CN112250753B - Method for free adsorption concentration of recombinant epidermal growth factor - Google Patents
Method for free adsorption concentration of recombinant epidermal growth factor Download PDFInfo
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- CN112250753B CN112250753B CN202011173896.5A CN202011173896A CN112250753B CN 112250753 B CN112250753 B CN 112250753B CN 202011173896 A CN202011173896 A CN 202011173896A CN 112250753 B CN112250753 B CN 112250753B
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- 101800003838 Epidermal growth factor Proteins 0.000 title claims abstract description 60
- 229940116977 epidermal growth factor Drugs 0.000 title claims abstract description 59
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 title claims abstract description 59
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- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 12
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- 239000003513 alkali Substances 0.000 description 1
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- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
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- 239000003102 growth factor Substances 0.000 description 1
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- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 230000006641 stabilisation Effects 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/02—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor with moving adsorbents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for concentrating recombinant epidermal growth factor by free adsorption, belonging to the technical field of biological medicine. The method for freely adsorbing and concentrating the recombinant epidermal growth factor in the fermentation liquor comprises the following steps: s1, preprocessing fermentation liquor of recombinant epidermal growth factor, adding reverse phase high performance liquid chromatography filler, stirring and free adsorption, enabling the recombinant epidermal growth factor to be adsorbed on the reverse phase high performance chromatography filler to form adsorption filler, removing upper layer liquor after the adsorption filler is settled, and collecting the adsorption filler; s2, eluting the adsorption filler by using an eluent, and collecting effluent, namely the recombinant EGF concentrate. The method has the characteristics of simple and rapid process, fewer steps, easy industrialized amplification and the like, and the product has higher activity and purity.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and relates to a method for free adsorption concentration of recombinant epidermal growth factor.
Background
Epidermal growth factor (Epidermal Growth Factor, EGF for short) is a class of polypeptide substances widely existing in mammals, and human epidermal growth factor (hEGF) was first purified from human urine in 1974. hEGF is composed of 53 amino acids, has a molecular weight of 6000 daltons, an isoelectric point of 4.6, and three pairs of disulfide bonds in the molecule, and is stable to physical and chemical factors such as acid, alkali, heat and the like.
hEGF is a multifunctional cell growth factor with a wide range of biological effects, which exert physiological effects by binding to hEGF receptors on cell membranes. Studies have shown that: the cell membranes of various cells such as epidermal cells, fibroblasts, endothelial cells, smooth muscle cells and the like all contain hEGF receptors, wherein the content of the epidermal cells is the highest. After approaching the cell, hEGF is combined with hEGF receptor on the cell membrane, so that a series of complex biochemical cascade reactions can be generated in the cell, RNA, DNA and protein synthesis can be increased, and finally, the growth and propagation of the cell can be promoted and the metabolism of the cell can be accelerated. hEGF has been used in medicine for the treatment of burns, ulcers, various wounds, corneal lesions, and the like. EGF can promote metabolism of normal epidermis cells, and can achieve the effects of whitening, anti-wrinkle and anti-aging by being added into the cosmetic skin care product.
The source of hEGF was mainly tissue and body fluid extraction before the 80 s of the 20 th century, and the application of hEGF is limited due to high extraction cost and low product purity caused by the extremely tiny content of hEGF in tissues and body fluids. After 80 s, with the development of genetic engineering technology, people succeed in producing hEGF by using escherichia coli, saccharomycetes and the like through genetic recombination technology, and a foundation is laid for industrially producing the genetic recombination human epidermal growth factor (rhEGF). However, the rhEGF fermentation broth prepared by the current gene recombination technology has low product concentration, complex extraction and purification processes and low hEGF activity.
Disclosure of Invention
The invention aims at solving the problems existing in the prior art, and provides a method for freely adsorbing and concentrating recombinant epidermal growth factor in fermentation liquor, which has the characteristics of simple and rapid process, few steps, easy industrialized amplification and the like, has higher product activity and purity, solves the problems of overlong sample injection time and increased column pressure caused by the separation of a large-volume fermentation liquor into a chromatographic column, and fully prepares the sample before further extraction and separation, and can directly separate a concentrated sample prepared by the method by using a gel molecular sieve.
