 7.0-9.0) accounting for 1/100 of the total volume of the sample. Calculating the total protein of the anion exchange chromatography sample according to the Lowry and RP-HPLC results, calculating the required Kex2 enzyme mass according to the ratio of the total protein to the Kex2 enzyme mass of 100:1-2500:1, adding Kex2 protease dilution buffer solution (10~100mM Tris,2~5mM CaCl 2, pH 8.0-9.0) into the Kex2 enzyme with the required calculated amount to ensure that the Kex2 enzyme concentration is 1mg/ml, lightly adding the obtained Kex2 enzyme solution into the mixture of the anion exchange chromatography sample and the protein dilution solution within 20-30 s, and carrying out enzyme digestion for 8-20 h at room temperature (18-30 ℃).
Step 12, performing fine purification by weak anion exchange chromatography
Mobile phase A is 20-50 mM PB (phosphate buffer solution) and pH is 7-10.
Mobile phase B20~50mM PB+100~500mM NaCl,pH 7~10.
Pumping the enzyme-digested product obtained in the step 11 into a container with proper size by adopting a peristaltic pump, pumping PB into the enzyme-digested product, and diluting the enzyme-digested product to be less than 3ms/cm in electric conduction. The equilibrium chromatographic column 3CV of mobile phase A is loaded, the loading flow rate is not more than 130cm/h, the mobile phase A is washed for 3CV, the mobile phase A is eluted for 4CV by 10% of mobile phase B, then gradient elution is carried out, namely, the mobile phase A90% at the beginning, the mobile phase B10% at the end, the mobile phase A50% at the end, the mobile phase B500% at the end, and the 4CV is operated. Collecting when UV is not less than 50mAU, stopping collecting when UV is less than 100mAU, combining samples, and collecting protein-containing eluent.
Step 13, ultra-filtration
And carrying out ultrafiltration concentration on the protein-containing eluent by 10 times by adopting a 10kDa membrane package to obtain concentrated solution.
Step 14, sterilizing and filtering
And (5) sterilizing, filtering and subpackaging the concentrated solution in an ultra-clean bench by using a 0.22 mu m filter head to obtain the growth hormone stock solution.
Step 15, freeze-drying and preserving
The method comprises the steps of freeze-drying a growth hormone stock solution, then placing the growth hormone stock solution in a container at 2-8 ℃ for preservation, freeze-drying, namely, after the container is placed in the container at 5 ℃, keeping the temperature for 1 hour, reducing the temperature to-40 ℃, keeping the temperature at-40 ℃ for 1 hour, heating the product to-18 ℃ and keeping the temperature for 1 hour, reducing the temperature of the product again, keeping the temperature at-35 ℃ for 1 hour, controlling the temperature of a plate layer at 10 ℃ and keeping the temperature for 15 hours until the frozen ice in the product sublimates, heating the product to the maximum allowable temperature of 30 ℃ and keeping the temperature until the freeze-drying is finished, recovering high vacuum, keeping the residual moisture of the product for more than 6 hours under the conditions of the maximum allowable temperature and the high vacuum, and finishing the freeze-drying.
The 50L fermentation system of the invention can obtain more than 75g of finished product growth hormone. The purification process can remove Kex2 enzyme residues, the removal rate reaches 99.99%, and the purified growth hormone contains almost no residues of cutting enzymes. Meanwhile, RP-HPLC and SEC-HPLC are carried out on the prepared recombinant human growth hormone stock solution to test the purity of the product, and the purity of the recombinant human growth hormone obtained by analysis and measurement is above 95%. The in vitro biological activity of the recombinant human growth hormone prepared according to the Nb2-11 cell proliferation method is above 3.0 IU/mg.
RP-HPLC method uses butyl silane bonded silica gel as filler (5-10 μm), 0.05mol/L tris buffer solution (1 mol/L hydrochloric acid solution is used for adjusting pH value to 7.5) and n-propanol (the volume ratio of tris buffer solution to n-propanol is 71:29) as mobile phase, and the proportion of n-propanol in the mobile phase is adjusted to ensure that the retention time of main peak of human growth hormone is 30-36 minutes, the flow rate is 0.5ml per minute, the column temperature is 45 ℃, and the detection wavelength is 220nm. Taking a human growth hormone reference substance, adding 0.05mol/L of tris buffer solution to dissolve and prepare a solution containing 2mg of the tris buffer solution per 1ml, filtering and sterilizing, and standing at room temperature for 24 hours to obtain a system applicability solution. And (3) taking 20 μl of the system applicability solution, injecting the solution into a liquid chromatograph, wherein the separation degree between the main peak of the human growth hormone and the deaminated main peak of the human growth hormone is not less than 1.0, and the tailing factor of the main peak of the human growth hormone is 0.9-1.8.
