CN1400313A - Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus - Google Patents
Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus Download PDFInfo
- Publication number
- CN1400313A CN1400313A CN 01124437 CN01124437A CN1400313A CN 1400313 A CN1400313 A CN 1400313A CN 01124437 CN01124437 CN 01124437 CN 01124437 A CN01124437 A CN 01124437A CN 1400313 A CN1400313 A CN 1400313A
- Authority
- CN
- China
- Prior art keywords
- growth factor
- epidermal growth
- human epidermal
- hours
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 title claims abstract description 61
- 229940116978 human epidermal growth factor Drugs 0.000 title claims abstract description 61
- 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 title claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 title claims description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 239000002609 medium Substances 0.000 claims abstract description 23
- 238000001035 drying Methods 0.000 claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 238000001179 sorption measurement Methods 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 40
- 239000007788 liquid Substances 0.000 claims description 35
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 238000011218 seed culture Methods 0.000 claims description 21
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 20
- 238000000859 sublimation Methods 0.000 claims description 20
- 230000008022 sublimation Effects 0.000 claims description 20
- 239000002054 inoculum Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 238000005227 gel permeation chromatography Methods 0.000 claims description 14
- 230000014759 maintenance of location Effects 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 5
- 239000000047 product Substances 0.000 abstract description 3
- 230000008014 freezing Effects 0.000 abstract 2
- 238000007710 freezing Methods 0.000 abstract 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 239000003480 eluent Substances 0.000 abstract 1
- 239000012530 fluid Substances 0.000 abstract 1
- 239000000499 gel Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 4
- 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 description 4
- 102000011632 Caseins Human genes 0.000 description 3
- 108010076119 Caseins Proteins 0.000 description 3
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000003531 protein hydrolysate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 206010043183 Teething Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003725 endotheliocyte Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000001913 submandibular gland Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000036346 tooth eruption Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The method for producing recombinant human epidermal growth factor by using genetically-engineered colibacillus includes the following steps: engineering bacterium fermentation: activating stored colibacillus by means of culture medium, fermenting main medium and introducing the human epidermal growth factor into fermentation cutlure liquor; expanding bed adsorption; centrifugalizing fermentation culture liquor, adding supernatant fluid into the expanding bed to make adsorption and collecting elution peak containing human epidermal growth factor; gel separation; adding collected eluent into gel chromatographic column, separating and collecting human epidermal growth factor elution peak; freezing, sublimating and drying; freezing collected liquor at -40 deg.C, sublimating and drying so as to obtain the invented product.
Description
Technical field
The present invention relates to a kind of method of producing recombinant human epidermal growth factor with gene engineering colibacillus.
Background technology
Last century, early forties was once found a kind of energy gastric acid inhibitory excretory material in pregnant woman's urine, and the title urogastrone (urogastrone, UG).When Cohen used the carboxymethyl cellulose post from mouse submandibular gland separation and purification of nerve growth factor (NGF) in 1962, found another kind of active substance, promoted newborn mice teething and the function of opening eyes according to it at that time, with its called after " Tooth-Lid Factor ", renamed as " Urogastron " afterwards.Cohen in 1975 and Carpenter have reported the composition of human epidermal growth factor (hEGF) amino-acid residue.In the same year, Gregory has described a kind of UG that separates from people's urine.Studies show that UG and hEGF are with a kind of material.Because hEGF can stimulate epidermis and various biological functions such as endotheliocyte, inoblast and capillary vessel growth, has now become one of the research focus in fields such as medical science and molecular biology.
HEGF production has three kinds of methods, chemosynthesis: people such as Marchi had at first finished the complete synthesis of EGF in 1988, because the existence of disulfide linkage and other multiple functional group in the hEGF molecule, thereby synthetic product purity and productive rate all can't satisfy the suitability for industrialized production requirement; Biogenetic derivation extracts: main Separation and Recovery from human urine, and hEGF concentration is low in the natural origin, be generally less than about 1 μ g/L, thereby it is low to separate purification efficiency; The present emphasis of the production of genetic engineering technique: hEGF is in the research of genetic engineering technique.
Although have the genetic engineering process for preparing of some hEGF at present, but these methods all exist production concentration low (being generally less than 100mg/L) in the fermented liquid, extraction process complexity (often needing the chromatography operation of 3～6 steps), the low shortcomings such as (20～30%) of running cost height and yield, therefore, present human epidermal growth factor's price is still up to US$210～1020/mg hEGF.Up to now, a comparatively sophisticated industrial fermentation production technique report is not arranged as yet.
