CN112209999B - Method for quickly separating pigment in recombinant epidermal growth factor fermentation liquor - Google Patents
Method for quickly separating pigment in recombinant epidermal growth factor fermentation liquor Download PDFInfo
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- 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 94
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- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 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
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
-
- 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
-
- 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)
- Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for quickly separating pigment in recombinant epidermal growth factor fermentation liquor, belonging to the technical field of biological medicines. The method for quickly separating the pigment in the recombinant epidermal growth factor fermentation liquid comprises the following steps: pretreating fermentation liquor of the recombinant epidermal growth factor, adding hydrophobic high-performance liquid chromatography packing, stirring, dissociating and adsorbing to enable the recombinant epidermal growth factor to be adsorbed on the hydrophobic high-performance liquid chromatography packing to form adsorption packing, removing supernatant after the adsorption packing is settled, and collecting the adsorption packing; eluting the adsorption filler by using a mobile phase I until no pigment flows out, then continuously eluting the adsorption filler by using a mobile phase II and collecting effluent liquid to obtain the recombinant epidermal growth factor with the pigment removed; the method for separating the pigment in the recombinant epidermal growth factor fermentation liquid has the characteristics of simple, convenient and quick process, few steps, easy industrial amplification and the like, and the product has higher activity and purity.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a method for quickly separating pigment in recombinant epidermal growth factor fermentation liquor.
Background
Epidermal Growth Factor (EGF) is a polypeptide substance widely present in mammals, and human Epidermal Growth Factor (hEGF) was first purified from human urine in 1974. hEGF is composed of small molecular polypeptide consisting of 53 amino acids, has molecular weight of about 6000 daltons, isoelectric point of about 4.6, and three pairs of disulfide bonds in molecules, and is stable to physicochemical factors such as acid, alkali and heat.
hEGF is a multifunctional cell growth factor with a wide range of biological effects that exerts physiological effects by binding to hEGF receptors on cell membranes. The research shows that: the hEGF receptor is contained in the cell membranes of various cells such as epidermal cells, fibroblasts, endothelial cells and smooth muscle cells, and the content of the epidermal cells is the highest. The hEGF is combined with an hEGEF receptor on a cell membrane after approaching the cell, so that a series of complex biochemical cascade reactions occur in the cell, RNA, DNA and protein synthesis is increased, cell growth and propagation are promoted, and cell metabolism is accelerated. hEGF has been used medically for the treatment of burns, ulcers, various wounds, corneal injuries, and the like. EGF also can promote the metabolism of normal epidermal cells, and can achieve the effects of whitening, resisting wrinkles and delaying aging when being added into a cosmetic skin care product.
The source of hEGF before the 80's in the 20 th century was mainly tissue and fluid extraction, and the application of hEGF was limited due to the high cost of extraction and low product purity of hEGF due to its very low content in tissues and fluids. After 80 s, with the development of genetic engineering technology, people succeeded in producing hEGF by using Escherichia coli, yeast and the like through genetic recombination technology, and laid a foundation for the industrial production of recombinant human epidermal growth factor (rhEGF). However, the rhEGF fermentation liquor prepared by the gene recombination technology at present has low product concentration, complex extraction and purification process and low activity of the obtained hEGF; wherein, the culture medium of the fermentation liquid generally contains pigment, and the metabolism also produces pigment molecules in the fermentation process, which become impurities in the recombinant epidermal growth factor fermentation liquid with larger proportion and influence the purity and the efficacy of the recombinant epidermal growth factor.
Disclosure of Invention
The invention aims to solve the problems in the prior art, provides a method for quickly separating pigment from recombinant epidermal growth factor fermentation liquor, has the characteristics of simple, convenient and quick process, few steps, easiness in industrial amplification and the like, and is high in product activity and purity.
