CN107987150A - A kind of method that hEGF can be prepared in the slave urine of industrialized production - Google Patents
A kind of method that hEGF can be prepared in the slave urine of industrialized production Download PDFInfo
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- CN107987150A CN107987150A CN201711379515.7A CN201711379515A CN107987150A CN 107987150 A CN107987150 A CN 107987150A CN 201711379515 A CN201711379515 A CN 201711379515A CN 107987150 A CN107987150 A CN 107987150A
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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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of method that hEGF can be prepared in the slave urine of industrialized production, Urine proteins concentrate including will be extracted from human urine obtains hEGF successively through metal chelate affinity chromatography column, dye affinity chromatography column, hydrophobic chromatography column, anion-exchange column, gel permeation chromatography column chromatography.Method provided by the invention is easy to operate, and cost is low, high income, the hEGF's non-immunogenicity being prepared, and bioactivity is high, and stable quality, is adapted to large-scale production operation.
Description
Technical field
The present invention relates to a kind of method that hEGF is prepared from urine, and more particularly to one kind can industrial metaplasia
The method that extraction prepares the epidermal growth factor of natural people source in the slave human urine of production.
Background technology
HEGF (Human Epidermal growth factor, hEGF) is American scientist in 1962
One kind that Cohen and Montalcini has found in purified mouse salivary gland nerve growth factor (NGF) can promote newborn mice
Widen the view ahead of time, long teeth and the component that dermal cell growth can be promoted.It is a kind of molecular weight being made of 53 amino acid residues
The small molecule single chain polypeptide of about 6kDa, isoelectric point (pI) is 4.6, containing 3 intrachain disulfide bonds.It is widely present in many organizers
In official and body fluid, as cell growth regulator, there is extensive biological function, growth, development with individual, cell turn
Change, the healing of wound or even the growth of hair are related.
Most significantly effect is to promote propagation and the differentiation of skin and corneal epithelium to EGF, this under overall condition and from
It is confirmed in the tissue cultures of body.EGF promotes skin tissue cell by being combined simultaneously activated receptor with acceptor on cell membrane
Propagation and growth, accelerate the process of skin revitalization cell replacement senile cell, make skin rejuvenation, and protection is played to skin and is made
With so as to play the functions such as anti-aging and skin-protection and health-care.So EGF make an addition in daily skin care item, cosmetics have anti-aging,
Increase the functions such as elasticity, to the skin being damaged, can automatically be nursed, raise and repair.EGF is specifically in cosmetics
Effect be mainly manifested in skin care, maintenance, crease-resistant, anti-aging, whitening, nti-freckle, sun-proof and after-sun, suppresses acne and youth
Acne growth, remove spot, hair care, hair care.Just because of EGF has skin in significant bioactivity, cosmetics of the in the market containing EGF
Also it is increasing, mainly there are aqua, gel and cream etc..Document report addition mass fraction is 1 × 10-6~1 × 10-4%'s
EGF can play the role of biological skin-active.
It is using recombinant human epidermal growth factor (rhEGF) mostly currently on the market.RhEGF is to pass through genetic engineering
Bacterium carrys out fermenting and producing and obtains rhEGF, but the production technology of each engineering bacteria is different, some fermentation tables
The rhEGF conformations reached are different from natural hEGF, it is necessary to could have bioactivity after renaturation, and renaturation ratio is extremely difficult to
100%, so can only be to evaluate the quality of recombinant human epidermal growth factor than living, specific activity is higher it may be considered that natural structure
The hEGF contents of elephant are higher, and the quality of product is better.
The hEGF researchs of native conformation are very deep, in addition to the gene of decoded human body coding hEGF, to this
The property of albumen, amino acid composition and structure etc. are also after measured.The content of EGF in normal human urine is about 17ug/L,
HEGF is made of 53 amino acid, the disulfide bond that intramolecular is made of six cysteines, forms 3 molecule inner annular structures.