According to the SEC-HPLC method, according to the method for detecting the content of human growth hormone high molecular protein for injection in 2020 edition of Chinese pharmacopoeia, hydrophilic modified silica gel suitable for separating spherical protein with molecular weight of 5000-60000 Da is used as a filler, isopropanol-0.063 mol/L phosphate buffer solution (5.18 g of anhydrous disodium hydrogen phosphate and 3.65g of sodium dihydrogen phosphate are taken, 950ml of water is added, pH value is regulated to 7.0 by phosphoric acid or sodium hydroxide test solution, 1000ml of water is prepared by water) (the volume ratio of isopropanol to phosphate buffer solution is 3:97) is used as a mobile phase, the flow rate is 0.6ml per minute, and the detection wavelength is 214nm. Taking a mixture reference substance of human growth hormone monomer and dimer, adding 0.025mol/L phosphate buffer solution (pH 7.0) to prepare a solution containing 1.0mg in each 1ml, and taking 20 μl to be injected into a liquid chromatograph, wherein the separation degree between the human growth hormone monomer peak and the dimer peak meets the requirement.
In the invention, the fusion protein is cut by recombinant double basic amino acid endopeptidase (Kex 2 enzyme) to form the free target recombinant human growth hormone and fragments thereof, the target recombinant human growth hormone and fragments thereof are not cut by the recombinant Kex2 enzyme, the N-terminal of the free target growth hormone and fragments thereof does not contain additional amino acid residues, the products before and after cutting have obvious difference, and the products before and after cutting can be distinguished, thereby being beneficial to subsequent purification.
Preferred embodiment 1 is as follows:
A recombinant human growth hormone fusion protein comprises a first peptide fragment and a second peptide fragment, wherein the first peptide fragment is target growth hormone and fragments thereof, the first peptide fragment has an amino acid sequence shown as SEQ ID NO.1, the second peptide fragment has an amino acid sequence MRRTGPR (M is methionine, namely A is RR, X is TG and B is PR in the formula (I)), and the N end of the first peptide fragment is connected with the C end of the second peptide fragment.
The amino acid sequence of the recombinant human growth hormone fusion protein is as follows:
MRRTGPRFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:2).
This example is based on the preparation of high purity recombinant human growth hormone from fusion proteins in the form of intracellular inclusion bodies expressed by E.coli, comprising the steps of:
step 1, preparing a genetically engineered bacterium plasmid vector and genetically engineered bacterium
The nucleotide sequence of the recombinant human growth hormone fusion protein is designed by using the amino acid sequence shown in SEQ ID NO. 2, the initiation codon ATG (the 4 th-6 th site in the SEQ ID NO. 3 sequence), the termination codon TAA (the 598 th-600 th site in the SEQ ID NO. 3 sequence), the sequence between the initiation codon ATG and the termination codon TAA is the gene sequence of the fusion protein (shown as SEQ ID NO. 3), the CATATG of the front end sequence of the fusion protein is Nde I restriction site, the CTCGAG is Xho I restriction site, the Nde I restriction site and the Xho I restriction site respectively correspond to the MCS region of the target gene cloning vector pET-30a (+), the target gene is synthesized into the cloning vector pET-30a (+), and the vector carrying the growth hormone fusion protein gene sequence and the genetic engineering bacteria for expressing the growth hormone fusion protein of the plasmid are obtained through transformation of host cells E.coli BL21 (DE 3), clone screening, plasmid extraction and restriction identification.
