CN112250756A - Purification method of recombinant human growth hormone protein - Google Patents
Purification method of recombinant human growth hormone protein Download PDFInfo
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- 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
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- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
A method for purifying recombinant human growth hormone protein. The invention provides a purification method of recombinant hGH protein, which comprises the following steps: through constructing yeast recombinant pPICZ alpha A-hGH-SMD1168, the recombinant is fermented under proper conditions, and methanol is used for inducing secretion to express the recombinant hGH protein with the complete amino acid sequence consistent with that of the human hGH protein. And filtering the supernatant of the fermentation liquid by using a 300kD membrane, intercepting the expressed recombinant hGH protein, passing the intercepted liquid through a molecular sieve Sephadex G200, and efficiently separating the target protein to obtain the recombinant hGH protein with the purity of more than 95%. Solves the problem of difficult purification of the recombinant protein without the label.
Description
Technical Field
The invention belongs to the field of bioengineering genes, and relates to a method for purifying recombinant human growth hormone eukaryotic expression.
Background
Human Growth Hormone (hereinafter abbreviated as hGH) contains 191 amino acid residues, has a molecular weight of 21.7 kDa and an isoelectric point of 4.9, is a single peptide chain protein Hormone secreted by anterior pituitary eosinophils, is the most important Hormone for promoting Growth after birth of Human beings, has multiple functions of regulating Growth and metabolism of Human bodies and the like, and plays an important role in the Growth and development process of the Human bodies. hGH promotes bone, visceral and systemic growth, protein synthesis and fat and mineral metabolism. Since the advent of hGH gene engineering products, the indications have expanded to adult growth hormone deficiency, Turner's syndrome, AIDS wasting, large area wound healing, and other symptoms. With the continuous and deep clinical research, the growth hormone has good curative effect in the aspects of anti-aging, osteoporosis and cardiovascular disease treatment.
The global growth hormone market is huge, at present, recombinant human growth hormone expressed in escherichia coli by using a DNA recombination technology is utilized, but exogenous protein expressed by escherichia coli recombination is added with an initiation codon at the N end of protein, compared with natural protein, the initiation codon is increased by initiating amino acid-methionine, sometimes for convenience of purification, a certain label is added into a protein sequence, and an antibody is easily generated after the growth hormone is applied; therefore, human growth hormone without Met or tag at the N-terminus is being studied. There are two methods for producing Met-free human growth hormone by using Escherichia coli, one is to remove Met molecule by enzyme method in the process of processing and purifying after producing Met-human growth hormone by intracellular expression; the other method is to use a secretion type vector to directly transfer the coding sequence of the mature protein of the human growth hormone into a high-expression high-secretion promoter and a signal sequence, and cut off a signal peptide in the secretion process to ensure that the expressed human growth hormone is accumulated in the periplasm of the cells. The pichia pastoris, as a lower eukaryote, has the characteristic of efficient secretory expression, and can obtain the Met-free human growth hormone by utilizing the characteristic of cutting of a signal peptide per se. In order not to produce antibodies, the same sequence as native hGH without the tag and Met is required, which presents difficulties in purification of hGH.
Disclosure of Invention
In order to overcome the above disadvantages, the inventors have optimized the hGH gene in the Pichia pastoris expression system and searched for purification of the expressed hGH, and surprisingly found that in this expression system, hGH secreted and expressed by yeast could be retained by an ultrafiltration membrane with a pore size of 300kD, and most of the protein secreted and expressed by yeast could be removed and desalted by a 300kD ultrafiltration membrane. And then the hGH protein with the purity of more than 96% can be obtained by one-step molecular sieve. The specific purification steps of hGH protein are as follows:
a method for purification of recombinant hGH protein comprising the steps of:
1) fermenting and expressing recombinant hGH protein by using yeast recon;
2) and (3) treating fermentation liquor: carrying out solid-liquid separation on the fermentation liquor obtained in the step 1), taking the supernatant, filtering the supernatant through a 2um filter membrane, and collecting filtrate;
3) and (3) ultrafiltration: ultrafiltering the filtrate obtained in the step 2) by using a 300kD ultrafiltration membrane, and taking trapped fluid;
4) passing the retentate obtained in step 3) through a molecular sieve to obtain purified hGH protein.