And hEGF does not have glycosylation site, so structure quite stable, heat-resisting acidproof, can preserve steadily in the long term.Fermenting and producing obtains
Although rhEGF easy purifications, yield it is high, if without fully purifying, a large amount of existing protease often remain in engineering bacteria
In the product, will degrade hEGF during preservation so that the shelf-life of hEGF greatly shortens, and in addition remains in hEGF products
In mycoprotein tend to that anaphylactogen can be become, cause reaction of the user to hEGF product allergy.Fermentation expression
RhEGF, therefore, at present can only external application compared to natural hEGF its security also existing defects.
The content of the invention
Goal of the invention：In order to easy to operate, cost is low, and the large-scale production of high income is obtained being not easy sensitization, more pacified
Complete reliable, stable structure, the hEGF for the natural people source that can be preserved steadily in the long term, the present invention provides one kind
The method that hEGF can be prepared in the slave urine of industrialized production.
Technical solution：The method of the present invention that hEGF can be prepared in the slave urine of industrialized production,
Including the Urine proteins concentrate that will be extracted from human urine successively through metal chelate affinity chromatography column, dye affinity chromatography column, hydrophobic
Chromatographic column, anion-exchange column, gel permeation chromatography column chromatography, obtain hEGF.
The Urine proteins concentrate is prepared by following steps：Adsorbed by the use of anion exchange resin as adsorbent
Urine proteins in urine, cleaning, desorption, elution, eluent is concentrated by ultrafiltration, up to Urine proteins concentrate.
The Urine proteins concentrate by further detail below the step of be prepared：Using anion exchange resin as absorption
Agent is placed in urinal or urine funnel, and absorption flows through the Urine proteins in urine or diluted urine；The resin of adsorbing urine protein is collected,
Concentration is cleaned, desorption, elution processing, eluent is concentrated by ultrafiltration, up to Urine proteins concentrate, wherein containing richness
The hEGF of collection.
The desorption is that the desorption of Urine proteins is carried out using the NaCl aqueous solutions of 0.5~1.5mol/L.
The metal chelate affinity chromatography balanced using equilibrium liquid, loading Urine proteins concentrate, is rinsed through flushing liquor, eluted
Eluting peak is collected after liquid elution；Need to adjust pH and conductance before Urine proteins concentrate loading metal chelant affinity column, adjust
Save pH 2.5~4.0,0.5~10mS/cm of conductance.The chelating metal ion of the metal chelant affinity column is Cu2+、
Zn2+、Ni2+、Fe3+In any one, preferably Cu2+, equilibrium liquid used is the acetic acid of 0.02~0.2mol/L when it is chromatographed
Salt or phosphate buffer, buffer solution NaCl containing 0.2~2.0mol/L；Flushing liquor is 0.02~0.2mol/L glycine
Aqueous solution, pH are 2.5~4.0；Eluent is 0.02~0.2mol/L glycine solutions, the aqueous solution containing 0.05~
0.2mol/L NH4Cl, pH are 2.5~4.0.The preferred 0.02mol/L acetate buffers of equilibrium liquid, the buffer solution contain
0.5mol/L NaCl；The preferred 0.05mol/L glycine solutions of flushing liquor, pH 3.0；The preferred 0.05mol/L of eluent is sweet
Propylhomoserin aqueous solution, the aqueous solution contain 0.1mol/LNH4Cl, pH 3.0.