Summary of the invention
The purpose of this invention is to provide a kind of method simple, be easy to grasp, not affected by environment, the cycle short, the method for producing recombinant human epidermal growth factor with gene engineering colibacillus of instant effect.
In order to achieve the above object, the present invention takes following measures:
With the method that gene engineering colibacillus is produced recombinant human epidermal growth factor, its production stage is as follows:
1) engineering bacterium fermentation
Picking list bacterium colony inserts 10～200ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 10～500mg/l, 28～37 ℃, 150～300rpm shaking table was cultivated about 18～30 hours, insert the nutrient solution of front in the seed culture medium once more by 0.1～10% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 10～500mg/l, 28～37 ℃, 150～300rpm shaking table was cultivated about 4～12 hours, by 0.1～10% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 10～500mg/ml, 28～37 ℃, 150～300rpm cultivated about 4～12 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.05～0.5mM, continue to cultivate about 8～20 hours.Recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 0～20 ℃ of condition with the centrifugal 5～30min of the speed of 1000～10000rpm, collecting supernatant liquor preserves standby under 0～20 ℃ of condition, in this supernatant liquor the linear velocity adding expanding bed with 50～300cm/h of chromatographic system with 50～1000ml, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 0～20 ℃ of condition;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above 1～2.5ml with 3～9cm/h is added in the SephadexG-50 Superfine gel chromatography column, then distilled water being carried out wash-out with the linear velocity of 3～9cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.2～19.7ml place, and collected volume is 3～5ml;
4) drying bu sublimation
To collect liquid in-35～-45 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Advantage of the present invention:
1) fermented liquid production concentration height, target product character are given prominence to
HEGF concentration is about 100～200mg/L in the fermented liquid, and the molecular weight of minimum heteroproteins molecular weight and hEGF differs about about 10000Da in the fermented liquid;
2) separating technology is simple, with short production cycle
This production separating technology is made up of Expanded Bed Adsorption and two operating units of gel chromatography.The expanding bed production cycle is about 3 hours, and gel chromatography is about 4 hours;
3) upstream and downstream compatibility, process integration
The molecular weight difference of heteroproteins and hEGFG provides the foundation for the gel chromatography separation and purification in the fermented liquid; Expanded Bed Adsorption plays the integrated effect that concentrates with initial gross separation; Gel chromatography all need not desalination in that upper prop is forward and backward, has reached the integrated effect of purifying and desalination;
4) production automation level height, easy to operate
This production technique is compared with other human epidermal growth factor's separation method, adopts equipment such as expanding bed STREAMLINE25 and chromatographic system KTA explorer100, and its separation detects with the human epidermal growth factor all can be carried out automatically;
4) occupation of land is little, cost is low
This separation production operation only needs more than 20 square metre laboratory to carry out;
5) mild condition, instant effect.
Embodiment
Below in conjunction with embodiment the present invention is elaborated:
With the method that gene engineering colibacillus is produced recombinant human epidermal growth factor, its production stage is as follows:
1) engineering bacterium fermentation
Picking list bacterium colony inserts 25～100ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 50～200mg/l, 30～35 ℃, 180～250rpm shaking table was cultivated about 20～28 hours, insert the nutrient solution of front in the seed culture medium once more by 0.5～5% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 50～200mg/l, 30～35 ℃, 180～250rpm shaking table was cultivated about 6～10 hours, by 0.5～5% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 50～200mg/ml, 30～35 ℃, 180～250rpm cultivated about 6～10 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.1～0.3mM, continue to cultivate about 10～18 hours.Recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 2～10 ℃ of conditions with the centrifugal 8～15min of the speed of 2000～8000rpm, collecting supernatant liquor preserves standby under 2～10 ℃ of conditions, with chromatographic system this supernatant liquor of 100～500ml linear velocity with 100～200cm/h is joined in the STREAMLINE25 expanding bed, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 2～10 ℃ of conditions;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above 1.5～2ml with 5～7cm/h is added in the SephadexG-50 Superfine gel chromatography column that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 5～7cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.5～19.2ml place, and collected volume is 3.5～4.5ml;
4) drying bu sublimation
To collect liquid in-37～-42 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Equipment used and material are as follows in engineering bacterium fermentation of the present invention:
1) bacterial classification
The host is Escherichia coli HB-101, and plasmid is LacUV5omp08hEGF, and engineering bacteria is Escherichia coli K-12HB-101, is preserved by Zhejiang University's bio-engineering research;
2) substratum
Seed culture medium (g/l): yeast extract 10.0, NaCl 5.0, casein protein hydrolyzate 20.0;
Fermention medium (g/l): glucose 1.0, lactose 4.0, yeast extract 20.0, Trypsin matter peptone 20.0, casein protein hydrolyzate 10.0, (NH
4)
3PO
43.5, KH
2PO
43.5, K
2HPO
45.0, MgSO
41.0 NaCl 3.0;
Solid medium (g/l): yeast extract 10.0, NaCl 5.0, casein protein hydrolyzate 20.0, agar 16.