The purpose of the invention can be realized by the following technical scheme:
a method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquor comprises the following steps:
s1, pretreating fermentation liquor of the recombinant epidermal growth factor, adding a hydrophobic high-performance liquid chromatography filler, stirring and dissociating for adsorption to enable the recombinant epidermal growth factor to be adsorbed on the hydrophobic high-performance liquid chromatography filler to form an adsorption filler, removing supernatant after the adsorption filler is settled, and collecting the adsorption filler;
s2, eluting the adsorption filler by using the mobile phase I until no pigment flows out, then continuously eluting the adsorption filler by using the mobile phase II and collecting effluent liquid to obtain the recombinant epidermal growth factor with the pigment removed.
Preferably, the step of preprocessing in step S1 is,
1) Acid precipitation: adjusting the pH value of the recombinant epidermal growth factor fermentation broth to 2-3 with acid liquor, standing, performing solid-liquid separation, discarding solid precipitate, and retaining supernatant fermentation broth.
2) Adding sodium chloride with final concentration of 0.6-0.8 mol/L into the supernatant fermentation liquor before adding the hydrophobic high performance liquid chromatography filler.
Preferably, the acid solution comprises one or two of dilute hydrochloric acid and trifluoroacetic acid.
In the pretreatment process, the pH value of the recombinant epidermal growth factor fermentation liquor is controlled to be 2-3 by an acid precipitation method (isoelectric precipitation method), most of proteins in the recombinant epidermal growth factor fermentation liquor can be removed, and the isoelectric points of most of hybrid proteins except the recombinant epidermal growth factor fermentation liquor are in an acidic range of pH3 +/-0.5.
According to the invention, sodium chloride with final concentration of 0.6-1.0 mol/L is added into the supernatant fermentation liquor at the later stage of pretreatment, so that the hydrophobicity of the recombinant epidermal growth factor can be increased, and the hydrophobic group is exposed on the protein surface of the recombinant epidermal growth factor.
Preferably, the stirring dissociative adsorption is magnetic stirring until the hydrophobic high performance liquid chromatography filler is suspended, then continuously stirring for 12-18 min, and then standing in an environment of 2-8 ℃ until the adsorbed filler is precipitated.
Preferably, the recombinant epidermal growth factor fermentation liquid in the step S1 is secreted and expressed by escherichia coli, and isopropyl- β -D-thiogalactoside (IPTG) is added to induce secretion of the epidermal growth factor into the extracellular fermentation liquid.
Preferably, the hydrophobic hplc packing material of step S1 is a silica gel matrix, and hydrophobic groups are bonded to the surface of the silica gel matrix.
Preferably, the hydrophobic group is a phenyl group.
The invention selects hydrophobic high performance liquid chromatography packing to process recombinant epidermal growth factor fermentation liquor, most of pigment molecules in the recombinant epidermal growth factor fermentation liquor have no hydrophobicity, the adsorption with the hydrophobic chromatography packing is not strong, the hydrophobic performance of the epidermal growth factor is greater than that of the pigment molecules, and the pigment molecules can generate stronger adsorption with the hydrophobic chromatography packing, so when the adsorption packing is eluted by adopting a proper mobile phase I, the pigment in the recombinant epidermal growth factor fermentation liquor can flow through when the hydrophobic chromatography is used for sampling, the recombinant epidermal growth factor can be retained, the effective separation of the pigment and the recombinant epidermal growth factor can be realized, then the adsorption packing is eluted by adopting a mobile phase II, the retained recombinant epidermal growth factor is eluted from the hydrophobic chromatography packing, and the recombinant epidermal growth factor with the pigment removed can be obtained. And the hydrophobic interaction between the recombinant epidermal growth factor and the hydrophobic chromatographic packing is mild interaction, and the molecular structure and the activity of the recombinant epidermal growth factor are not damaged after elution.
According to the invention, phenyl groups are preferably selected as bonding groups of the hydrophobic high performance liquid chromatography packing, the phenyl groups have strong hydrophobic action force, and the separation effect on recombinant epidermal growth factors and pigments in fermentation liquor is optimal.