The eluting peak that previous step metal chelate affinity chromatography obtains adjusts and liquid pH4.0- is concentrated by ultrafiltration by being concentrated by ultrafiltration
6.0,0.2~2mS/cm of conductance, on dye affinity chromatography column that equilibrium liquid has balanced.The dye affinity chromatography column is adopted
Dyestuff is any one in Red dyestuffs, Blue dyestuffs, preferably Blue dyestuffs, its equilibrium liquid used when chromatographing is
The acetate or phosphate buffer of 0.02~0.2mol/L, pH are 4.0~6.0；Flushing liquor is the vinegar of 0.02~0.2mol/L
Hydrochlorate or phosphate buffer, the buffer solution NaCl containing 0.1~0.5mol/L, pH are 4.0~6.0；Eluent is 0.02
The acetate or phosphate buffer of~0.2mol/L, pH are 5.0~8.0.The preferred 0.05mol/L acetate salt buffers of equilibrium liquid
Liquid, pH 5.0；The preferred 0.05mol/L acetate buffers of flushing liquor, the buffer solution NaCl containing 0.4mol/L, pH are
5.0；The preferred 0.05mol/L phosphate buffers of eluent, pH 6.2.
The eluting peak that dye affinity chromatography step obtains is by being concentrated by ultrafiltration, adjusting pH4.0~8.0, and conductance 5.0~
The hydrophobic chromatography column balanced on 10mS/cm.The hydrophobic medium of the hydrophobic chromatography column coagulates for Phenyl- agaroses
Glue, equilibrium liquid used is the acetate buffer of 0.02~0.2mol/L when it is chromatographed, the buffer solution containing 0.5~
2.0mol/L (NH4)2SO4, pH is 4.0~8.0；Flushing liquor is 0.5~1.0mol/L (NH4)2SO4Aqueous solution, pH for 5.0~
8.0；Eluent is 0.2~0.5mol/L (NH4)2SO4Aqueous solution, pH are 5.0~8.0.The preferred 0.05mol/L acetic acid of equilibrium liquid
Salt buffer, the buffer solution (NH containing 1.1mol/L4)28O4, pH 5.0；Preferred 0.7mol/L (the NH of flushing liquor4)2SO4It is water-soluble
Liquid, pH 7.0；Preferred 0.3mol/L (the NH of eluent4)2SO4Aqueous solution, pH 7.0.
The eluting peak that hydrophobic chromatography step obtains adjusts and liquid pH7.0~9.0, conductance 0.1 is concentrated by ultrafiltration by being concentrated by ultrafiltration
The anion exchange chromatography balanced on~5.0 mS/cm.The anion-exchange chromatography is preferably weak anionic
Displacement chromatography.Equilibrium liquid used is 0.02~0.1mol/L Tris buffer solutions during described anion exchange chromatography,
0.02~0.1mol/L NaCl, pH7.0~9.0；Eluent is 0.02~0.1mol/L Tris buffer solutions, 0.1~0.5mol/
L NaCl, pH7.0~9.0.The preferred 0.02mol/L Tris buffer solutions of equilibrium liquid, 0.05mol/L NaCl, pH8.0；Eluent
It is preferred that 0.02mol/L Tris buffer solutions, 0.2mol/LNaCl, pH8.0.
The eluting peak that anion-exchange chromatography step obtains carries out that gel permeation chromatography is concentrated by ultrafiltration.The gel mistake
It is preferably Sephadex G-50 to filter chromatographic column.Flushing liquor used is 0.02-0.05mol/L during gel permeation chromatography column chromatography
Phosphate buffer or acetate buffer, pH 4.0-8.0.It is preferred that 0.05mol/L acetate buffers, pH 5.0,
It is hEGF highly finished product to collect purpose peak.
Virus removal step is further included between the chromatographic step, which can add any chromatographic step in technique
Between, it is specially regulatory protein matter concentration.Preferably add between hydrophobic chromatography and anion-exchange chromatography.Described prevents or cure a disease
Malicious step is specially that regulatory protein matter concentration to 3-7mg/ml, addition 5-50g/L glycine adjusts pH4.5- as protective agent
When 7.0,60 DEG C of heating 10 are small.