The nucleotide sequence of the recombinant human growth hormone fusion protein is as follows:
CATATGCGTCGTACCGGCCCCCGTTTTCCGACCATTCCGCTGAGCCGTCTGTTTGATAATGCAATGCTGCGCGCCCATCGTCTGCATCAGCTGGCATTTGATACCTATCAGGAATTTGAAGAGGCATATATCCCGAAAGAACAGAAATATAGCTTCCTGCAGAACCCGCAGACCAGTCTGTGTTTTAGTGAAAGTATTCCGACCCCGAGCAATCGTGAAGAAACCCAGCAGAAAAGCAATCTGGAACTGCTGCGTATTAGCCTGCTGCTGATTCAGAGCTGGCTGGAACCGGTTCAGTTTCTGCGTAGCGTGTTTGCCAATAGTCTGGTGTATGGCGCAAGCGATAGTAATGTGTATGATCTGCTGAAAGACCTGGAAGAAGGTATTCAGACCCTGATGGGTCGCCTGGAAGATGGCAGCCCTCGTACAGGTCAGATTTTTAAACAGACCTATAGCAAGTTCGACACCAATAGTCATAACGATGATGCACTGCTGAAAAATTACGGTCTGCTGTATTGCTTTCGCAAAGATATGGATAAGGTGGAAACCTTTCTGCGCATTGTTCAGTGTCGTAGTGTTGAAGGTAGCTGTGGCTTTTAACTCGAG(SEQ ID NO:3)
Step 2, working seed culture
1ML of working seeds of the genetically engineered bacteria are taken and all inoculated into LB shake flask culture medium (180 mL) containing kanamycin monosulfate (50 mug/mL) for shake flask culture, wherein the culture temperature is 37.0 ℃ and the rotation speed is 220rpm. When the concentration OD600 of the thalli is more than or equal to 2 and the form of the thalli detected by an optical microscope is the typical form of escherichia coli, the culture of the working seeds is ended without other microbial contamination.
Step 3, culturing in a fermentation tank
The culture scale is further enlarged by a two-stage stepwise amplified thallus fermentation system. The primary fermentation culture step comprises the steps of inoculating working seeds cultured in a shake flask into a primary fermentation tank (5L) filled with 1.8L of sterile culture medium according to an inoculation amount of 1 percent, and carrying out primary fermentation culture until the concentration OD600 of the thalli is more than or equal to 20, the thalli form is detected to be a typical escherichia coli form by an optical microscope, and the primary fermentation culture is finished when no other microorganisms pollute, wherein the culture temperature is 30.0 ℃, the rotating speed is 400rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation amount is 40L/min, the dissolved oxygen is more than or equal to 20.0%, and the culture time is 6h. The secondary fermentation culture step comprises the steps of transferring all fermentation liquor in a primary fermentation tank to a secondary fermentation tank (50L) filled with 18L of sterile culture medium for secondary fermentation culture, wherein the culture temperature is 30.0 ℃, the rotation speed is 600rpm, the pH value is 7.0 (ammonia water adjustment), the ventilation amount is 500.0L/min, the dissolved oxygen is 60.0%, feeding and adjusting the feeding rate when a carbon source (glycerin) is consumed, the pH value is rising and the dissolved oxygen rises in the culture process, so that the carbon source, the pH value and the dissolved oxygen in the secondary fermentation tank meet the secondary fermentation culture condition, adding 300g of lactose inducer for induction for 6h at the induction temperature of 26 ℃ for 15 ℃, and sampling and performing SDS-PAGE electrophoresis test to detect the expression condition of recombinant human growth hormone fusion protein.
The aseptic culture medium and the feed supplement culture medium adopted in the primary fermentation culture and the secondary fermentation culture comprise the following components:
the primary fermentation tank culture medium comprises 5g/L of peptone, 5g/L of yeast powder, 4g/L of disodium hydrogen phosphate, 3.4g/L of sodium dihydrogen phosphate, 1g/L of magnesium sulfate and 21g/L of glycerin.
The secondary fermentation tank culture medium comprises 5.8g/L of yeast powder, 4g/L of dipotassium hydrogen phosphate, 4g/L of monopotassium phosphate, 7g/L of disodium hydrogen phosphate, 1.2g/L of magnesium sulfate, 0.2g/L of ammonium chloride, 1.2g/L of ammonium sulfate and 0.02g/L of calcium chloride.
The feed medium comprises 30g/L peptone, 15g/L yeast powder, 7g/L dipotassium hydrogen phosphate, 7g/L potassium dihydrogen phosphate, 10g/L disodium hydrogen phosphate, 2.5g/L magnesium sulfate and 10g/L ammonium chloride.
Step4, recovering the thalli
And (3) centrifuging the secondary fermentation culture solution to obtain thalli, wherein the centrifugation parameter is 15000rpm/min and 1 hour.