The aim of the invention can be achieved by the following technical scheme:
a method for freely adsorbing and concentrating recombinant epidermal growth factor in fermentation liquor comprises the following steps:
s1, preprocessing fermentation liquor of recombinant epidermal growth factor, adding reverse phase high performance liquid chromatography filler, stirring and free adsorption, enabling the recombinant epidermal growth factor to be adsorbed on the reverse phase high performance chromatography filler to form adsorption filler, removing upper layer liquor after the adsorption filler is settled, and collecting the adsorption filler;
s2, eluting the adsorption filler by using an eluent, and collecting effluent, namely the recombinant EGF concentrate.
Preferably, the pretreatment in step S1 is performed by,
1) Acid precipitation: regulating the pH of the recombinant epidermal growth factor fermentation liquor to 2-3 by using acid liquor, standing, performing solid-liquid separation, discarding solid precipitate, and reserving supernatant fermentation liquor;
2) Sodium chloride with the final concentration of 0.6-0.8 mol/L is added into the supernatant fermentation broth before the reversed-phase high performance liquid chromatography packing is added.
Preferably, the acid solution comprises one or two of dilute hydrochloric acid and trifluoroacetic acid.
In the pretreatment process, the PH of the recombinant EGF fermentation liquor is controlled to be 2-3 by acid precipitation (isoelectric point precipitation method), so that most proteins of the recombinant EGF fermentation liquor can be removed, and the isoelectric points of most hybrid proteins of the recombinant EGF fermentation liquor are all within the acid range of PH 3+/-0.5.
According to the invention, sodium chloride with the final concentration of 0.6-1.0 mol/L is added into the supernatant fermentation liquor in the later pretreatment stage, so that the hydrophobicity of the recombinant epidermal growth factor can be increased, the surface of the recombinant epidermal growth factor protein is exposed out of hydrophobic groups, and the adsorptivity and the retentivity of the recombinant epidermal growth factor on reversed phase chromatographic packing are further improved.
Preferably, the stirring free adsorption in the step S1 is magnetic stirring until the reversed-phase high performance liquid chromatography filler is suspended, stirring is continued for 12-18 min, and then the filler to be adsorbed is kept stand in an environment of 2-8 ℃ for precipitation.
Preferably, the recombinant epidermal growth factor fermentation broth in the step S1 is secreted and expressed by adopting escherichia coli, and isopropyl-beta-D-thiogalactoside (IPTG) is added to induce the secretion of the epidermal growth factor into the extracellular fermentation broth.
Preferably, the reversed-phase high performance liquid chromatography packing in step S1 is a silica gel matrix, and the surface of the silica gel matrix is bonded with reversed-phase C4 groups.
Preferably, the granularity of the reversed-phase high-performance liquid chromatography packing in the step S1 is 16-25 μm.
The invention selects the reversed phase high performance liquid chromatography filling material with the surface bonded with the reversed phase C4 group to adsorb the recombinant epidermal growth factor in the fermentation liquor, the C4 group has proper retention strength on the recombinant epidermal growth factor, and the recombinant epidermal growth factor can be eluted by adopting a solvent with lower concentration as eluent in the follow-up process, thereby being beneficial to the activity stabilization of the recombinant epidermal growth factor. If the retention of the recombinant EGF is too strong by adopting C8 groups and C18 groups, the recombinant EGF can be eluted by adopting a high-concentration organic solvent, and the recombinant EGF can be caused by the high-concentration organic solvent.
According to the invention, the granularity of the chromatographic packing is controlled, so that the packing can be quickly, effectively and naturally settled after recombining the epidermal growth factor in the suspension adsorption fermentation liquor, the next operation can be carried out without waiting for too long, and the supernatant is poured out for elution. If the particles are too small, the sedimentation time is long, and the elution speed is slow.
Preferably, the eluent in step S2 comprises the following components: 18 to 25% by volume of ethanol and 0.07 to 0.13% by volume of trifluoroacetic acid (TFA).
Preferably, the elution in step S2 is performed in an elution device comprising a buchner funnel, a suction flask and a circulating vacuum pump.
Further preferably, the elution in step S2 is to place the collected adsorption packing in a buchner funnel, and to feed the eluent into the buchner funnel while evacuating.
The elution device is formed by combining a Buchner funnel, a suction flask and a circulating vacuum pump, adding the collected adsorption filler into the Buchner funnel, vacuumizing, flowing and removing liquid, and collecting effluent liquid to finish concentration.
Compared with the prior art, the invention has the following beneficial effects: .