The present invention efficiently separates hEGF and other based on patent CN103694334 A, using metal chelate chromatography
Urine proteins and for follow-up hEGF purifying preparing raw material.It is dense in low pH and less salt according to the specificity of dye affinity chromatography
Column chromatography in the case of degree, in loading penetrates, destination protein hEGF is incorporated on column chromatography most foreign protein, then is led to
Cross change pH or salinity elutes.This step of dye affinity chromatography, which can live the ratio of hEGF, improves 15 times or so, greatly
The big pressure for alleviating following protein purification.Hydrophobic chromatography is mainly the pigment removed in high molecular weight protein and protein solution,
Its property is similar with reversed phase chromatography, but compares reversed phase chromatography, and hydrophobic chromatography is more easy to operate, also avoids having used organic solvent.
Anion-exchange chromatography can replace the ammonium sulfate of previous step, and the step can also remove some micro miscellaneous eggs in addition
In vain, and final step gel permeation chromatography be hEGF polishing purification, mainly displace salt in protein solution and some
The foreign protein of trace, the purity for making destination protein reach more than 95%.
Metal chelate chromatography and dye affinity chromatography are mainly the preparation of the purified feed stock of hEGF and thick purification step,
This two step can efficiently separate hEGF and other Urine proteins and can be greatly improved than work.Hydrophobic chromatography mainly removes
Pigment in macromolecular foreign protein and protein solution, and anion-exchange chromatography can remove some micro foreign proteins.By with
The purity that the upper obtained hEGF of step there are about 90% passes through gel permeation chromatography polishing purification again, and final sample is ensure that with this
Purity, the technique be connected smooth, high income.
Beneficial effect：Compared with the prior art, the present invention has the following advantages：
(1) the EGF products that the present invention is prepared are pure humanized's product, and no heterologous protein pollution, is not easy sensitization, does not deposit
In the worry of security, therefore hEGF prepared by this method more trusts reliably applied to skin care in cosmetics.
(2) the hEGF product structures in the present invention are very stablized, and acidproof heat-proof, controls endotoxic content, and
It can preserve steadily in the long term.
(3) chromatographic technique is combined in present invention utilization realizes the purifying of hEGF, and the equipment used is low pressure chromatography column, is compared
Mesohigh tomographic system is simple to operate, and cost is low；In addition, what is used during purifying is common inorganic salts
Class, avoids the use of organic solvent, safe operation, suitable for large-scale industrial production.
(4) present invention is based on the online adsorption technology of Urine proteins, it is possible to achieve the efficient richness of a variety of Urine proteins such as hEGF
Collection, can carry out coproduction with the preparation of other Urine proteins, not only realize the comprehensive utilization of urine, but also reduce cost, fit
Industrialized production is closed, considerable economic benefit can be obtained, it is therefore prevented that the pollution to environment, has good economy and society
It can be worth.
Brief description of the drawings
Fig. 1 prepares hEGF's flow chart for the present invention；
Fig. 2 schemes for hEGF SDS-PAGE produced by the present invention；
Fig. 3 schemes for hEGF HPLC produced by the present invention.
Embodiment
The preparation of embodiment 1, Urine proteins concentrate
100 kilograms of the resin of adsorbing urine protein is collected, is rinsed well with water, fills column；With the NaCl aqueous solutions of 0.5mol/L
Eluted, collect eluting peak, be concentrated by ultrafiltration with the ultrafiltration membrane of molecular cut off 30K, obtain Urine proteins concentrate, used
It is about 2.16mg that Elisa detection kits, which measure Egf Content therein,.
Embodiment 2, the preparation of hEGF
(1) metal chelant affinity chromatography
The Urine proteins concentrate adjusting pH to 4.0 that embodiment 1 is prepared, conductance to 0.5mS/cm, loading are flat
The metal ion to have weighed is Cu2+Metal chelant affinity column (Chelating sepharose FF)；It is (flat with equilibrium liquid again
Weigh formula of liquid：0.02mol/L acetate buffers, NaCl containing 0.5mol/L, pH4.0) metal chelant affinity column is rinsed,
Collection loading penetrates liquid and flushing penetrates liquid, is rinsed with 0.05mol/L glycine solutions (pH3.0), sweet with 0.05mol/L
Propylhomoserin aqueous solution, containing 0.1mol/LNH4Cl (pH3.0) elutes metal chelant affinity column, collects eluting peak (A liquid).