Preferably, the step 1) of fermenting and expressing the recombinant hGH protein by the yeast recombinant comprises the following steps: A. constructing pPICZ alpha A-hGH recombinant vector containing the gene sequence coding hGH protein; B. b, linearizing the pPICZ alpha A-hGH recombinant vector in the step A, and transferring the linearized pPICZ alpha A-hGH recombinant vector into an SMD1168 pichia pastoris strain to obtain a yeast recombinant pPICZ alpha A-hGH-SMD 1168; C. the yeast recon pPICZ alpha A-hGH-SMD1168 is cultured and supplemented at proper time, and when the wet weight of yeast is more than 150 mg/ml, methanol is induced for 36-60 h.
The sequence of the gene encoding the hGH protein is preferably as shown in SEQ ID NO: 1, and the DNA sequence of the pPICZ alpha A-hGH vector is shown as SEQ ID: 1.
Preferably, the culture conditions of the yeast recon pPICZ alpha A-hGH-SMD1168 are as follows: the temperature is 30 ℃, the pH value is 5.0, and the dissolved oxygen is controlled to be 25-45%; the methanol induction conditions were: the temperature is 28 ℃, the pH value is 5.6, and the dissolved oxygen is controlled to be 25-45%.
Preferably, the solid-liquid separation in the step 2) is to centrifuge the fermentation liquid obtained in the step 1) at 5000rpm for 5-10 min.
Preferably, in step 3), the ultrafiltration is tangential ultrafiltration.
As a preferred step 3) the ultrafiltration comprises the following steps: adjusting the pH of the filtrate obtained in the step 2) to 6.9-7.5, and performing ultrafiltration on the filtrate through a 300kD tangential ultrafiltration membrane under the pressure of 0.1-0.2 MPa, adding ddH2O with the volume of 5-10 times of that of the original volume when the trapped fluid is 1/8-1/15 of the original volume, and continuing ultrafiltration until the original volume is 1/8-1/15; repeating the steps for 3-8 times.
Preferably, in step 4), the molecular sieve is: sephadex G200.
The preferable step 4) specifically comprises the following steps: balancing 2-3 column volumes of the molecular sieve with deionized water, concentrating a sample, eluting with deionized water, detecting with a nucleic acid protein instrument, collecting a human growth hormone protein peak, concentrating, and freeze-drying.
The invention has the beneficial effects that: the vast majority of proteins less than 100 kD in the fermentation supernatant can be removed by a 300kD tangential flow ultrafiltration membrane package, so that the subsequent purification steps are greatly simplified, and the separation efficiency and the protein purity of the proteins are improved. Although the molecular weight of hGH is only 21.7kD, it was unexpected that recombinant hGH obtained by our fermentation could permeate 300kD of ultrafiltration tubes, but not 300kD of tangential flow ultrafiltration membranes.
Drawings
FIG. 1 is an electrophoretogram of 300kD filter ultrafiltration proteins, where M is protein molecular weight Marker, 1 is 300kD tangential flow filter ultrafiltration retentate, and 2 is 300kD tangential flow filter ultrafiltration permeate. The arrow indicates the expressed recombinant hGH.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: obtaining recombinant yeast capable of expressing hGH.
1, constructing pPICZ alpha A-hGH recombinant plasmid containing an hGH coding gene by artificial synthesis and molecular cloning technology, wherein the DNA sequence of the hGH coding gene is shown as SEQ ID: 1, and the DNA sequence of the pPICZ alpha A-hGH recombinant plasmid is shown as SEQ ID: 2.