3) equipment
Expanding bed (STREAMLINE25), chromatographic system ( KTA explorer100), chromatography column (XK16/20), sample introduction post (Superloop
TM50), gel (STREAMLINE DEAE) is available from AmershamPharmacia Biotech AB.Sweden; Electrophoresis apparatus (Mini VE complete) is available from AmershamPharmacia Biotech Inc., USA; Normal man's Urogastron is available from Pepro Tech EC Ltd., England; Other reagent is commercially available analytical reagent.
Embodiment 1
The step of producing the recombinant human epidermal growth factor method with gene engineering colibacillus is as follows:
1) engineering bacterium fermentation
The single bacterium colony of one of picking inserts the seed culture medium of 50ml (triangular flask of 500ml) from solid medium, add penbritin simultaneously, make its concentration in substratum reach 100mg/l, 32 ℃, the 220rpm shaking table was cultivated 24 hours, insert once more in the seed culture medium of 50ml by the nutrient solution of 1% inoculum size the front, add penbritin simultaneously, make its concentration in substratum reach 100mg/l, 32 ℃, the 220rpm shaking table was cultivated 8 hours, by 1% inoculum size the activatory seed liquor is joined in the 50ml fermention medium again, add penbritin simultaneously and make concentration reach 100mg/ml, 32 ℃, 220rpm cultivated 8 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.2mM, continue to cultivate 14 hours.Recombinant human epidermal growth factor enters (concentration is 198mg/L) in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 1Omin of the speed of 4000rpm, collecting supernatant liquor preserves standby under 4 ℃ of conditions, with chromatographic system this supernatant liquor of 300ml linear velocity with 183cm/h is joined in the STREAMLINE25 expanding bed that contains STREAMLINE DEAE that the post height is 13.8cm, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and (volume is 20ml, retention volume is from 150～170ml), and elutriant is preserved standby under 4 ℃ of conditions;
3) gel separation
With the automatic chromatographic system of KTA explorer100 the linear velocity of elutriant above the 1.5ml with 6cm/h is added in the SephadexG-50 Superfine gel chromatography column (XK16/20) that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 6cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 19ml place, and collected volume is 4ml;
4) drying bu sublimation
To collect liquid in-40 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.Purity is 94.87%, and total recovery is 57.6%.
Embodiment 2
The step of producing the recombinant human epidermal growth factor method with gene engineering colibacillus is as follows:
1) engineering bacterium fermentation
The single bacterium colony of one of picking inserts the seed culture medium of 25ml (triangular flask of 500ml) from solid medium, add penbritin simultaneously, make its concentration in substratum reach 120mg/l, 33 ℃, the 200rpm shaking table was cultivated 22 hours, insert once more in the seed culture medium of 25ml by the nutrient solution of 2% inoculum size the front, add penbritin simultaneously, make its concentration in substratum reach 120mg/l, 33 ℃, the 200rpm shaking table was cultivated 7.5 hours, by 2% inoculum size the activatory seed liquor is joined in the 50ml fermention medium again, add penbritin simultaneously and make concentration reach 120mg/ml, 33 ℃, 200rpm cultivated 7.5 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.15mM, continue to cultivate 15 hours.Recombinant human epidermal growth factor enters (concentration is 178mg/L) in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 30min of the speed of 4000rpm, collecting supernatant liquor preserves standby under 4 ℃ of conditions, with chromatographic system this supernatant liquor of 500ml linear velocity with 200cm/h is joined in the STREAMLINE25 expanding bed that contains STREAMLINE DEAE that the post height is 13.8cm, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and (volume is 30ml, retention volume is from 145～175ml), and elutriant is preserved standby under 4 ℃ of conditions;
3) gel separation
With the automatic chromatographic system of KTA explorer100 the linear velocity of elutriant above the 2ml with 5cm/h is added in the SephadexG-50 Superfine gel chromatography column (XK16/20) that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 5cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 19.5ml place, and collected volume is 3ml;
4) drying bu sublimation
To collect liquid in-40 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Embodiment 3
The step of producing the recombinant human epidermal growth factor method with gene engineering colibacillus is as follows:
1) engineering bacterium fermentation