(2) dye affinity chromatography
A liquid is concentrated by ultrafiltration, it is 5.0, conductance 1.0mS/cm to adjust pH, and the Blue that loading equilibrium liquid has balanced is close
And chromatographic column, flushing liquor rinse, elution, collects eluting peak (B liquid).Wherein, equilibrium liquid is 0.05mol/L acetate
Buffer solution, pH 5.0；Flushing liquor be 0.05mol/L acetate buffer, NaCl containing 0.4mol/L, pH 5.0；Elution
Liquid be 0.05mol/L phosphate buffer, pH6.2.
(3) hydrophobic chromatography
B liquid is concentrated by ultrafiltration, adjusts and liquid pH to 5.0, conductance 8.0mS/cm is concentrated by ultrafiltration, upper equilibrium liquid has balanced hydrophobic
Medium is the hydrophobic chromatography column of Phenyl- Ago-Gels.Equilibrium liquid is 0.05mol/L acetate buffers, containing 1.1mol/L
(NH4)2SO4, pH5.0；Flushing liquor is 0.7mol/L (NH4)2SO4Aqueous solution, pH 7.0；, eluent is 0.3mol/L (NH4)2SO4Aqueous solution, pH 7.0.Collect eluting peak (C liquid).
(4) virus removal
C liquid is concentrated by ultrafiltration, regulatory protein concentration is 5mg/ml, adds 30g/L glycine as protein protective agent, adjusts
PH5.0, when 60 DEG C of heating 10 are small.
(5) anion-exchange chromatography
Sample after heating is adjusted liquid pH8.0 is concentrated by ultrafiltration, conductance 0.20mS/cm, upper anion exchange chromatography, puts down
The liquid that weighs rinses, and elution, collects eluting peak (D liquid).Wherein, equilibrium liquid is 0.05mol/L Tris buffer solutions, is contained
0.05mol/LNaCl, pH8.0；Eluent is 0.05mol/L Tris buffer solutions, containing 0.2mol/LNaCl, pH8.0.
(5) gel permeation chromatography
D liquid is concentrated by ultrafiltration to protein concentration 5mg/ml, loading gel permeation chromatography column chromatography, is rushed using flushing liquor
Wash, the flushing liquor is the acetate buffer of 0.05mol/L, and pH 5.0, collects destination protein peak, obtain human epidermal growth
Factor sterling 0.97mg, electrophoresis purity 98%.Obtained hEGF's sterling is subjected to SDS-PAGE electrophoresis,
HPLC and biological activity mtt assay are detected.
3. small molecule SDS-PAGE of embodiment measures hEGF purity
(1) 16.5% separation gel configuration
Distilled water：1.67mL；AB-6：2mL；Gel buffer liquid (pH8.45)：2mL；Urea：2.16g；Glycerine：0.3g；
10%APS：30ul；TEMED：3ul；Cumulative volume 6mL, adds in the glass plate crack of electrophoresis tank to certain altitude after mixing, uses
Distilled water binds.
(2) 12% squeegee configuration
Distilled water：1.5mL；AB-3：1.22mL；Gel buffer liquid (pH8.45)：2mL；Glycerine：0.26g；10%APS：
26ul；TEMED：2ul cumulative volumes 5mL.After gelling to be separated is solid, incline upper strata distilled water, and the squeegee after mixing is added
To certain altitude in the glass plate crack of electrophoresis tank, bound with distilled water.
(3) 7% concentration glue configuration
Distilled water：1mL；AB-3：0.3mL；Gel buffer liquid (pH8.45)：0.93mL；10%APS：17ul； TEMED：
2ul；Cumulative volume 2.6mL.After squeegee solidification, upper strata purified water of inclining, the concentration glue after mixing is filled above squeegee,
Careful insertion sample comb.
(4) solution allocation
1. AB-6 separation gels：48g acrylamides, 1.5g methylene diacrylamides, add water to be settled to 100ml；
2. AB-3 separation gels：46.5g acrylamides, 3.0g methylene diacrylamides, add water to be settled to 100ml；
3. gel buffer liquid (3 ×)：By 181.5g Tris, 1.5SDS is dissolved in 400ml distilled waters, and pH is adjusted with HCl
To 8.45, water is added to be settled to 500mL；
4. anode buffer liquid (10 ×)：Tris 121.14g are weighed to be dissolved in 400ml distilled waters, with HCl adjust pH to
8.9, add water to be settled to 500mL；
5. Cathode buffer (10 ×)：Tris 60.55g are weighed, 89.58g Tricine and 5g SDS are dissolved in 400ml
In distilled water, water is added to be settled to 500mL, pH need not be adjusted；
6. sample buffer (4 ×)：Contain 4%SDS, 12% glycerine, 0.05mol/L Tirs, 2% mercaptoethanol (v/v)
And a little bromophenol blue, pH6.8.
7. coomassie brilliant blue staining liquid weighs coomassie brilliant blue R250 1g, methanol 200mL, glacial acetic acid 50mL, water are added
250mL is mixed.
8. destainer：Ethanol 100mL, glacial acetic acid 100mL and water 800mL is taken to mix.
(5) sample preparation
HEGF samples are mixed with sample buffer 3: 1 respectively, 4-5min is kept the temperature in 100 DEG C of boiling water, is taken out stand-by.
(6) sample measures
After the sample and standard protein that embodiment 2 is prepared add loading wells, power on, constant pressure electrophoresis to bromine phenol
When indigo plant is arrived from bottom about 0.5cm, stop electrophoresis.Separation gel is removed from glass plate, 30min is dyed in dyeing liquor, is decolourized
2h, changes and preserves liquid preservation.As shown in Fig. 2, hEGF samples have no obvious impurity band, SDS-PAGE purity is 98%, in being higher than
" hEGF's purity should be not less than 95.0% " standard to electrophoresis as defined in state's pharmacopeia.
Embodiment 4：HEGF RP-HPLC purity testings
Instrument：DIONEX UltiMate 3000HPLC；Chromatographic column：Sepax BR-C18 (5um, 4.6*150mm)；Flowing
Phase：+ 30% acetonitrile solution of 0.1% trifluoroacetic acid；Flow velocity 0.7mL/min；Type of elution：0-10min, isocratic elution；Detect ripple
It is long：280nm.The hEGF samples for taking the 20 μ L present invention to be prepared, carry out purity testing, measurement result is shown in Fig. 3 by above-mentioned condition.
As shown in figure 3, four main peaks are the hEGF naturally extracted.
Embodiment 5：HEGF biological activity determinations
The bioactivity for the hEGF being prepared using cell proliferation method/MTT colorimetric method for determining present invention.
Balb/c 3T3 cell lines are cultivated with complete culture solution under 37 DEG C, 5% carbon dioxide conditions, control cell concentration
For 1 × 105-5×105A cell/ml, is used for Determination of biological activity when 24-36 is small after passage.Discard the culture in blake bottle
Liquid carries, and digests and collects cell, and being made into every 1ml with complete culture solution contains 5 × 104-8×104The cell suspension of a cell, connects
Kind, per hole 100ul, is cultivated in 96 porocyte culture plates under 37 DEG C, 5% carbon dioxide conditions.Change maintenance training after 24h into
Nutrient solution, 24h is cultivated under 37 DEG C, 5% carbon dioxide conditions.The Tissue Culture Plate of preparation discards maintaining liquid, and it is molten to add standard items
Liquid and test solution, per hole 100ul, 37 DEG C, cultivate 64-72h under 5% carbon dioxide conditions.MTT solution is added per hole
20ul, 5h is cultivated under 37 DEG C, 5% carbon dioxide conditions.Operation aseptically carries out above.Discard the liquid in culture plate
After body, dimethyl sulfoxide (DMSO) 100ul is added into every hole, after mixing in microplate reader, using 630nm as reference wavelength, at 570nm
Light absorption value is measured, records measurement result.
Computational methods：
Test sample biological activity
Wherein：Pr is standard items biological activity, IU/ml；
Ds is test sample pre-dilution multiple；
Dr is standard items pre-dilution multiple；
Es is extension rate of the test sample equivalent to standard items median effective dose；
Er is the extension rate of standard items median effective dose.
The bioactivity testing result of hEGF produced by the present invention is shown in Table 1.
Table 1
Comparative example 1, the preparation of hEGF
(1) metal chelant affinity chromatography
The Urine proteins concentrate adjusting pH to 3.5 that embodiment 1 is prepared, conductance to 2.0mS/cm, loading are flat
The metal ion to have weighed is Cu2+Metal chelant affinity column (Chelating sepharose FF)；It is (flat with equilibrium liquid again
Weigh formula of liquid：0.05mol/L acetate buffers, NaCl containing 1.0mol/L, pH3.5) metal chelant affinity column is rinsed,
Collection loading penetrates liquid and flushing penetrates liquid, is rinsed with 0.02mol/L glycine solutions (pH2.5), sweet with 0.02mol/L
Propylhomoserin aqueous solution, containing 0.2mol/LNH4Cl (pH3.5) elutes metal chelant affinity column, collects eluting peak (A liquid).
(2) anion-exchange chromatography
A liquid is concentrated by ultrafiltration, adjusts pH7.0, conductance 0.5mS/cm, upper anion exchange chromatography, equilibrium liquid flushing, is washed
De- liquid elution, collects eluting peak (B liquid).Wherein, equilibrium liquid is 0.02mol/L Tris buffer solutions, NaCl containing 0.02mol/L,
pH7.0；Eluent is 0.02mol/L Tris buffer solutions, containing 0.15mol/LNaCl, pH7.0.
(3) hydrophobic chromatography
B liquid is concentrated by ultrafiltration, adjusts and liquid pH to 4.0, conductance 10.0mS/cm is concentrated by ultrafiltration, what upper equilibrium liquid had balanced dredges
Aqueous medium be Phenyl- Ago-Gels hydrophobic chromatography column, equilibrium liquid 0.05mol/L acetate buffers, containing 1.5mol/L
(NH4)2SO4Aqueous solution, pH4.0；Flushing liquor 0.9mol/L (NH4)2SO4Aqueous solution, pH5.0；, eluent 0.5mol/L
(NH4)2SO4Aqueous solution, pH5.0.Collect eluting peak (C liquid).
(4) virus removal
C liquid is concentrated by ultrafiltration, regulatory protein concentration is 6mg/ml, adds 50g/L glycine as protein protective agent, adjusts
PH5.5, when 60 DEG C of heating 10 are small,
(5) dye affinity chromatography
By the sample after heating, 5.5 are adjusted to, conductance 0.5mS/cm, the Blue affinity chromatographys that loading equilibrium liquid has balanced
Column, flushing liquor rinse, and elution, collects eluting peak (D liquid).Wherein, equilibrium liquid is 0.05mol/L acetate buffers,
PH is 5.5；Flushing liquor be 0.05mol/L acetate buffer, NaCl containing 0.2mol/L, pH 5.5；Eluent is
The phosphate buffer of 0.05mol/L, pH7.0.
(5) gel permeation chromatography
D liquid is concentrated by ultrafiltration to protein concentration 5mg/ml, loading gel permeation chromatography column chromatography, is rushed using flushing liquor
Wash, the flushing liquor is the acetate buffer of 0.05mol/L, and pH 5.0, collects destination protein peak, obtain human epidermal growth
Factor sterling 0.67mg, electrophoresis purity 85%.