(1) The whole adsorption-elution flow is rapid, the operation is simple, the concentration speed is high, the efficiency is high, the cost is low, and the method is suitable for industrialized mass production;
(2) The method has the purification function while concentrating, thereby greatly improving the purity of the final product;
(3) According to the invention, the recombinant epidermal growth factor can be eluted from the chromatographic packing by preferably selecting the reversed-phase high-performance liquid chromatographic packing bonded with the C4 group and using a solvent with lower concentration as an eluent, so that the activity of the recombinant epidermal growth factor is ensured;
(4) According to the invention, the components of the eluent are optimized, so that the recombinant epidermal growth factor can be rapidly eluted from the chromatographic packing;
(5) The invention improves the sedimentation velocity of the chromatographic packing by controlling the particle size range of the chromatographic packing.
Detailed Description
The following are specific examples of the present invention, and the technical solutions of the present invention are further described, but the present invention is not limited to these examples.
Example 1
The method for free adsorption concentration of recombinant EGF in fermentation broth in this embodiment comprises the following steps:
(1) The escherichia coli is taken as a genetic engineering bacterium to secrete and express the recombinant epidermal growth factor to prepare a recombinant epidermal growth factor fermentation broth, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into the extracellular fermentation broth;
(2) Pretreatment of fermentation broth of recombinant epidermal growth factor: regulating pH of the recombinant EGF fermentation broth to 2.5 with acid solution (dilute hydrochloric acid, trifluoroacetic acid), standing, performing solid-liquid separation, discarding solid precipitate, retaining supernatant fermentation broth, and adding sodium chloride with final concentration of 0.7mol/L into supernatant fermentation broth;
(3) Stirring and adsorbing: adding reverse-phase high-performance liquid chromatography filler into the pretreated supernatant fermentation liquor, wherein the reverse-phase high-performance liquid chromatography filler is a silica gel matrix, reverse-phase C4 groups are bonded on the surface of the reverse-phase high-performance liquid chromatography filler, the granularity is 20 mu m, stirring free adsorption is magnetic stirring until the reverse-phase high-performance liquid chromatography filler is suspended, continuing stirring for 15min, standing in a 5 ℃ environment until the filler to be adsorbed is precipitated, adsorbing the recombinant epidermal growth factor on the reverse-phase high-performance liquid chromatography filler to form an adsorption filler, removing the upper layer liquid after the adsorption filler is settled, and collecting the adsorption filler;
(4) Placing the collected adsorption filling material in a buchner funnel of an elution device (the elution device comprises a buchner funnel, a suction filtration bottle and a circulating vacuum pump), and simultaneously, feeding eluent into the buchner funnel to elute the adsorption filling material while vacuumizing, and collecting the effluent, namely, recombinant epidermal growth factor concentrated solution, wherein the eluent comprises the following components: 20v% ethanol, 0.1v% TFA.
Measuring activity of the concentrated solution by cell proliferation method (MTT method), calculating to obtain concentrated solution with activity recovery rate of 87% and concentration specific activity of 3×10 5 The specific activity of the pure product is 1x10 per mg 6 /mg。
Example 2
The method for free adsorption concentration of recombinant EGF in fermentation broth in this embodiment comprises the following steps:
(1) The escherichia coli is taken as a genetic engineering bacterium to secrete and express the recombinant epidermal growth factor to prepare a recombinant epidermal growth factor fermentation broth, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into the extracellular fermentation broth;
(2) Pretreatment of fermentation broth of recombinant epidermal growth factor: regulating pH of the recombinant EGF fermentation broth to 2 with acid solution (dilute hydrochloric acid, trifluoroacetic acid), standing, performing solid-liquid separation, discarding solid precipitate, retaining supernatant fermentation broth, and adding sodium chloride with final concentration of 0.8mol/L into the supernatant fermentation broth;
(3) Stirring and adsorbing: adding reverse-phase high-performance liquid chromatography filler into the pretreated supernatant fermentation liquor, wherein the reverse-phase high-performance liquid chromatography filler is a silica gel matrix, reverse-phase C4 groups are bonded on the surface of the reverse-phase high-performance liquid chromatography filler, the granularity is 16 mu m, stirring free adsorption is magnetic stirring until the reverse-phase high-performance liquid chromatography filler is suspended, continuing stirring for 18min, standing in a 2 ℃ environment until the filler to be adsorbed is precipitated, adsorbing the recombinant epidermal growth factor on the reverse-phase high-performance liquid chromatography filler to form an adsorption filler, removing the upper layer liquid after the adsorption filler is settled, and collecting the adsorption filler;
(4) Placing the collected adsorption filling material in a buchner funnel of an elution device (the elution device comprises a buchner funnel, a suction filtration bottle and a circulating vacuum pump), and washing the adsorption filling material by adding an eluent into the buchner funnel while vacuumizing, and collecting the effluent, namely a recombinant epidermal growth factor concentrated solution, wherein the eluent comprises the following components: 25v% ethanol, 0.13v% TFA.
Measuring activity of the concentrated solution by cell proliferation method (MTT method), calculating to obtain concentrated solution with activity recovery rate of recombinant EGF of 85% and concentration specific activity of 2.8x10 5 Per mg, the specific activity of the pure product is 0.9x10 6 /mg。
Example 3
The method for free adsorption concentration of recombinant EGF in fermentation broth in this embodiment comprises the following steps:
(1) The escherichia coli is taken as a genetic engineering bacterium to secrete and express the recombinant epidermal growth factor to prepare a recombinant epidermal growth factor fermentation broth, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into the extracellular fermentation broth;
(2) Pretreatment of fermentation broth of recombinant epidermal growth factor: regulating the pH of the recombinant epidermal growth factor fermentation liquor to 3 by dilute hydrochloric acid, standing, performing solid-liquid separation, discarding solid precipitate, reserving supernatant fermentation liquor, and adding sodium chloride with the final concentration of 06mol/L into the supernatant fermentation liquor;
(3) Stirring and adsorbing: adding reverse-phase high-performance liquid chromatography filler into the pretreated supernatant fermentation liquor, wherein the reverse-phase high-performance liquid chromatography filler is a silica gel matrix, reverse-phase C4 groups are bonded on the surface of the reverse-phase high-performance liquid chromatography filler, the granularity is 25 mu m, stirring free adsorption is magnetic stirring until the reverse-phase high-performance liquid chromatography filler is suspended, continuing stirring for 12min, standing in an environment of 8 ℃ until the filler to be adsorbed is precipitated, adsorbing the recombinant epidermal growth factor on the reverse-phase high-performance liquid chromatography filler to form an adsorption filler, removing the upper layer liquid after the adsorption filler is settled, and collecting the adsorption filler;
(4) Placing the collected adsorption filling material in a buchner funnel of an elution device (the elution device comprises a buchner funnel, a suction filtration bottle and a circulating vacuum pump), and washing the adsorption filling material by adding an eluent into the buchner funnel while vacuumizing, and collecting the effluent, namely a recombinant epidermal growth factor concentrated solution, wherein the eluent comprises the following components: 25v% ethanol, 0.07v% TFA.
Measuring activity of the concentrated solution by cell proliferation method (MTT method), calculating to obtain concentrated solution with activity recovery rate of 83% and concentration specific activity of 2.7x10 5 The specific activity of the pure product is 0.8x10 per mg 6 /mg。
Example 4
The method for free adsorption concentration of recombinant EGF in fermentation broth in this embodiment comprises the following steps:
(1) The escherichia coli is taken as a genetic engineering bacterium to secrete and express the recombinant epidermal growth factor to prepare a recombinant epidermal growth factor fermentation broth, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into the extracellular fermentation broth;
(2) Pretreatment of fermentation broth of recombinant epidermal growth factor: regulating the pH of the recombinant epidermal growth factor fermentation liquor to 2.5 by using trifluoroacetic acid, standing, performing solid-liquid separation, discarding solid precipitate, reserving supernatant fermentation liquor, and adding sodium chloride with the final concentration of 0.7mol/L into the supernatant fermentation liquor;
(3) Stirring and adsorbing: adding reverse-phase high-performance liquid chromatography filler into the pretreated supernatant fermentation liquor, wherein the reverse-phase high-performance liquid chromatography filler is a silica gel matrix, reverse-phase C4 groups are bonded on the surface of the reverse-phase high-performance liquid chromatography filler, the granularity is 20 mu m, stirring free adsorption is magnetic stirring until the reverse-phase high-performance liquid chromatography filler is suspended, continuing stirring for 16min, standing in a 6 ℃ environment to precipitate the filler to be adsorbed, adsorbing the recombinant epidermal growth factor on the reverse-phase high-performance liquid chromatography filler to form an adsorption filler, removing the upper layer liquid after the adsorption filler is settled, and collecting the adsorption filler;
(4) Placing the collected adsorption filling material in a buchner funnel of an elution device (the elution device comprises a buchner funnel, a suction filtration bottle and a circulating vacuum pump), and washing the adsorption filling material by adding an eluent into the buchner funnel while vacuumizing, and collecting the effluent, namely a recombinant epidermal growth factor concentrated solution, wherein the eluent comprises the following components: 20v% ethanol, 0.1% TFA.
Measuring activity of the concentrated solution by cell proliferation method (MTT method), calculating to obtain concentrated solution with activity recovery rate of 87% and concentration specific activity of 3.1x10 5 The specific activity of the pure product is 1x10 per mg 6 /mg。
Comparative example 1
Adsorption treatment was performed using a silica gel filler having a reversed phase C8 group bonded to the surface, and the same procedure as in example 1 was repeated.
The activity of the collected concentrated solution is measured by adopting a cell proliferation method (MTT method), and the activity recovery rate of the recombinant EGF in the collected concentrated solution is only 46% through calculation, because the surfactant is difficult to be eluted smoothly by adopting the eluent with the same concentration when the silica gel filler with the reverse phase C8 group bonded on the surface is adopted for adsorption treatment.
Comparative example 2
Adsorption treatment was performed using a silica gel filler having a reversed phase C18 group bonded to the surface, and the same procedure as in example 1 was repeated.
The activity of the collected concentrated solution is measured by a cell proliferation method (MTT method), and the activity recovery rate of the recombinant EGF in the collected concentrated solution is only 38% through calculation, and the reason is that the surfactant is difficult to be eluted smoothly by adopting the eluent with the same concentration when the silica gel filler with the reverse phase C18 group bonded on the surface is adopted for adsorption treatment.
The point values in the technical scope of the present invention are not exhaustive, and the new technical solutions formed by equivalent substitution of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the present invention; meanwhile, in all the listed or unrecited embodiments of the present invention, each parameter in the same embodiment represents only one example of the technical scheme (i.e. a feasibility scheme), and no strict coordination and limitation relation exists between each parameter, wherein each parameter can be replaced with each other without violating axiom and the requirement of the present invention, except what is specifically stated.
The technical means disclosed by the scheme of the invention is not limited to the technical means disclosed by the technical means, and also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.
Claims (3)
1. A method for freely adsorbing and concentrating recombinant epidermal growth factor in fermentation liquor, which is characterized by comprising the following steps:
s1, preprocessing fermentation liquor of recombinant epidermal growth factor, adding reverse phase high performance liquid chromatography filler, stirring and free adsorption, enabling the recombinant epidermal growth factor to be adsorbed on the reverse phase high performance chromatography filler to form adsorption filler, removing upper layer liquor after the adsorption filler is settled, and collecting the adsorption filler;
s2, eluting the adsorption filler by using an eluent, and collecting effluent, namely a recombinant epidermal growth factor concentrated solution;
the preprocessing step in step S1 is that,
1) Acid precipitation: regulating pH of the recombinant EGF fermentation broth to 2.5 with acid liquor, standing, performing solid-liquid separation, discarding solid precipitate, retaining supernatant fermentation broth,
2) Adding sodium chloride with the final concentration of 0.7mol/L into the supernatant fermentation broth before adding the reversed-phase high performance liquid chromatography filler;
the reversed phase high performance liquid chromatography filler in the step S1 is a silica gel matrix, the surface of which is bonded with reversed phase C4 groups, and the granularity of which is 20 mu m;
s1, the recombinant epidermal growth factor fermentation broth adopts escherichia coli to secrete and express, and isopropyl-beta-D-thiogalactoside (IPTG) is added to induce and secrete the epidermal growth factor into the extracellular fermentation broth;
the eluent in the step S2 comprises the following components: 20v% ethanol, 0.1v% trifluoroacetic acid (TFA).
2. The method for freely adsorbing and concentrating recombinant epidermal growth factor in fermentation broth according to claim 1, wherein in step S1, the stirring and free adsorption is magnetic stirring until the reverse phase high performance liquid chromatography packing is suspended, stirring is continued for 15min, and then the packing to be adsorbed is allowed to settle in an environment of 5 ℃.
3. The method of claim 1, wherein the eluting in step S2 is performed in an eluting device comprising a buchner funnel, a suction flask, and a circulating vacuum pump.
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