CN106222222B - A kind of preparation method of recombinant human leukemia inhibitory - Google Patents
A kind of preparation method of recombinant human leukemia inhibitory Download PDFInfo
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- CN106222222B CN106222222B CN201610642110.7A CN201610642110A CN106222222B CN 106222222 B CN106222222 B CN 106222222B CN 201610642110 A CN201610642110 A CN 201610642110A CN 106222222 B CN106222222 B CN 106222222B
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- 208000032839 leukemia Diseases 0.000 title claims abstract description 8
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 7
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 12
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000000945 filler Substances 0.000 claims description 26
- 101000942967 Homo sapiens Leukemia inhibitory factor Proteins 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 18
- 102000046645 human LIF Human genes 0.000 claims description 18
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 239000008363 phosphate buffer Substances 0.000 claims description 14
- 238000000746 purification Methods 0.000 claims description 14
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 12
- 239000006166 lysate Substances 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 238000010521 absorption reaction Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 8
- 229920002684 Sepharose Polymers 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 6
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 5
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 5
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims description 4
- 239000013504 Triton X-100 Substances 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 4
- 229920004929 Triton X-114 Polymers 0.000 claims description 4
- 150000001768 cations Chemical class 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- 239000002736 nonionic surfactant Substances 0.000 claims description 4
- 238000005498 polishing Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000012149 elution buffer Substances 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 2
- 150000001412 amines Chemical group 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims 1
- 230000003111 delayed effect Effects 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract 1
- 230000008521 reorganization Effects 0.000 abstract 1
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 6
- 102100032352 Leukemia inhibitory factor Human genes 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000003009 phosphonic acids Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 150000003141 primary amines Chemical group 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5415—Leukaemia inhibitory factor [LIF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to field of medicaments, the specially a kind of application method of preparation with high-purity recombinant human leukemia inhibitory from Bacillus coli cells expression product.Specially apply Bacillus coli expression LIF ELISA albumen, and makes LIF ELISA albumen in solubility expression using low temperature induction, there is high-purity and high activity using the LIF ELISA albumen that cation-exchange chromatography, anion-exchange chromatography and three step purifying method of cation-exchange chromatography obtain later.This method has the characteristics that easy to operate, at low cost, can be used for scale preparation and reorganization LIF ELISA.
Description
Technical field
The present invention relates to field of medicaments, specially a kind of application preparation from Bacillus coli cells expression product has high-purity
Spend the method for recombinant human leukemia inhibitory.
Background technique
LIF ELISA (Leukemia inhibitory factor, LIF) belongs to IL- by 180 Amino acid profiles
6 families.Its intramolecule contains 6 cysteines, can be formed 3 pairs of disulfide bond (cys12-cys134, cys18-cys131,
cys60-cys163).Its receptor includes two subunits, respectively LIFR and gp130, all has intracellular region and transmembrane region, it is known that
It can include JAK/STAT3, PI3K/AKT, ERK1/2 and mTOR signal path by the LIF downstream signaling pathway activated；At present
Know that LIF can promote the proliferation of stem cell and prevent its differentiation, while to embryonic development, nervous system, immune and endocrine system
Function especially tumour cell proliferation and occur have significant impact, in scientific research field, LIF is in embryonic stem cell etc.
That applies in culture is relatively broad, is mainly used for preventing stem cell from Spontaneous Differentiation occurs and maintains its vegetative state.
Hilton DJ et al. applies DEAE sepharose, Lentil Lectin sepharose, CM sepharose
And the reversed chromatography method of Phenyl silica gel purifies LIF albumen from the Krebs ascites cells culture supernatant stimulated through endotoxin
(Hilton DJ et al.Anal Biochem, 1988,173:359-367, WO8807548), but by the resulting LIF of this method
Its expression quantity of albumen is lower, and purification step is relatively complicated, is not appropriate for a large amount of preparations of LIF albumen.
Since the LIF albumen of the aglycosylated modification of procaryotic cell expression is lived compared with the LIF albumen of eukaryotic cell expression
Property it is suitable, therefore also there is researcher to attempt to express LIF albumen using prokaryotic cell such as Escherichia coli.Samal BB et al. is big
The LIF albumen expressed in enterobacteria exists with inclusion bodies, with reference to the method for above-mentioned Hilton DJ et al. after refolding strategy
(Samal BB et al.Biochim Biophys Acta, 1995,1260:27-34) is purified to LIF albumen, still
Since inclusion body needs a large amount of denaturant in denaturation renaturation process, and the efficiency of renaturation is lower, therefore this method is not yet
It is suitble to a large amount of preparations of LIF albumen.In addition there are researchers to attempt in the form of fusion protein in Bacillus coli expression LIF egg
It is white, it usually can promote solubility expression (CN103981205, Jung the AS et of LIF albumen with the albumen of LIF fusion
Al.PLoS ONE, 2013,8:e83781), but since the albumen expressed in the form of fusion is needed by subsequent digestion step
The destination protein of no label can be obtained, process is relatively complicated, and be readily incorporated other albumen (as the enzyme for digestion with
And the protein tag of amalgamation and expression) pollution, while since it is desired that using affinity chromatography in purification process, cost often compared with
It is high.
Therefore, invent it is a kind of can prepare high-purity, high activity and recombination human leukemia easy to operate, at low cost inhibit because
The preparation method of son becomes the task of top priority.
Summary of the invention
Preparation that the purpose of the present invention is to provide a kind of from Bacillus coli cells have the leukaemia of high-purity inhibit because
The method of son.Itself the following steps are included:
(1) Tris-HCL, NaCl, EDTA are mixed, the concentration of the three in mixed liquor respectively reaches 20mM, 30-150mM
And 1mM, one of lauryl sodium sulfate, Triton X-100 or Tween-20 is then added, reaches its concentration
0.5%-0.05% obtains lysate；
(2) to the Escherichia coli for including human leukemia inhibitory factor expression vector be put into LB culture medium, SOB culture medium,
In SOC culture medium, 2 × YT culture medium or TB culture medium, is cultivated at 25-37 DEG C, added when OD600nm reaches 0.5-0.8
The isopropyl-β-D-thiogalactoside (IPTG) for entering 0.5-1.0mM is induced, and adjusts the cultivation temperature of Escherichia coli extremely
16-25 DEG C continue culture 16-30 hour after harvest bacterium, thallus is crushed after lysate is added, separating thallus precipitating with
Supernatant uses NaH2PO4The pH to 6.0 of solution adjustment supernatant；
(3) preliminary purification: using the chromatographic column of cation-exchange chromatography filler, with phosphoric acid, NaCl, EDTA according to 25mM,
50mM and 1mM prepares buffer solution A, buffer solution B is prepared according to 25mM, 500mM and 1mM with phosphoric acid, NaCl, EDTA, with buffer solution A
Balance chromatographic column, the supernatant of acquisition be subjected to loading, then balance chromatographic column using buffer solution A, then with phosphoric acid, NaCl,
EDTA prepares solution according to three's concentration 25mM, 100mM and 1mM, and nonionic surfactant is added, obtains containing 0.01%-
The cleaning buffer solution of 0.1% nonionic surfactant is answered after baseline stability combining the albumen on filler to clean
The elution buffer prepared to buffer solution A and B volume ratio 1:4 elutes protein sample of the combination on chromatographic stuffing,
Eluting peak is collected, ultrafiltration is carried out to the eluting peak of collection and changes liquid processing, is pH's 7.0 by the buffer exchange of elution fraction
25mM phosphate buffer；
(4) moderate purifies: flat with 25mM phosphate buffer (pH7.0) using the chromatographic column of anion-exchange chromatography filler
After the chromatographic column that weighs, the eluant component that liquid is changed after merging is subjected to loading, use is with 25mM phosphate buffer (pH 7.0) balance
Chromatographic column is increased and is fallen after rise to 20mAU-200mAU in UV absorption and collects the component containing sample flowed through；
(5) polishing purification: flat with the 25mM phosphate buffer of pH 7.0 using the chromatographic column of cation-exchange chromatography filler
After the chromatographic column that weighs, above-mentioned collection is flowed through into component loading, reuses the 25mM phosphate buffer balance chromatographic column of pH 7.0,
The buffer C for preparing pH 7.0 according to 25mM, 500mM with phosphoric acid, NaCl, later according to the scale linear transformation pH of Sample Purification on Single
7.0 25mM phosphate buffer and the relative scale of buffer C, using the mode of linear gradient elution in conjunction on filler
Sample is eluted, and is increased and is fallen after rise to 20mAU-200mAU in UV absorption and collect eluting peak component, obtains final weight
Group human leukemia inhibitory factor eluent.
The filler aglucon of cation-exchange chromatography used in preliminary purification can be weak cation class filler, the phosphonic acids of carboxyl type
The middle strong cation class exchange filler of fundamental mode or the strong cation class exchange chromatography filler of sulfonic acid fundamental mode, the nonionic table used
Face activating agent is Triton X-100, Triton X-114 or Tween 20.
Moderate purifying anion-exchange chromatography filler used is the strong base anion exchange chromatography filler of quaternary amine fundamental mode,
Or the weak base type anion-exchange chromatography filler for primary amine groups, secondary amine and tertiary amine fundamental mode.
The filler aglucon of strong cation chromatography used in polishing purification is the cation-exchange chromatography with phosphate groups
Filler preferentially selects hydroxyapatite (Hydroxyapatite).
Detailed description of the invention
Fig. 1 is to identify human leukemia after inducing the Escherichia coli for including human leukemia inhibitory factor expression vector
The gel electrophoresis scanning result of inhibitory factor expression.
Fig. 2 is the chromatographic column using filling cation exchange filler SP Sepharose Fast Flow to pre-processing mistake
E. coli supernatant carry out resulting elution curve after purification.
Fig. 3 is the chromatographic column using filling anion exchange filler Q Sepharose Fast Flow to from SP
The eluent that Sepharose Fast Flow chromatographic column is collected carries out resulting elution curve after purification.
Fig. 4 is the chromatographic column using filling cation exchange filler Hydoxyapatite to from Q Sepharose Fast
The eluent that Flow chromatographic column is collected carries out resulting elution curve after purification.
Fig. 5 is using SDS-PAGE to the purity detecting of the resulting human leukemia inhibitory factor protein sample of each purification step
As a result.
Fig. 6 is the determination of activity result that experiment in vitro is carried out to resulting human leukemia inhibitory factor protein sample.
Specific embodiment
1. the preparation of E. coli lysate
Tris-HCL, NaCl, EDTA are mixed, the concentration of the three in mixed liquor respectively reaches 20mM, 60mM and 1mM,
Then Triton X-100 is added, so that its concentration is reached 0.5%, it is spare to obtain lysate.
The Escherichia coli containing human leukemia inhibitory factor expression vector are inoculated with into LB culture medium, are turned over 37 DEG C, 200
Night culture, is inoculated in the LB culture medium of Fresh with the volume ratio of 1:50 later, shakes culture extremely in 37 DEG C of degree, 200 rotational oscillations
When bacterium OD600nm testing result reaches 0.6, the IPTG that final concentration of 0.5mM is added is induced, and then reduces Bacteria Culture
The temperature of liquid is to 16 DEG C, and the cultivation temperature for changing simultaneously bacterium continues to harvest after 200 rotational oscillations shake culture 20 hours thin to 16 DEG C
Bacterium.
After the weighing of harvested bacterium, lysate (such as bacterium weight in wet base is added according to the volume ratio of 1:10 according to bacterium weight in wet base
For 1g, then the lysate being added is 10mL) it is crushed, supernatant is separated after broken bacterium, uses the NaH of 0.2M2PO4Solution tune
The pH to 6.0 of whole lysate supernatant is spare.Expression application to human leukemia inhibitory factor in cracking liquid precipitate and supernatant
SDS-PAGE is detected, as a result as shown in Figure 1, arrow show the human leukemia inhibitory factor albumen of expression in figure.
2. being captured using cation-exchange chromatography to the LIF ELISA in lysate
With phosphoric acid, NaCl, EDTA according to 25mM, 50mM and 1mM prepare buffer solution A, with phosphoric acid, NaCl, EDTA according to
25mM, 500mM and 1mM prepare buffer solution B, are then filled with cation-exchange chromatography filler SP with buffer solution A balance
Lysate supernatant is carried out loading after baseline stability by the chromatographic column of sepharose fast flow, then uses buffer solution A
Chromatographic column is balanced, then solution is prepared according to three's concentration 25mM, 100mM and 1mM with phosphoric acid, NaCl, EDTA, Triton is added
X-114 obtains the cleaning buffer solution containing 0.1%Triton X-114, to combining the albumen on filler to clean, in base
Line applies protein sample of the elution buffer prepared with buffer solution A and B volume ratio 1:4 to combination on chromatographic stuffing after stablizing
It is eluted, collects eluting peak.Acquisition phase is carried out to the LIF ELISA in lysate using cation-exchange chromatography
The chromatographic results of purifying are as shown in Fig. 2, wherein UV1_280 is the ultraviolet absorption curve of 280nm, and Cond is conductance profile, yin
Shadow show the eluant component of collection.
Ultrafiltration is carried out to the eluting peak of collection and changes liquid processing, the 25mM phosphorus for being pH 7.0 by the buffer exchange of elution fraction
Acid buffer.
3. application anion-exchange chromatography carries out moderate purifying to the LIF ELISA of capture
Anion-exchange chromatography filler Q sepharose fast is filled with 25mM phosphate buffer (pH 7.0) balance
After the chromatographic column of flow, the eluant component for changing liquid after above-mentioned merging is subjected to loading after baseline stability, uses 25mM phosphoric acid
Buffer (pH 7.0) balances chromatographic column, increases and falls after rise to what 20mAU-200mAU collection flowed through in UV absorption and contains sample
The component of product.Using anion-exchange chromatography to LIF ELISA carry out moderate purifying chromatographic results as shown in figure 3, its
Middle UV1_280 is the ultraviolet absorption curve of 280nm, and Cond is conductance profile, and Conc B is the eluent proportional curve of setting；
Shade show the component containing human leukemia inhibitory factor of collection.
4. application cation-exchange chromatography carries out polishing purification to LIF ELISA
The chromatographic column of hydroxyapatite (Hydroxyapatite) is filled with the 25mM phosphate buffer balance of pH 7.0
Afterwards, the component collected above-mentioned steps after baseline stability carries out loading, applies 25mM phosphate buffer (pH 7.0) after loading
Chromatographic column is balanced, the buffer C of pH 7.0 is prepared according to 25mM, 500mM with phosphoric acid, NaCl, later according to three times chromatography cylinder
Long-pending elution liquor capacity is converted into 100% buffer C from the 25mM phosphate buffer of 100% pH 7.0, using linear
The mode of gradient elution in UV absorption raising and is fallen after rise to 20mAU- to combining the sample on filler to elute
Eluting peak component is collected when 200mAU, and is identified.LIF ELISA is carried out using cation-exchange chromatography fine
For the chromatographic results of purifying as shown in figure 4, wherein UV1_280 is the ultraviolet absorption curve of 280nm, Cond is conductance profile,
Conc B is the eluent proportional curve of setting；Shade show the component of the eluent of collection.
The purity of the resulting human leukemia inhibitory factor protein sample of each purification step is detected, as a result such as Fig. 5 institute
Show, is the human leukemia inhibitory factor albumen of expression indicated by arrow in figure.
5. the active determination in vitro of human leukemia inhibitory factor
By M1 cell inoculation to 96 orifice plates, 40000/hole, 50 μ L culture medium (DMEM+10%Fetal calf are added
Serum), 50 μ L proportionally diluted purified obtained human leukemia inhibitory factor protein sample is then added；Using city
The human leukemia inhibitory factor sold is diluted, and keeping final volume also is 50 μ as control according to identical dilution process
L, addition have been inoculated in 96 orifice plates of M1 cell.Using CCK-8 detection reagent according to producer after 37 DEG C of culture different times
The method measurement of recommendation is to the inhibition situation of M1 cell Proliferation, with the expression activitiy of commercial goods human leukemia inhibitory factor,
As a result as shown in fig. 6, wherein Puri-LIF represents human leukemia inhibitory factor egg prepared by application institute's procedures set forth of the present invention
It is white to obtain as a result, Mill-LIF is is obtained result using commercially available human leukemia inhibitory factor albumen.
Beneficial effects of the present invention now are further described by embodiment of above and in conjunction with attached drawing, it is thus understood that real
The mode of applying is only used for the purpose of illustration, does not limit the scope of the invention, while those of ordinary skill in the art institute according to the present invention
The obvious change and modification made are also contained within the scope of the invention.
Claims (1)
1. a kind of preparation method of recombinant human leukemia inhibitory, comprising the following steps:
(1) Tris-HCL, NaCl, EDTA are mixed, the concentration of the three in mixed liquor respectively reaches 20mM, 60mM and 1mM, so
Triton X-100 is added afterwards, so that its concentration is reached 0.5%, obtains lysate；
(2) Escherichia coli for including human leukemia inhibitory factor expression vector are put into culture medium and are cultivated at 37 DEG C,
The isopropyl-β-D-thiogalactoside that 0.5mM is added when OD600nm reaches 0.6 is induced, and the training of Escherichia coli is adjusted
It supports after temperature continues culture 20 hours to 16 DEG C and harvests bacterium, thallus is crushed after lysate is added, separating thallus precipitating
And supernatant, use NaH2PO4The pH to 6.0 of solution adjustment supernatant；
(3) preliminary purification: being the strong cation class exchange chromatography filler SP sepharose fast of sulfonic acid fundamental mode using aglucon
Flow load chromatographic column, with phosphoric acid, NaCl, EDTA according to 25mM, 50mM and 1mM prepare buffer solution A, with phosphoric acid, NaCl,
EDTA prepares buffer solution B according to 25mM, 500mM and 1mM, then balances chromatographic column with buffer solution A, and the supernatant of acquisition is carried out
Loading then proceedes to balance chromatographic column using buffer solution A, then with phosphoric acid, NaCl, EDTA according to three's concentration 25mM, 100mM and
1mM prepares solution, and nonionic surfactant Triton X-114 is added, obtains containing 0.1% nonionic surfactant
The cleaning buffer solution of Triton X-114, to combining the albumen on filler to clean, using with buffering after baseline stability
The elution buffer that liquid A and B volume ratio 1:4 is prepared collects elution to combining the protein sample on chromatographic stuffing to elute
Peak carries out ultrafiltration to the eluting peak of collection and changes liquid processing, the 25mM phosphoric acid that the buffer exchange of elution fraction is pH 7.0 is delayed
Fliud flushing；
(4) moderate purifies: being the strong base anion exchange chromatography filler Q sepharose fast of quaternary amine fundamental mode using aglucon
The chromatographic column that flow is loaded will change the eluent group of liquid after balancing chromatographic column with the 25mM phosphate buffer of pH 7.0 after merging
Divide and carry out loading, continue to use and chromatographic column is balanced with the 25mM phosphate buffer of pH 7.0, increases and fall after rise in UV absorption
The component containing sample flowed through is collected to 20mAU-200mAU；
(5) polishing purification: being what the cation-exchange chromatography filler hydroxyapatite with phosphate groups loaded using aglucon
Above-mentioned collection is flowed through component loading, reused by chromatographic column after balancing chromatographic column with the 25mM phosphate buffer of pH 7.0
The 25mM phosphoric acid buffer of pH 7.0 balances chromatographic column, prepares the buffer C of pH 7.0 according to 25mM, 500mM with phosphoric acid, NaCl,
Later according to the relative scale of the 25mM phosphate buffer of the scale linear transformation pH 7.0 of Sample Purification on Single and buffer C, application
The mode of linear gradient elution in UV absorption raising and is fallen after rise to 20mAU- to combining the sample on filler to elute
200mAU collects eluting peak component, obtains final recombinant human leukemia inhibitory eluent.
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| CN1119018A (en) * | 1993-03-05 | 1996-03-20 | 先灵公司 | Purification of human interleukin-10 |
| CN1616489A (en) * | 2004-09-30 | 2005-05-18 | 中国科学技术大学 | Method for purifying and recombining human iterleukin-12 |
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| CN102477098A (en) * | 2010-11-29 | 2012-05-30 | 清华大学深圳研究生院 | Fusion protein and application thereof in preparation of human leukemia inhibitory factor |
| CN103981205A (en) * | 2014-05-23 | 2014-08-13 | 上海同科生物科技有限公司 | Method for recombinant expression of human leukemia inhibitory factor |
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| CN1119018A (en) * | 1993-03-05 | 1996-03-20 | 先灵公司 | Purification of human interleukin-10 |
| CN1616489A (en) * | 2004-09-30 | 2005-05-18 | 中国科学技术大学 | Method for purifying and recombining human iterleukin-12 |
| WO2009058812A1 (en) * | 2007-10-30 | 2009-05-07 | Genentech, Inc. | Antibody purification by cation exchange chromatography |
| CN102477098A (en) * | 2010-11-29 | 2012-05-30 | 清华大学深圳研究生院 | Fusion protein and application thereof in preparation of human leukemia inhibitory factor |
| CN103981205A (en) * | 2014-05-23 | 2014-08-13 | 上海同科生物科技有限公司 | Method for recombinant expression of human leukemia inhibitory factor |
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