Claims (9)
- A kind of 1. method that hEGF can be prepared in the slave urine of industrialized production, it is characterised in that including will be from The Urine proteins concentrate extracted in human urine is successively through metal chelate affinity chromatography column, dye affinity chromatography column, hydrophobic chromatography column, the moon Ion exchange column, gel permeation chromatography column chromatography, obtain hEGF.
- 2. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is that the Urine proteins concentrate is prepared by following steps：Adsorbed by the use of anion exchange resin as adsorbent Urine proteins in urine, cleaning, desorption, elution, eluent is concentrated by ultrafiltration, up to Urine proteins concentrate.
- 3. the method according to claim 2 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is that the desorption is that the desorption of Urine proteins is carried out using the NaCl aqueous solutions of 0.5~1.5mol/L.
- 4. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is that the chelating metal ion of the metal chelant affinity column is Cu2+、Zn2+、Ni2+、Fe3+In any one, Equilibrium liquid used is the acetate or phosphate buffer of 0.02~0.2mol/L when it is chromatographed, the buffer solution containing 0.2~ 2.0mol/L NaCl；Flushing liquor is 0.02~0.2mol/L glycine solutions, and pH is 2.5~4.0；Eluent is 0.02- 0.2mol/L glycine solutions, the aqueous solution NH containing 0.05-0.2mol/L4Cl, pH 2.5-4.0.
- 5. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is that the dyestuff that the dye affinity chromatography column uses is any one in Red dyestuffs, Blue dyestuffs, when it is chromatographed Equilibrium liquid used is the acetate or phosphate buffer of 0.02~0.2mol/L, and pH is 4.0~6.0；Flushing liquor is 0.02 The acetate or phosphate buffer of~0.2mol/L, the buffer solution NaCl containing 0.1~0.5mol/L, pH are 4.0~6.0； Eluent is the acetate or phosphate buffer of 0.02~0.2mol/L, and pH is 5.0~8.0.
- 6. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is that the hydrophobic medium of the hydrophobic chromatography column is Phenyl- Ago-Gels, its equilibrium liquid used when chromatographing is The acetate buffer of 0.02~0.2mol/L, the buffer solution (NH containing 0.5~2.0mol/L4)2SO4, pH is 4.0~8.0； Flushing liquor is 0.5~1.0mol/L (NH4)2SO4Aqueous solution, pH are 5.0~8.0；Eluent is 0.2~0.5mol/L (NH4)2SO4Aqueous solution, pH are 5.0~8.0.
- 7. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is, virus removal step is further included between hydrophobic chromatography column chromatography and anion exchange chromatography.
- 8. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is, when anion exchange chromatography, equilibrium liquid used was 0.02~0.1mol/L Tris buffer solutions, described slow The fliud flushing NaCl containing 0.02~0.1mol/L, pH are 7.0~9.0；Eluent is 0.02~0.1mol/L Tris buffer solutions, described The buffer solution NaCl containing 0.1~0.5mol/L, pH are 7.0~9.0.
- 9. the method according to claim 1 that hEGF can be prepared in the slave urine of industrialized production, it is special Sign is, when gel permeation chromatography column chromatography flushing liquor used be 0.02-0.05mol/L phosphate buffer or Acetate buffer, pH 4.0-8.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111285929A (en) * | 2018-12-10 | 2020-06-16 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human epidermal growth factor from genetically engineered rice seeds |
| CN112175063A (en) * | 2020-10-28 | 2021-01-05 | 宁波博睿瀚达生物科技有限公司 | Process for preparing high-purity recombinant epidermal growth factor by high performance liquid chromatography |
| CN112209999A (en) * | 2020-10-28 | 2021-01-12 | 宁波博睿瀚达生物科技有限公司 | Method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquid |
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