Step 5, crushing thalli to obtain inclusion bodies
Adding the thalli into a lysis buffer (containing 20mM Tris, 3mM metal ion chelating agent EDTA, 1.2M denaturant urea and 2.6% nonionic surfactant Triton X-100) according to the mass volume ratio of the thalli to the lysis buffer of 1:10, dispersing the thalli by a high-speed dispersing machine at the speed of 850bar for 3 times, centrifuging at 4 ℃ and 9000rpm for 30min, and collecting inclusion body precipitates.
Step 6, washing inclusion bodies
Adding an inclusion body washing buffer solution I (containing 20mM Tris, 3mM metal ion chelating agent EDTA and 2.6% nonionic surfactant Triton X-100 and having a pH value of 7.0) into the inclusion body precipitate, adding the inclusion body washing buffer solution I according to the mass-volume ratio of the thalli added in the step 5 to the inclusion body washing buffer solution I of 1:3, dispersing the inclusion body precipitate by using a high-speed dispersing machine for washing for the 1 st time, and centrifuging at 4 ℃ and 9000rpm for 30min, and collecting the inclusion body precipitate. And then adding the inclusion body washing buffer solution II (20 mM Tris, 3mM metal ion chelating agent EDTA, pH value is 7.0) according to the mass-volume ratio of the thalli to the inclusion body washing buffer solution II of 1:3 in the step 5, dispersing the inclusion body sediment by using a high-speed dispersing machine to carry out the 2 nd washing on the inclusion body sediment, and collecting the inclusion body sediment after centrifugation for 30min at 4 ℃ and 9000 rpm. And then adding pure water according to the mass-volume ratio of the thalli to the pure water of 1:3 in the step 5, dispersing the inclusion body sediment by using a high-speed dispersing machine, washing the inclusion body sediment for the 3 rd time, centrifuging for 30min at 4 ℃ and 9000rpm, collecting the inclusion body sediment, and weighing the inclusion body.
Step 7, inclusion body is dissolved
Crushing the inclusion body sediment obtained in the step 6 into small blocks, calculating the volume of an inclusion body dissolution buffer (containing 25mM glycine, 7M urea and 1mM metal ion chelating agent EDTA, with the pH value of 10.2) according to the mass-volume ratio of the inclusion body sediment to the inclusion body dissolution buffer of 1:15, adding beta-mercaptoethanol into the inclusion body dissolution buffer to enable the final concentration to be 50mM, adding inclusion body fragments, and using a high-speed dispersing machine to accelerate dissolution of the inclusion body, wherein the dissolution temperature is required to be controlled at 4 ℃. And after the inclusion body is completely dissolved, continuously adding an inclusion body dissolving buffer solution, and slowly diluting, wherein the inclusion body dissolving buffer solution is added according to the volume ratio of a dissolving system obtained after the inclusion body is completely dissolved to the inclusion body dissolving buffer solution of 1:3, so as to obtain an inclusion body mixture I.
Step 8, inclusion body renaturation
Adding the inclusion body mixture I into a newly prepared inclusion body dilution buffer solution with the volume ratio of the inclusion body mixture I to the inclusion body dilution buffer solution with the volume ratio of 1:3 by adopting a peristaltic pump (containing 25mM glycine and 1mM metal ion chelating agent EDTA and the pH value of 10.2), controlling the flow rate of the inclusion body mixture I to be 5ml/min by adopting the peristaltic pump, stirring (80 rpm) the mixture at a low speed by using a magnetic stirrer in the adding process, adding the mixture into a newly prepared inclusion body renaturation buffer solution with the volume ratio of 1:1 after the dilution is completed under the ice bath (the temperature of 0-4 ℃), putting the inclusion body mixture into a4 ℃ refrigerator, and renaturating for 12h at a low speed of 100rpm to obtain the inclusion body mixture II.
Step 9, filtering and ultra-filtering
Filtering the inclusion body mixture II by using a 0.45 mu m capsule filter, and then performing ultrafiltration by using a10 KD ultrafiltration membrane bag to obtain concentrated solution, wherein ice bath is needed before and after filtration.
Step 10, performing crude purity by anion exchange chromatography
Mobile phase A20 mM Tris, pH 8.
Mobile phase B20mM Tris+50mM NaCl,pH 8.
Elution buffer 20mM Tris+150mM NaCl,pH 8.