The single bacterium colony of one of picking inserts the seed culture medium of 100ml (triangular flask of 500ml) from solid medium, add penbritin simultaneously, make its concentration in substratum reach 80mg/l, 31 ℃, the 240rpm shaking table was cultivated 26 hours, insert once more in the seed culture medium of 100ml by the nutrient solution of 0.5% inoculum size the front, add penbritin simultaneously, make its concentration in substratum reach 80mg/l, 31 ℃, the 240rpm shaking table was cultivated 8.5 hours, by 0.5% inoculum size the activatory seed liquor is joined in the 100ml fermention medium again, add penbritin simultaneously and make concentration reach 80mg/ml, 31 ℃, 240rpm cultivated 8.5 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.25mM, continue to cultivate 10 hours.Recombinant human epidermal growth factor enters (concentration is 126mg/L) in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 5min of the speed of 3000rpm, collecting supernatant liquor preserves standby under 4 ℃ of conditions, with chromatographic system this supernatant liquor of 100ml linear velocity with 150cm/h is joined in the STREAMLINE25 expanding bed that contains STREAMLINE DEAE that the post height is 13.8cm, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and (volume is 20ml, retention volume is from 150～17ml), and elutriant is preserved standby under 4 ℃ of conditions;
3) gel separation
With the automatic chromatographic system of AKTA explorer100 the linear velocity of elutriant above the 1ml with 5cm/h is added in the SephadexG-50 Superfine gel chromatography column (XK16/20) that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 3cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.2ml place, and collected volume is 3ml;
4) drying bu sublimation
To collect liquid in-40 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Embodiment 4
The step of producing the recombinant human epidermal growth factor method with gene engineering colibacillus is as follows: 1) engineering bacterium fermentation
Picking list bacterium colony inserts the 10ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 10mg/l, 28 ℃, the 150rpm shaking table was cultivated about 18 hours, insert the nutrient solution of front in the seed culture medium once more by 0.1% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 10mg/l, 28 ℃, the 150rpm shaking table was cultivated about 12 hours, by 0.1% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 10mg/ml, 28 ℃, 150rpm cultivated about 4 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.05mM, continue to cultivate about 8 hours.Recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 0 ℃ of condition with the centrifugal 30min of the speed of 1000rpm, collecting supernatant liquor preserves standby under 0 ℃ of condition, in this supernatant liquor the linear velocity adding expanding bed with 50cm/h of chromatographic system with 50ml, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 0 ℃ of condition;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above the 1ml with 3cm/h is added in the SephadexG-50Superfine gel chromatography column, then distilled water being carried out wash-out with the linear velocity of 3cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.2ml place, and collected volume is 3ml;
4) drying bu sublimation
To collect liquid in-35 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Embodiment 5
The step of producing the recombinant human epidermal growth factor method with gene engineering colibacillus is as follows: 1) engineering bacterium fermentation
Picking list bacterium colony inserts the 200ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 500mg/l, 37 ℃, the 300rpm shaking table was cultivated about 30 hours, connecing the sharp nutrient solution of measuring the front by 10% inserts in the seed culture medium once more, add penbritin simultaneously, make its concentration in substratum reach 500mg/l, 37 ℃, the 300rpm shaking table was cultivated about 4 hours, by 10% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 500mg/ml, 37 ℃, 300rpm cultivated about 12 hours.Add comfort property inductor isopropyl-then and induce, make its concentration in substratum reach 0.5mM, continue to cultivate about 20 hours.Recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 20 ℃ of conditions with the centrifugal 5min of the speed of 10000rpm, collecting supernatant liquor preserves standby under 20 ℃ of conditions, in this supernatant liquor the linear velocity adding expanding bed with 300cm/h of chromatographic system with 1000ml, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 20 ℃ of conditions; 3) gel separation
With automatic chromatographic system the linear velocity of elutriant above the 2.5ml with 9cm/h is added in the SephadexG-50Superfine gel chromatography column, then distilled water being carried out wash-out with the linear velocity of 9cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 19.7ml place, and collected volume is 5ml; 4) drying bu sublimation