The method for removing the pigment in the recombinant epidermal growth factor fermentation liquid has high efficiency and good effect, is obviously superior to other methods, such as the method for separating the pigment in the recombinant epidermal growth factor fermentation liquid by adopting the anion exchange filler, and has the advantages that the anion exchange filler can adsorb and retain most of the pigment, but can adsorb and retain the recombinant epidermal growth factor, and the adsorption strength of the pigment and the recombinant epidermal growth factor in the anion exchange filler is not greatly different, so that the pigment and the recombinant epidermal growth factor are difficult to be effectively separated; in addition, if anion filler is adopted for separation, the fermentation liquor needs to be desalted, so that the separation cost is increased, and the large-scale production is not facilitated.
Preferably, the particle size of the hydrophobic high performance liquid chromatography packing material in the step S1 is 16-25 μm.
According to the invention, by controlling the granularity of the chromatographic packing, the packing can be rapidly, effectively and naturally settled after stirring and adsorption, the next operation can be carried out without waiting for too long time, 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 mobile phase I in the step S2 comprises the following components: adjusting the pH value to 0.6-0.8 mol/L sodium chloride of 2.5-3.5; and the mobile phase II is ultrapure water with the PH value adjusted to 2.5-3.5.
The pigment in the recombinant epidermal growth factor fermentation liquid has small adsorption force with hydrophobic chromatographic packing under the hydrophobic action condition, and most of the pigment which is not adsorbed or weakly adsorbed is eluted by the mobile phase 1 under the flushing action of the high-strength mobile phase I, so that the aim of preferentially removing the pigment is fulfilled; then the recombinant epidermal growth factor is eluted by the mobile phase II, so that the purposes of separating the recombinant epidermal growth factor and the pigment and concentrating the recombinant epidermal growth factor are achieved. The elution order of the mobile phase Il and the mobile phase 1 cannot be reversed in the present invention, and if the elution is performed first by the mobile phase Il, part of the pigment and EGF remaining on the surface of the filler are eluted simultaneously, and the removal effect of the pigment is relatively poor.
Preferably, the elution in step S2 is performed in an elution apparatus comprising a buchner funnel, a filtration flask and a circulation vacuum pump.
Further preferably, the elution in step S2 is to place the collected adsorption packing in a buchner funnel, and to flow the eluent into the buchner funnel while vacuumizing.
The elution device of the invention is composed of a Buchner funnel, a suction flask and a circulating vacuum pump, the collected adsorption filler is added into a Buchner funnel for vacuum pumping, and the liquid is added in a flowing manner for liquid removal and the effluent liquid is collected, thereby completing the concentration.
Compared with the prior art, the invention has the following beneficial effects:
(1) The whole adsorption-elution process is quick and simple to operate, and the method has the advantages of high separation speed, high efficiency and low cost for pigment in the recombinant epidermal growth factor fermentation broth, and is suitable for industrial large-scale production;
(2) The method of the invention has the functions of concentration and purification while removing the pigment in the recombinant epidermal growth factor fermentation liquor, thereby greatly improving the purity of the final product;
(3) According to the invention, the hydrophobic high performance liquid chromatography filler bonded with phenyl groups is preferably selected, so that a better separation effect of the recombinant epidermal growth factor and the pigment in the fermentation liquor is realized;
(4) The components of the eluent are optimized, so that the pigment recombinant epidermal growth factors can be rapidly and sequentially eluted from the chromatographic packing, and the rapid and efficient separation of the pigment recombinant epidermal growth factors and the chromatographic packing is realized;
(5) The invention improves the sedimentation speed 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 further describe the technical solutions of the present invention, but the present invention is not limited to these examples.
Example 1
The method for rapidly separating the pigment in the recombinant epidermal growth factor fermentation broth in the embodiment comprises the following steps:
(1) Escherichia coli is taken as a genetic engineering bacterium to secrete and express recombinant epidermal growth factor to prepare recombinant epidermal growth factor fermentation liquid, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into extracellular fermentation liquid;
(2) Pretreating fermentation liquor of the recombinant epidermal growth factor, specifically, firstly carrying out acid precipitation: adjusting the pH value of the recombinant epidermal growth factor fermentation liquor to 2.5 by using dilute hydrochloric acid, standing for 2 hours, centrifuging, removing solid precipitates, and keeping supernatant fermentation liquor; adding sodium chloride with the final concentration of 0.8mol/L into the supernatant fermentation liquor before use;
(3) Adding hydrophobic high performance liquid chromatography filler (the hydrophobic high performance liquid chromatography filler is composed of silica gel matrix and phenyl group bonded on the surface of the silica gel matrix, the granularity is 20 mu m) into the pretreated supernatant fermentation liquor, stirring for 15min after the hydrophobic high performance liquid chromatography filler is suspended by magnetic stirring, adsorbing the recombinant epidermal growth factor on the hydrophobic high performance liquid chromatography filler to form an adsorption filler, standing in an environment at 5 ℃ until the adsorption filler is settled, discarding supernatant, and collecting the adsorption filler;
(4) Placing the collected adsorption filler in a Buchner funnel of an elution device (the elution device comprises the Buchner funnel, a suction flask and a circulating vacuum pump), firstly adding a mobile phase I to elute the adsorption filler until no pigment flows out while vacuumizing, then adding a mobile phase II to continuously elute the adsorption filler and collecting the effluent liquid to obtain the recombinant epidermal growth factor without the pigment,
the mobile phase I is 0.7mol/L sodium chloride, and the pH value is adjusted to 3 by using 2mol/L hydrochloric acid; the mobile phase II is ultrapure water, and the pH value is adjusted to 3 by using 2mol/L hydrochloric acid.
The recovery rate of the activity of the recombinant epidermal growth factor collected in example 1 was 89%.
Example 2
The method for rapidly separating the pigment in the recombinant epidermal growth factor fermentation broth in the embodiment comprises the following steps:
(1) Escherichia coli is taken as a genetic engineering bacterium to secrete and express recombinant epidermal growth factor to prepare recombinant epidermal growth factor fermentation liquid, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into extracellular fermentation liquid;
(2) Pretreating fermentation liquor of the recombinant epidermal growth factor, specifically, firstly carrying out acid precipitation: regulating the pH value of the recombinant epidermal growth factor fermentation liquor to 2.0 by using dilute hydrochloric acid, standing for 2.5 hours, centrifuging, removing solid precipitates, and keeping supernatant fermentation liquor; adding sodium chloride with the final concentration of 0.6mol/L into the supernatant fermentation liquor before use;
(3) Adding hydrophobic high performance liquid chromatography filler (the hydrophobic high performance liquid chromatography filler is composed of silica gel matrix and phenyl group bonded on the surface of the silica gel matrix, the granularity is 20 mu m) into the pretreated supernatant fermentation liquor, stirring for 18min after the hydrophobic high performance liquid chromatography filler is suspended by magnetic stirring, adsorbing the recombinant epidermal growth factor on the hydrophobic high performance liquid chromatography filler to form an adsorption filler, standing in an environment at 3 ℃ until the adsorption filler is settled, discarding supernatant, and collecting the adsorption filler;
(4) Placing the collected adsorption filler in a Buchner funnel of an elution device (the elution device comprises the Buchner funnel, a filter flask and a circulating vacuum pump), feeding a mobile phase I to elute the adsorption filler until no pigment flows out of the Buchner funnel while vacuumizing, feeding a mobile phase II to continuously elute the adsorption filler and collecting the effluent to obtain the recombinant epidermal growth factor without the pigment,
the mobile phase I is 0.6mol/L sodium chloride, and the pH value is adjusted to 2.5 by using 2mol/L hydrochloric acid; the mobile phase II is ultrapure water, and the pH value of the ultrapure water is adjusted to 2.5 by using 2mol/L hydrochloric acid.
The recovery rate of the activity of the recombinant epidermal growth factor collected in example 2 was 86%.
Example 3
The method for rapidly separating the pigment in the recombinant epidermal growth factor fermentation broth in the embodiment comprises the following steps:
(1) Escherichia coli is taken as a genetic engineering bacterium to secrete and express recombinant epidermal growth factor to prepare recombinant epidermal growth factor fermentation liquid, and isopropyl-beta-D-thiogalactoside (IPTG) is added in the preparation process to induce and secrete the epidermal growth factor into extracellular fermentation liquid;
(2) Pretreating the fermentation liquor of the recombinant epidermal growth factor, specifically, firstly carrying out acid precipitation: regulating the pH value of the recombinant epidermal growth factor fermentation broth to 3 by using dilute hydrochloric acid, standing for 1.5 hours, centrifuging, removing solid precipitate, and keeping supernatant fermentation broth; adding sodium chloride with the final concentration of 1.0mol/L into the supernatant fermentation liquor before use;
(3) Adding hydrophobic high performance liquid chromatography filler (the hydrophobic high performance liquid chromatography filler consists of a silica gel substrate and phenyl groups bonded on the surface of the silica gel substrate, and has a granularity of 20 mu m) into the pretreated supernatant fermentation liquor, stirring by magnetic force until the hydrophobic high performance liquid chromatography filler is suspended, continuing stirring for 12min to make the recombinant epidermal growth factor adsorbed on the hydrophobic high performance liquid chromatography filler to form an adsorption filler, then standing in an environment at 8 ℃ until the adsorption filler is settled, discarding supernatant, and collecting the adsorption filler;
(4) Placing the collected adsorption filler in a Buchner funnel of an elution device (the elution device comprises the Buchner funnel, a filter flask and a circulating vacuum pump), feeding a mobile phase I to elute the adsorption filler until no pigment flows out of the Buchner funnel while vacuumizing, feeding a mobile phase II to continuously elute the adsorption filler and collecting the effluent to obtain the recombinant epidermal growth factor without the pigment,
the mobile phase I is 0.8mol/L sodium chloride, and the pH value is adjusted to 3.5 by using 2mol/L hydrochloric acid; the mobile phase II is ultrapure water, and the pH is adjusted to 3.5 by using 2mol/L hydrochloric acid.
The recovery rate of the activity of the recombinant epidermal growth factor collected in example 3 was 83%.
Comparative example 1
Adopting DEAE weak-base anion exchange filler to carry out adsorption treatment, carrying out desalination pretreatment on recombinant epidermal growth factor fermentation liquor, and adopting eluent to elute the main active part of the recombinant epidermal growth factor, wherein the eluent comprises the following components: 150mmol/L sodium chloride solution 10mmol/L LTris-HCl and PH7.0, and the anion exchange packing is regenerated by adopting a regeneration liquid, wherein the regeneration liquid comprises the following components: 1mol/L NaCl, 10mmol/L Tris-HCl, pH7.0, and the rest is the same as example 1.
Observing the test process of the comparative example 1, after stirring and free adsorption, the observation shows that the pigment molecules are almost completely adsorbed by the anion exchange filler, the pigment is simultaneously eluted and concentrated after elution, the pigment and the recombinant epidermal growth factor are difficult to be effectively separated by mobile phase elution due to the small difference of the adsorption strength of the pigment and the recombinant epidermal growth factor in the anion exchange filler, and the activity recovery rate of the recombinant epidermal growth factor is about 50-60% after a plurality of tests. On anion exchange packing, the pigment molecules are relatively more strongly retained than EGF, but are difficult to separate due to the small difference, and part of the more strongly retained pigment can be washed off during the regeneration of the packing.
Comparative example 2
The recombinant epidermal growth factor fermentation broth was not pretreated, and the rest was the same as in example 1.
The recovery rate of the activity of the collected recombinant epidermal growth factor is 52%.
In the examples and comparative examples of the present invention, the activity recovery rate of recombinant epidermal growth factor = (number of activity units per ml product x volume)/number of total activity units of fermentation broth x 100%.
The whole adsorption-elution process is quick and simple to operate, pigment is removed, the pigment has the functions of concentration and purification, the activity recovery rate can reach more than 80%, and the electrophoresis purity can reach more than 75%.
The technical scope of the invention claimed by the embodiments of the present application is not exhaustive, and new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the invention claimed by the present application; in all the embodiments of the present invention, which are listed or not listed, each parameter in the same embodiment only represents an example (i.e., a feasible embodiment) of the technical solution, and there is no strict matching and limiting relationship between the parameters, wherein the parameters may be replaced with each other without departing from the axiom and the requirements of the present invention, unless otherwise specified.
The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and the technical scheme also comprises the technical scheme formed by any combination of the technical characteristics. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.
Claims (2)
1. A method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquor is characterized by comprising the following steps:
s1, pretreating fermentation liquor of the recombinant epidermal growth factor, adding a hydrophobic high-performance liquid chromatography filler, stirring and dissociating for adsorption to enable the recombinant epidermal growth factor to be adsorbed on the hydrophobic high-performance liquid chromatography filler to form an adsorption filler, removing supernatant after the adsorption filler is settled, and collecting the adsorption filler;
s2, eluting the adsorption filler by using the mobile phase I until no pigment flows out, then continuously eluting the adsorption filler by using the mobile phase II and collecting effluent liquid to obtain the recombinant epidermal growth factor with the pigment removed;
the step of preprocessing in step S1 is,
1) Acid precipitation: adjusting the pH value of the recombinant epidermal growth factor fermentation broth to 2.5 by using acid liquor, standing, performing solid-liquid separation, removing solid precipitates, and keeping supernatant fermentation broth;
2) Adding sodium chloride with final concentration of 0.8mol/L into the supernatant fermentation liquor before adding hydrophobic high performance liquid chromatography filler;
the hydrophobic high performance liquid chromatography filler in the step S1 consists of a silica gel matrix and a hydrophobic group bonded on the surface of the silica gel matrix; the hydrophobic group is a phenyl group; the granularity of the hydrophobic high performance liquid chromatography packing in the step S1 is 20 mu m;
s1, collecting the adsorption filler, namely, continuously stirring for 15min after the hydrophobic high-performance liquid chromatography filler is suspended by magnetic stirring to enable the recombinant epidermal growth factor to be adsorbed on the hydrophobic high-performance liquid chromatography filler to form the adsorption filler, standing in an environment at 5 ℃ until the adsorption filler is settled, removing supernatant, and collecting the adsorption filler;
s2, the mobile phase I is 0.7mol/L sodium chloride with the PH adjusted to 3, and the mobile phase II is ultrapure water with the PH adjusted to 3;
and (3) carrying out elution in the step S2 in an elution device, wherein the elution device comprises a Buchner funnel, a suction filtration bottle and a circulating vacuum pump.
2. The method for rapidly separating pigment from recombinant epidermal growth factor fermentation broth according to claim 1, wherein the recombinant epidermal growth factor in step S1 is secreted and expressed by fermentation broth escherichia coli, and isopropyl- β -D-thiogalactoside (IPTG) is added to induce secretion of epidermal growth factor into extracellular fermentation broth.
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| PT97321A (en) * | 1990-04-11 | 1992-01-31 | Indu Parikh | EPIDERMIC GROWTH FACTOR PURIFICATION PROCESS |
| CN1279288A (en) * | 2000-07-19 | 2001-01-10 | 深圳市华生元基因工程发展有限公司 | Recombination human epidermal growth factor gene, its expression product and its application |
| CN100489096C (en) * | 2005-04-21 | 2009-05-20 | 甘人宝 | Escherichia expression system for secreting and expressing recombinant human epidermal growth factor |
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| CN102020710B (en) * | 2010-09-03 | 2012-09-05 | 深圳市华生元基因工程发展有限公司 | Novel mutant EN-46 of human epidermal growth factor |
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