Washing 3CV with mobile phase A balance chromatographic column, directly loading sample with flow rate not greater than 210cm/h, washing 3CV with mobile phase A balance chromatographic column 2CV with mobile phase B, eluting weak binding protein, eluting target protein with mobile phase A balance chromatographic column 2CV, collecting when UV is not less than 50mAU, stopping collecting when UV is less than 100mAU, mixing samples, and collecting anion exchange chromatographic sample at 4deg.C for temporary storage. RP-HPLC was performed and Lowry was performed.
Step 11, kex2 enzyme digestion
The pH of the anion exchange chromatography sample in step 10 was slowly adjusted to 8.0 with 2M HCl and then a protein dilution (50 mM Tris, 500mM CaCl 2, pH 7.5) was slowly added to 1/100 of the total sample volume. Calculating total protein of anion exchange chromatography sample according to Lowry and RP-HPLC results, calculating required Kex2 enzyme mass according to the ratio of total protein to Kex2 enzyme mass of 1000:1, adding Kex2 protease dilution buffer (20mM Tris,5mM CaCl 2, pH 8.5) to the Kex2 enzyme with required calculated amount to make Kex2 enzyme concentration be 1mg/ml, lightly adding the obtained Kex2 enzyme solution into the mixture of anion exchange chromatography sample and protein dilution solution within 30s, and placing the mixture at room temperature (23 ℃) for enzyme digestion for 10 hours. The content of related protein in the sample after enzyme digestion is 8.17% by RP-HPLC detection, and the content of high molecular protein in the sample after enzyme digestion is 10.44% by SEC-HPLC detection.
Step 12, performing fine purification by weak anion exchange chromatography
Mobile phase a 50mM PB (phosphate buffer), pH 7.5 (NaOH adjusted).
Mobile phase B20mM PB+220mM NaCl,pH 7.5.
Pumping the enzyme-digested product obtained in the step 11 into a container with proper size by adopting a peristaltic pump, pumping PB into the enzyme-digested product, and diluting the enzyme-digested product to be less than 3ms/cm in electric conduction. The equilibrium chromatographic column 3CV of mobile phase A is loaded, the loading flow rate is not more than 130cm/h, the mobile phase A is washed for 3CV, the mobile phase A is eluted for 4CV by 10% of mobile phase B, then gradient elution is carried out, namely, the mobile phase A90% at the beginning, the mobile phase B10% at the end, the mobile phase A50% at the end, the mobile phase B500% at the end, and the 4CV is operated. Collecting when UV is not less than 50mAU, stopping collecting when UV is less than 100mAU, combining samples, and collecting protein-containing eluent. The content of the related protein of the sample after the weak anion exchange chromatography is 2.65 percent by RP-HPLC detection, and the content of the high molecular protein of the sample after the weak anion exchange chromatography is 0.04 percent by SEC-HPLC detection.
Step 13, ultra-filtration
And carrying out ultrafiltration concentration on the protein-containing eluent by 10 times by adopting a 10kDa membrane package to obtain concentrated solution.
Step 14, sterilizing and filtering
And (5) sterilizing, filtering and subpackaging the concentrated solution in an ultra-clean bench by using a 0.22 mu m filter head to obtain the growth hormone stock solution.
Step 15, freeze-drying and preserving
The freeze-drying step includes the steps of feeding the product into a box at 5 ℃ for 1 hour, cooling to-40 ℃ and keeping the temperature at-40 ℃ for 1 hour, heating the product to-18 ℃ and keeping the temperature for 1 hour, cooling the product again and keeping the temperature at-35 ℃ for 1 hour, controlling the temperature of the plate layer at 10 ℃ and keeping the temperature for 15 hours until the frozen ice in the product sublimates, heating the product to the maximum allowable temperature of 30 ℃ and keeping the temperature until the freeze-drying is finished, recovering high vacuum, and keeping the temperature for more than 6 hours under the conditions of the maximum allowable temperature and the high vacuum, so that the residual moisture of the product reaches the requirement of less than 2%, and the freeze-drying is completed.
Through detection, 100.2g of finished product growth hormone is obtained by a 50L fermentation system in the embodiment, kex2 enzyme residues can be removed by a purification process, the removal rate is 99.99%, the purity of the recombinant human growth hormone is 99.6%, and the in vitro biological activity of the prepared recombinant human growth hormone is 3.98IU/mg according to the Nb2-11 cell proliferation method.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (4)
1. A method for preparing recombinant human growth hormone is characterized by comprising the following steps of constructing genetically engineered bacteria, culturing working seeds, culturing in a fermentation tank, recovering thalli, crushing thalli, washing inclusion bodies, dissolving the inclusion bodies, obtaining inclusion bodies comprising fusion proteins, performing ultrafiltration operation to obtain inclusion body concentrated solution, performing crude purification on the inclusion body concentrated solution by adopting an anion exchange chromatography to obtain an anion exchange chromatography sample, performing Kex2 enzyme digestion to obtain an enzyme digestion product, performing fine purification by adopting a weak anion exchange chromatography to obtain protein-containing eluent, performing ultrafiltration, sterilization filtration, freeze-drying and storage to obtain recombinant human growth hormone;
the fusion protein consists of a first peptide fragment and a second peptide fragment, wherein the first peptide fragment is target growth hormone, the amino acid sequence of the first peptide fragment is shown as SEQ ID NO. 1, the amino acid sequence of the second peptide fragment is shown as formula (I), and the N end of the first peptide fragment is connected with the C end of the second peptide fragment;
The amino acid sequence of the second peptide fragment is:
MAXB(I)
in the formula (I), M is methionine, A is RR, X is TG, B is PR;
The specific method for enzyme digestion of Kex2 comprises the steps of slowly adjusting the pH of an anion exchange chromatography sample to 8.0 by using 2M HCl, slowly adding protein diluent accounting for 1/100 of the total volume of the sample, calculating the total protein of the anion exchange chromatography sample according to the results of Lowry and RP-HPLC, calculating the mass of Kex2 enzyme according to the ratio of the total protein to Kex2 enzyme of 100:1-2500:1, adding Kex2 protease diluent buffer to the Kex2 enzyme with the calculated required amount to enable the Kex2 enzyme concentration to be 1mg/ml, adding the obtained Kex2 enzyme solution into a mixture of the anion exchange chromatography sample and the protein diluent, and carrying out enzyme digestion at room temperature;
The protein diluent is a solution containing 10-100 mM Tris and 200-500 mM CaCl 2, the pH value is 7.0-9.0, and the Kex2 protease diluent buffer is a solution containing 10-100 mM Tris and 2-5 mM CaCl 2, and the pH value is 8.0-9.0.
2. The method for preparing recombinant human growth hormone according to claim 1, wherein the anion exchange chromatography method comprises the specific steps of washing 3CV with a mobile phase A equilibrium chromatographic column, directly loading samples after balancing, wherein the loading flow rate is not more than 210cm/h, washing 3CV with a mobile phase A equilibrium chromatographic column 2CV and a mobile phase B, eluting weakly bound proteins, eluting target proteins with a mobile phase A equilibrium chromatographic column 2CV and an elution buffer, starting collection when UV is not less than 50mAU, stopping collection when UV is not less than 100mAU, merging samples, and obtaining anion exchange chromatographic samples, and temporarily storing at 4 ℃.
3. The method for preparing recombinant human growth hormone according to claim 2, wherein the mobile phase A is a solution of 10-50 mM Tris, the pH is 7-10, the mobile phase B is a solution of 10-50 mM Tris, 30-80 mM NaCl, the pH is 7-10, and the elution buffer is a solution of 10-50 mM Tris, 100-200 mM NaCl, the pH is 7-10.
4. The method for preparing recombinant human growth hormone according to claim 1, wherein the weak anion exchange chromatography comprises the specific steps of:
Pumping the enzyme-digested product into a container with proper size by adopting a peristaltic pump, pumping PB into the enzyme-digested product, diluting the enzyme-digested product to be less than 3ms/cm in electric conduction, carrying out the sample loading by using a mobile phase A equilibrium chromatographic column 3CV, carrying out the sample loading by using a sample loading flow rate not more than 130cm/h, washing 3CV by using 10% of mobile phase B, eluting 4CV by using the mobile phase A, then carrying out gradient elution, wherein the mobile phase A90%, the mobile phase B10%, the mobile phase A50% and the mobile phase B500% are started at the beginning, the collection is started when the UV is not less than 50mAU, the collection is stopped when the UV is less than 100mAU, and the sample is combined to collect protein-containing eluent;
the pH of the solution with the mobile phase A of 20-50 mM PB is 7-10;
the mobile phase B is a solution containing 20-50 mM PB and 100-500 mM NaCl, and the pH is 7-10.
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