To collect liquid in-45 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Claims (3)
1. method of producing recombinant human epidermal growth factor with gene engineering colibacillus is characterized in that its production stage is as follows:
1) engineering bacterium fermentation
Picking list bacterium colony inserts 10～200ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 10～500mg/l, 28～37 ℃, 150～300rpm shaking table was cultivated 18～30 hours, insert the nutrient solution of front in the seed culture medium once more by 0.1～10% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 10～500mg/l, 28～37 ℃, 150～300rpm shaking table was cultivated 4～12 hours, by 0.1～10% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 10～500mg/ml, 28～37 ℃, 150～300rpm cultivated 4～12 hours, adding comfort property inductor isopropyl-then induces, make its concentration in substratum reach 0.05～0.5mM, continue to cultivate 8～20 hours, recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 0～20 ℃ of condition with the centrifugal 5～30min of the speed of 1000～10000rpm, collecting supernatant liquor preserves standby under 0～20 ℃ of condition, in this supernatant liquor the linear velocity adding expanding bed with 50～300cm/h of chromatographic system with 50～1000ml, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 0～20 ℃ of condition;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above 1～2.5ml with 3～9cm/h is added in the SephadexG-50 Superfine gel chromatography column, then distilled water being carried out wash-out with the linear velocity of 3～9cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.2～19.7ml place, and collected volume is 3～5ml;
4) drying bu sublimation
To collect liquid in-35～-45 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
2. a kind of method of producing recombinant human epidermal growth factor with gene engineering colibacillus according to claim 1 is characterized in that its production stage is as follows:
1) engineering bacterium fermentation
Picking list bacterium colony inserts 25～100ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 50～200mg/l, 30～35 ℃, 180～250rpm shaking table was cultivated 20～28 hours, insert the nutrient solution of front in the seed culture medium once more by 0.5～5% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 50～200mg/l, 30～35 ℃, 180～250rpm shaking table was cultivated 6～10 hours, by 0.5～5% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 50～200mg/ml, 30～35 ℃, 180～250rpm cultivated 6～10 hours, adding comfort property inductor isopropyl-then induces, make its concentration in substratum reach 0.1～0.3mM, continue to cultivate 10～18 hours, recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 2～10 ℃ of conditions with the centrifugal 8～15min of the speed of 2000～8000rpm, collecting supernatant liquor preserves standby under 2～10 ℃ of conditions, with chromatographic system this supernatant liquor of 100～500ml linear velocity with 100～200cm/h is joined in the STREAMLINE25 expanding bed, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 2～10 ℃ of conditions;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above 1.5～2ml with 5～7cm/h is added in the SephadexG-50 Superfine gel chromatography column that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 5～7cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 18.5～19.2ml place, and collected volume is 3.5～4.5ml;
4) drying bu sublimation
To collect liquid in-37～-42 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
3. a kind of method of producing recombinant human epidermal growth factor with gene engineering colibacillus according to claim 1 and 2 is characterized in that its production stage is as follows:
1) engineering bacterium fermentation
Picking list bacterium colony inserts the 50ml seed culture medium from solid medium, add penbritin simultaneously, make its concentration in substratum reach 100mg/l, 32 ℃, the 220rpm shaking table was cultivated 24 hours, insert the nutrient solution of front in the seed culture medium once more by 1% inoculum size, add penbritin simultaneously, make its concentration in substratum reach 100mg/l, 32 ℃, the 220rpm shaking table was cultivated 8 hours, by 1% inoculum size the activatory seed liquor is joined in the fermention medium again, add penbritin simultaneously and make concentration reach 100mg/ml, 32 ℃, 220rpm cultivated 8 hours, adding comfort property inductor isopropyl-then induces, make its concentration in substratum reach 0.2mM, continue to cultivate 14 hours, recombinant human epidermal growth factor enters in the fermented liquid;
2) Expanded Bed Adsorption