2 the pPICZ alpha A-hGH recombinant plasmid of step 1 was linearized with the restriction enzyme SacI and recovered. And (3) electrically transferring the recovered linearized plasmid into a SMD1168 pichia pastoris strain to obtain a recombinant strain pPICZ alpha A-hGH-SMD 1168. Mixing 10 μ g of the linearized pPICZ α A-hGH plasmid with 80 μ L of Pichia pastoris SMD1168 competent cells, transferring to an electric conversion cup with ice pre-cooling of 0.2 cm, and keeping the voltage at 1 kV; a capacitance of 30 μ F; a resistor is 200 omega; and (3) electric shock is carried out for 4-10 milliseconds, 1 mL of ice-precooled 1 mol/L sorbitol solution is added to uniformly mix the thalli, and the thalli are placed in a water bath kettle at the temperature of 30 ℃ and subjected to water bath for 30 min to complete conversion. And (3) screening the transformed yeast by using a selective culture medium to obtain a positive clone pPICZ alpha A-hGH-SMD1168 recombinant strain.
Example 2: and (3) fermenting the recombinant human growth hormone protein.
The selected pPICZ alpha A-hGH-SMD1168 recombinant strain is inoculated into 300 mL BMGY (yeast extract 10 g/L, peptone 20 g/L, K2HPO 43 g/L, KH2PO411.8 g/L, YNB 13.4 g/L, biotin 4X 10-4 g/L, glycerol 10 g/L) culture medium, shaking culture is carried out at 30 ℃ for 24 hours, the recombinant strain is transferred into a 5L fermentation tank filled with 3L BMGY culture medium as seeds, the temperature is set to be 30 ℃, the pH value is 5.0, the dissolved oxygen is controlled to be 25-45 percent, when the dissolved oxygen rises suddenly, the glycerol in the basic salt culture medium is predicted to be exhausted, 10-20 mL/h/L glycerol with the mass percent concentration of 50 percent (preferably 12 mL/h/L), the trace elements PTM1 with the mass percent concentration of 12 mL/L (the trace elements in PTM1 contain CuSO4. H2O 6 g/L in the PTM 1), 0.08 g of NaI, 0.08 g of MnSO4.H2O 3, 0.2 g of Na2MoO4.H2O, H3BO30.02 g, H2SO 45 ml, 0.5 g of CoCl2.6H2O, 220 g of ZnCl, 75 g of FeSO4.7H2O and 0.2 g of biotin) to maintain the dissolved oxygen at 25-45 percent. Stopping feeding when the wet bacteria weight of the fermentation liquid reaches 150-. And (3) adjusting the rotating speed, the tank pressure and the ventilation quantity to enable the dissolved oxygen to be more than 20%, and performing induced fermentation for 36-48 hours. The induction temperature is 28 ℃, and the pH value is 5-5.5.
Example 3: and (5) purifying the recombinant human growth hormone.
(1) And (4) performing ultrafiltration. After fermentation, the fermentation liquor is centrifugally separated at 5000rpm for 10min, and supernatant is taken. Filtering with 2um filter membrane, and collecting filtrate. Adjusting the pH value of the filtrate to 6.9-7.5 (preferably 7) by using sodium hydroxide, and performing ultrafiltration by using a tangential flow ultrafiltration membrane package with the molecular cut-off of 300kD, wherein the ultrafiltration pressure is 0.1-0.2 MPa: when the volume of the trapped fluid is reduced to 1/8-1/15 (preferably 1/10) of the original volume, adding 5-10 times (preferably 5) of ddH2O, and continuously performing ultrafiltration until the original volume is 1/8-1/15; repeating for 3-8 times (preferably 5 times); the retentate was collected. A large amount of protein generated by fermentation of pichia pastoris can be removed by the one-step ultrafiltration method, and desalting is carried out at the same time, so as to obtain the human growth hormone protein concentrated solution.
The results of SDS-PAGE electrophoresis of the ultrafiltrate, i.e., the growth hormone protein concentrate and the permeate, are shown in FIG. 1 (wherein M is protein molecular weight Marker, the first band has a molecular weight of 15 kD from bottom to top, the second band has a molecular weight of 35 kD, 300kD membrane ultrafiltrate retentate in lane 1, 300kD membrane ultrafiltrate permeate in lane 2, and expressed recombinant hGH as indicated by the arrow). We surprisingly found that the recombinant expressed human growth hormone protein can not penetrate through a tangential flow ultrafiltration membrane with the molecular cut-off of 300kD, but most of the protein with the molecular weight of less than 300kD secreted and expressed by yeast in the supernatant of the fermentation liquor penetrates through the membrane, so that the step not only concentrates and desalts, but also removes most of the foreign protein in the supernatant of the fermentation liquor in one step, and the protein purity can reach more than 80%. The purification steps are greatly simplified.
(2) And (5) performing molecular sieve chromatography. And (3) separating the concentrated solution of the human growth hormone by chromatography by using a Sephadex G200 molecular sieve. Balancing 2-3 column volumes with deionized water, concentrating a sample, wherein the sample loading amount is not more than one tenth of the column volume, the flow rate is 10 mL/min, eluting with deionized water, detecting with a nucleic acid protein instrument, collecting a protein peak of the human growth hormone, concentrating, and freeze-drying to obtain the human growth hormone.
And carrying out mass spectrum detection on the obtained recombinant human growth hormone to determine the recombinant human growth hormone as a human growth hormone coding sequence. The purity of the recombinant human growth hormone purified by the method can reach more than 96%.
Most of the hetero protein in the trapped fluid after ultrafiltration in the step 1 is high molecular weight protein, and the molecular weight of hGH is only 21.7kD, and the molecular weight difference with the hetero protein is large, so that the recombinant expressed hGH protein can be effectively separated by a one-step molecular sieve.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention.
Sequence listing
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tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960
tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020
agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080
tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140
tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1200
ttttccaact attcctttgt ctagattgtt cgataacgct atgttgagag ctcatagatt 1260
gcaccaattg gcttttgata cttaccaaga attcgaagag gcttacatcc caaaggagca 1320
aaagtactct ttcttgcaaa accctcaaac ttctttgtgt ttctctgaat ctattccaac 1380
tccttctaac agagaagaga ctcaacaaaa gtctaatttg gaattgttga gaatctcttt 1440
gttgttgatc caatcttggt tggagccagt tcaatttttg agatctgttt tcgctaactc 1500
tttggtttat ggtgcttctg attctaacgt ttacgatttg ttgaaggatt tggaagaggg 1560
tattcaaact ttgatgggta gattggagga tggttctcct agaactggtc aaatttttaa 1620
gcaaacttac tctaagttcg atactaactc tcataacgat gatgctttgt tgaaaaacta 1680
cggtttgttg tactgtttca gaaaggatat ggataaggtt gaaactttct tgagaatcgt 1740
tcaatgtaga tctgttgagg gttcttgtgg tttttaagaa gctgaattca cgtggcccag 1800
ccggccgtct cggatcggta cctcgagccg cggcggccgc cagctttcta gaacaaaaac 1860
tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt 1920
gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt 1980
gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt 2040
tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct 2100
tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg 2160
ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag 2220
tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat agcttcaaaa 2280
tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac 2340
ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta gggtgtcgtt 2400
aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg 2460
tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt tttttttagt 2520
ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct tcaatttctc 2580
aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg ttcattagaa 2640
agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata 2700
tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg 2760
ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg 2820
ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc 2880
tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg 2940
tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg 3000
acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc 3060
tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc 3120
gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc 3180
atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa 3240
aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta 3300
gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac 3360
gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta 3420
atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 3480
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 3540
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3600
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3660
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 3720
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3780
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3840
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3900
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 3960
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 4020
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 4080
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 4140
tcacgttaag ggattttggt catgagatc 4169
Claims (9)
1. A process for the purification of recombinant hGH protein, characterized in that it comprises the steps of:
1) fermenting and expressing recombinant hGH protein by using yeast recon;
2) and (3) treating fermentation liquor: carrying out solid-liquid separation on the fermentation liquor obtained in the step 1), taking the supernatant, filtering the supernatant through a 2um filter membrane, and collecting filtrate;
3) and (3) ultrafiltration: ultrafiltering the filtrate obtained in the step 2) by using a 300kD ultrafiltration membrane, and taking trapped fluid;
4) ion exchange chromatography: and (3) passing the trapped fluid obtained in the step 3) through a Q ion exchange column to obtain the purified hGH protein.
2. The method for purifying recombinant hGH protein according to claim 1, wherein the step 1) of expressing recombinant hGH protein by yeast recombinant fermentation comprises the steps of: A. constructing pPICZ alpha A-hGH vector containing the gene sequence coding hGH protein; B. b, linearizing the pPICZ alpha A-hGH vector in the step A, and transferring the linearized pPICZ alpha A-hGH vector into an SMD1168 pichia pastoris strain to obtain a yeast recombinant pPICZ alpha A-hGH-SMD 1168; C. and culturing and timely supplementing the yeast recon pPICZ alpha A-hGH-SMD1168, and inducing for 36-60 h by using methanol when the wet weight of the yeast is more than 150 mg/ml.
3. The method for purifying recombinant hGH protein according to claim 2, wherein the DNA sequence of the gene encoding hGH protein is shown in SEQ ID: 1, and the DNA sequence of pPICZalphaA-hGH vector is shown in SEQ ID: 2.
4. The method for purifying recombinant hGH protein according to claim 2, wherein the culture conditions of the yeast recombinant pPICZ α A-hGH-SMD1168 are: the temperature is 30 ℃, the pH value is 5.0, and the dissolved oxygen is controlled to be 25-45%; the methanol induction conditions were: the temperature is 28 ℃, the pH value is 5.6, and the dissolved oxygen is controlled to be 25-45%.
5. The method for purifying recombinant hGH protein according to claim 1, wherein the solid-liquid separation in step 2) is performed by centrifuging the fermentation broth obtained in step 1) at 5000rpm for 5-10 min.
6. The process for the purification of recombinant hGH protein according to claim 1, wherein the ultrafiltration of step 3) is tangential ultrafiltration.
7. The process for the purification of recombinant hGH protein according to claim 1, wherein the ultrafiltration of step 3) comprises the steps of: adjusting the pH of the filtrate obtained in the step 2) to 6.9-7.5, and performing ultrafiltration with 300kD tangential ultrafiltration membrane under the pressure of 0.1-0.2 MPa when the trapped fluid is 1/8E1/15, adding ddH with the volume 5-10 times that of the mixture2Continuously performing ultrafiltration until the original volume is 1/8-1/15; repeating the steps for 3-8 times.
8. The process for the purification of recombinant hGH protein of claim 1, wherein in step 4) the molecular sieve is: sephadex G200.
9. The method for purifying recombinant hGH protein according to claim 1, wherein the step 4) comprises the following steps: balancing 2-3 column volumes of the molecular sieve with deionized water, concentrating a sample, eluting with deionized water, detecting with a nucleic acid protein instrument, collecting a human growth hormone protein peak, concentrating, and freeze-drying.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN111041033A (en) * | 2018-10-12 | 2020-04-21 | 江苏众红生物工程创药研究院有限公司 | Recombinant human growth hormone and eukaryotic system expression method thereof |
| CN111647607A (en) * | 2020-07-21 | 2020-09-11 | 浙江理工大学绍兴生物医药研究院有限公司 | Method for efficiently expressing and secreting human growth hormone by using escherichia coli |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN111041033A (en) * | 2018-10-12 | 2020-04-21 | 江苏众红生物工程创药研究院有限公司 | Recombinant human growth hormone and eukaryotic system expression method thereof |
| CN111647607A (en) * | 2020-07-21 | 2020-09-11 | 浙江理工大学绍兴生物医药研究院有限公司 | Method for efficiently expressing and secreting human growth hormone by using escherichia coli |
Non-Patent Citations (1)
| Title |
|---|
| 闫立论: "重组人生长激素(rhGH)的毕赤酵母工程菌构建、表达、分离纯化及其生物学作用研究", 中国优秀硕士学位论文全文数据库 基础科学辑, no. 02, pages 006 - 204 * |
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