With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 10min of the speed of 4000rpm, collecting supernatant liquor preserves standby under 4 ℃ of conditions, with chromatographic system this supernatant liquor of 300ml linear velocity with 183cm/h is joined in the STREAMLINE25 expanding bed that contains STREAMLINE DEAE that the post height is 13.8cm, the uv-absorbing peak value that washs when wavelength is 280nm with the identical linear velocity of sample introduction with the phosphoric acid buffer of 0.02M is zero, use the phosphoric acid elutriant wash-out of the 0.02M that contains 2M NaCl again, collection contains human epidermal growth factor's elution peak, and elutriant is preserved standby under 4 ℃ of conditions;
3) gel separation
With automatic chromatographic system the linear velocity of elutriant above the 1.5ml with 6cm/h is added in the SephadexG-50 Superfine gel chromatography column that the post bed height is 16cm, then distilled water being carried out wash-out with the linear velocity of 6cm/h finishes until going out the peak, detect under wavelength 280nm with Ultraviolet Detector, collecting retention volume is the elutriant at second peak at 19ml place, and collected volume is 4ml;
4) drying bu sublimation
To collect liquid in-40 ℃ of following drying bu sublimations, get white dry powder human epidermal growth factor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01124437 CN1400313A (en) | 2001-07-27 | 2001-07-27 | Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01124437 CN1400313A (en) | 2001-07-27 | 2001-07-27 | Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1400313A true CN1400313A (en) | 2003-03-05 |
Family
ID=4665675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01124437 Pending CN1400313A (en) | 2001-07-27 | 2001-07-27 | Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1400313A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212596A (en) * | 2010-04-01 | 2011-10-12 | 孙民富 | Preparation method of human epidermal growth factor |
| CN106319002A (en) * | 2015-06-19 | 2017-01-11 | 烟台华昕生物科技有限公司 | Preparation method using Escherichia coli to express oligopeptide-1 |
| CN110564661A (en) * | 2019-09-23 | 2019-12-13 | 杭州纽龙生物科技有限公司 | Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor |
-
2001
- 2001-07-27 CN CN 01124437 patent/CN1400313A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212596A (en) * | 2010-04-01 | 2011-10-12 | 孙民富 | Preparation method of human epidermal growth factor |
| CN106319002A (en) * | 2015-06-19 | 2017-01-11 | 烟台华昕生物科技有限公司 | Preparation method using Escherichia coli to express oligopeptide-1 |
| CN110564661A (en) * | 2019-09-23 | 2019-12-13 | 杭州纽龙生物科技有限公司 | Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100354409C (en) | Method for preparing hickory chick by liquid deep fermentation and product thereof | |
| CN1840684A (en) | Process for fermentation preparation of nosiheptide by using streptomycete | |
| CN103233053B (en) | Production method for recombinant human granulocyte colony-stimulating factor | |
| CN1159447C (en) | Microbe fermenting process of producing perfume phenylethanol | |
| CN1300305C (en) | Adenosme generation bacteria and method for generating adenosine by fermentation | |
| CN1966705A (en) | Process for preparing soybean isoflavone aglycon by microorganism enzyme method | |
| CN105670940A (en) | Fungus strain with high-efficiency expression of huperzine A and application thereof | |
| CN1554240A (en) | Bacillus subtilis lipo-peptide biological pesticide and use | |
| CN1515678A (en) | Preparation method of natamycin | |
| CN1683547A (en) | Liquid deep layer fermenting process for producing extracellular polysaccharide using medicinal fungus long root mushroom | |
| CN113308378B (en) | Ganoderma lucidum strain for high-yield ergothioneine and application thereof | |
| CN1400313A (en) | Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus | |
| CN107604026A (en) | A kind of method for improving cordyceps liquid fermentation cordycepin output | |
| CN110832066B (en) | Ivermectin B1B producing strain and application thereof | |
| CN1718735A (en) | Utilize the bacillus natto solid fermentation to produce polyglutamic acid and product application | |
| CN1123577C (en) | Ocean thalassiomycete hypoxylon polyose and its extracting process and application | |
| CN1827771A (en) | Microorganism polysaccharide and its preparation method and application | |
| CN1212404C (en) | Fermentation process for raising ebomycin A yield | |
| CN1190497C (en) | Preparation technology of geldanamycin and its derivative | |
| CN1291031C (en) | Method for preparing recombined thymosin alpha 1 | |
| CN1177051C (en) | Recombinant thymin alpha-1 and its prepn | |
| CN1137139C (en) | Process for producing recombined lymphotoxin derivative | |
| NL2030498B1 (en) | Method for producing p-coumaric acid esterases by degrading sorghum straw with the natural anaerobic fungus-methanogen co-culture | |
| CN1313489C (en) | GP thymosin alpha 1 and preparation method | |
| CN113980821B (en) | Aspergillus niger capable of converting hesperidin and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |