CN104961820B - A kind of recycling chromatography refolding method of recombinant human bone morphogenesis protein-2 - Google Patents
A kind of recycling chromatography refolding method of recombinant human bone morphogenesis protein-2 Download PDFInfo
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- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 37
- 238000004064 recycling Methods 0.000 title claims abstract description 25
- 230000034127 bone morphogenesis Effects 0.000 title claims description 45
- 238000004153 renaturation Methods 0.000 claims abstract description 123
- 239000003480 eluent Substances 0.000 claims abstract description 46
- 239000000539 dimer Substances 0.000 claims abstract description 42
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 41
- 238000004925 denaturation Methods 0.000 claims abstract description 24
- 230000036425 denaturation Effects 0.000 claims abstract description 24
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 16
- 239000012149 elution buffer Substances 0.000 claims description 63
- 108090000623 proteins and genes Proteins 0.000 claims description 43
- 102000004169 proteins and genes Human genes 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 238000010828 elution Methods 0.000 claims description 35
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 20
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- 229920000642 polymer Polymers 0.000 claims description 20
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- 239000006167 equilibration buffer Substances 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
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- 235000019799 monosodium phosphate Nutrition 0.000 claims description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical class [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 6
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- 150000001413 amino acids Chemical group 0.000 claims description 3
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 3
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- 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 description 1
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- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of recycling chromatography refolding methods of rhBMP2.The present invention provides a kind of refolding methods of rhBMP2, include the following steps：1) rhBMP2 inclusion body, albumen after being denaturalized are denaturalized；2) albumen after the denaturation is subjected to affinity chromatography renaturation, collects eluent, obtains rhBMP2 dimer after renaturation；The rhBMP2 amino acid sequence is sequence 2 in sequence table.The experiment proves that 2 albumen of method renaturation rhBMP of the invention, albumen concentration is high, and reagent consumption is few, and up to 70% or more, the method for the present invention produces renaturation yield suitable for industrial scale；The development ＆ production also for similar recombinant protein medicine provides reference simultaneously.
Description
Technical field
The invention belongs to biotechnologies, particularly belong to gene recombinant protein drug field, are related to a kind of recombinant human bone
The recycling chromatography refolding method of morphogenesis protein-2.
Background technology
Bone morphogenetic protein (BMP) is a kind of hydrophobic proteins secreted by bone matrix, belongs to transforming growth factor
(TGF-β) superfamily member is put forward for the first time by Urist et al. in nineteen sixty-five, is defined within 1972, and in nineteen eighty-two for the first time from decalcification
Isolated a kind of reactive protein in bone matrix extract.BMP families include BMP-1,2,4,6,7,9,12,14 etc.,
Middle BMP-2 has the function of that it is osteoblast obviously to induce undifferentiated fibroblast directed differentiation, and can adjust osteoblast
With the differentiation of chondroblast, induced ectopic bon e formation and the function of promoting union.BMP-2 be ostosis startup because
Son can accelerate the reconstruction of bone, be the ideal target gene of Bone Defect Repari gene therapy, be that currently the only can induce ectopic osteogenesis
Cell factor, thus as most important growth factor in bone tissue engineer research.Currently, BMP-2 has been successfully used to control
Treat the reparation of bone nonunion and bone defect.BMP generally without species specificity, has across kind induced osteogenesis ability, thus it is clinical
Application prospect is very wide.
BMP-2 can be by isolating and purifying two methods system after natural ox bone extraction purification and genetic recombination induced expression
It is standby.Wherein natural extraction method is of high cost, low yield, and easy in inactivation is unsuitable for industrialized production, is gradually replaced by method of gene recombination.
The advantages that escherichia expression system relies on technology maturation, expression quantity high, at low cost becomes current method of gene recombination production BMP-
2 hot spot technology.However, the BMP-2 through Bacillus coli expression is present in intracellular with inactive inclusion bodies, how
Inclusion body is obtained into biologically active BMP-2 dimers by renaturation, is the key that in recombinant protein medicine development ＆ production
Step.
Common BMP-2 renaturing inclusion bodies technologies are dilution refolding and dialysis renaturation technology, such as studies in China person Yao is few
China had studied recombination BMP-2 dilution refoldings, dialysis renaturation technique equal to 2005, and had studied the hydroxy-apatite stone of lysozyme
Compose renaturation technique；Sun Fenyong has carried out recombination BMP-2 renaturation technical studies equal to 2006, using dilution refolding technique；Wang Fu
It is beautiful be equal to have studied within 2008 high concentration renaturation technique, using dialysis renaturation technique carry out renaturation.Current renaturation technique exists
It albumen and easily precipitates the problems such as renaturation yield is low, the process route loaded down with trivial details production cycle is long, the big cost height of renaturation solution consumption, be not suitable for
Industrial scale produces.
Invention content
The object of the present invention is to provide a kind of refolding methods of recombinant human bone morphogenesis protein-2 inclusion body.
Method provided by the invention, includes the following steps：
1) recombinant human bone morphogenesis protein-2 inclusion body, albumen after being denaturalized are denaturalized；
2) affinity chromatography renaturation is carried out to albumen after the denaturation, collects eluent successively, renaturation is chromatographed after being recycled
Liquid, recombinant human bone morphogenesis protein-2 monomer, recombinant human bone morphogenesis protein-2 dimer and recombinant human bone form occur
- 2 polymer of albumen；
3) it is denaturalized the recombinant human bone morphogenesis protein-2 polymer, obtains recombinant human bone morphogenesis protein-2 poly
Albumen after body denaturation；
4) albumen and the recombinant human bone form occur after being denaturalized to the recombinant human bone morphogenesis protein-2 polymer
- 2 monomer of albumen carries out affinity chromatography renaturation again, collects eluent, and the recombinant human bone form for obtaining recycling chromatography renaturation occurs
- 2 dimer of albumen；
5) merge the recombinant human bone morphogenesis protein-2 dimer that step 2) obtains and the cycle that step 4) obtains
The recombinant human bone morphogenesis protein-2 dimer of renaturation is chromatographed, realizes answering for recombinant human bone morphogenesis protein-2 inclusion body
Property.
In the above method,
Further include the steps that prepurification between step 1) and step 2)：Albumen after the denaturation is exchanged through cation
Chromatographic purifying collects elution buffer, obtains albuminate after prepurification.
In the above method,
Step 2) includes the following steps：
2) albumen after the denaturation -1), is loaded to affinity column, obtains affinity column after loading, then with chromatography
Renaturation solution flows through affinity column after the loading, stands renaturation；
The chromatography renaturation solution is by final concentration of 0.1mol/L Tris-HCl, final concentration of 0.5mol/L urea, final concentration
For 0.1mol/L 2- Cyclohexylaminos ethanesulfonic acid, final concentration of 0.2mol/L arginine, final concentration of 0.5mmol/L oxidized forms paddy
The sweet peptide of Guang, final concentration of 1mmol/L reduced glutathiones and water composition；
2) -2, with A pairs of elution buffer, 2) -1 treated that chromatographic column elutes, and eluent is collected, after being recycled
Chromatograph renaturation solution；The eluent A is elution buffer FH-A；
2) -3, with B pairs of elution buffer, 2) -2 treated that chromatographic column elutes, and collects eluent, obtains recombined human
Bone morphogenesis protein-2 monomer；The eluent B is the equilibration buffer FH-A and volume fraction by volume fraction 75%
The mixed liquor of 25% elution buffer FH-B-1 compositions；
2) -4, with C pairs of elution buffer, 2) -3 treated that chromatographic column elutes, and collects eluent, obtains recombined human
Bone morphogenesis protein-2 dimer；The eluent C is to be by 55% equilibration buffer FH-A of volume fraction and volume fraction
The mixed liquor of 45% elution buffer FH-B-2 compositions；
2) -5, with D pairs of elution buffer, 2) -4 treated that chromatographic column elutes, and collects eluent, obtains recombined human
Bone morphogenesis protein-2 polymer；The elution buffer D is elution buffer FH-B-3；
The elution buffer FH-A is by final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea and water
Composition；
The elution buffer FH-B-3 is by final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea, end
A concentration of 1mol/L NaCl and water composition；
The elution buffer FH-B-2 is to be adjusted to the NaCl final concentrations in the elution buffer FH-B-3
The solution that 0.45mol/L is obtained；
The elution buffer FH-B-1 is to be adjusted to the NaCl final concentrations in the elution buffer FH-B-3
The solution that 0.25mol/L is obtained.
In the above method,
It is described 2) -2, it is described collect eluent be collection 0.8 column volume eluent；The time of the elution is 25 points
Clock
It is described 2) -3, it is described collect eluent be collection 3 column volumes eluent；The time of the elution is 70 points
Clock,
It is described 2) -4, it is described collect eluent be collection 1.5 column volumes eluent；The time of the elution is 38 points
Clock,
It is described 2) -5, it is described collect eluent be collection 1.5 column volumes eluent；The time of the elution is 25 points
Clock.
In the above method,
Step 4) includes the following steps：
4)-1：Albumen and recombinant human bone form hair after the recombinant human bone morphogenesis protein-2 polymer is denaturalized
- 2 monomer loading affinity column of raw albumen obtains affinity column after loading, then recycling chromatography renaturation solution is flowed through institute
Chromatographic column after loading is stated, renaturation is stood；
4)-2：4) -1 is washed with the elution buffer B) treated chromatographic column；The washing flow velocity is 80 ± 20ml/
min；
4)-3：With elution buffer C elutions 4) -2) treated chromatographic column, eluent is collected, recycling chromatography is obtained
The recombinant human bone morphogenesis protein-2 dimer of renaturation.
In the above method,
4) in -3, the eluent of collecting is the eluent for collecting 0.8 column volume, and the time of the elution is 20 points
Clock.
In the above method,
The bed volume 1L of the affinity column, column internal diameter 100mm, filled media heparin-agarose (Heparin
Sepharose)；
The temperature for standing renaturation is 4 degrees Celsius, the time is 4 days；
The flow velocity that elution is often walked in the affinity chromatography is 80 ± 20ml/min.
In the above method,
In the prepurification, it is described by albumen after the denaturation through cation-exchange chromatography purifying be egg after by the denaturation
Cation-exchange chromatography post is flowed through in vain, then is eluted with elution buffer, the eluent of 1 column volume is collected, obtains prepurification change
Property albumen；
The elution buffer is to be delayed by the elution of the equilibration buffer SP-A and volume fraction 75% of volume fraction 25%
The mixed liquor of fliud flushing SP-B compositions；
The equilibration buffer SP-A is by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea and water
Composition；
The elution buffer SP-B is by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea, end
A concentration of 1mol/L NaCl and water composition；
The time of the elution is 45 minutes；
The flow velocity of the elution is 80 ± 20ml/min；
Chromatographic column bed volume 1.8L, column internal diameter 100mm, the chromatography media of the cation-exchange chromatography be
SPSepharose。
In the above method,
It is described denaturation use denaturing soln by 0.1mol/L Tris-HCl, 8mol/L urea, 0.1mol/L DTT,
5mmol/L EDTA and water composition；
The condition of the denaturation is 25 DEG C, 100r/min vibrates 6 hours.
In the above method,
The amino acid sequence of the recombinant human bone morphogenesis protein-2 is sequence 2；
The chromatography media of the cation-exchange chromatography is SPSepharose；
The chromatography media of the affinity chromatography is Heparin Sepharose.
The experiment of the invention proves that recombinant human bone morphogenesis protein-2 refolding method of the present invention, compared with the conventional method,
Advantage is：
1, compared with the method for common dilution refolding and dialysis renaturation, this technique has place using chromatography renaturation technique
The superiority such as small, protein concentration is big, renaturation yield is high, reagent consumption is few are managed, the reagent consumption of this technique is commonly to dilute
The 10% of renaturation technique, due to expensive reagents in renaturation solution, therefore the technique greatly reduces reagent cost；
2, due to being the purpose that can realize preliminary purification albumen in chromatography on-column refolding, elution samples, this renaturation technique
The dimer sample purity of acquisition reaches 87%, therefore can be able to renaturation and protein purification be simultaneously the another big excellent of this technique
Gesture；
3, renaturation solution can be recycled recycling, to reduce the consumption of reagent, while protein monomers and polymer
It can also recycle and carry out secondary renaturation, to realize that 100% renaturation yield provides possibility.By the monomer of the generation of first time renaturation and
After poly body circulation renaturation, renaturation yield reaches 60%, and more common dilution refolding technique improves about 20%.
4, ion-exchange chromatography prepurification step is increased before renaturing inclusion bodies, removes most polymer, miscellaneous egg
It is white to wait impurity, it is possible to reduce albumen precipitation when renaturation, to improve renaturation yield.
In short, using method provided by the present invention to recombinant human bone morphogenesis protein-2 into renaturation, technique linking is closed
Reason, with short production cycle, gained protein yield is high, of low cost, is very suitable for industrial scale production；The renaturation technique simultaneously
Development ＆ production for similar recombinant protein medicine provides reference.
Description of the drawings
Fig. 1 is albuminate prepurification figure, and P0 is to penetrate peak, predominantly nucleic acid；P1, P2 are foreign protein peak, for the purpose of P3
Protein peak.
Fig. 2 is cycle renaturation figure, and P1 is recombinant human bone morphogenesis protein-2 monomer, and P3 is recombinant human bone morphogenetic protein
- 2 polymer in vain, P2 are purpose Protein reconstitution human bone morphogenesis protein-2 dimer peak.
Fig. 3 is affinity chromatography renaturation and recycles the destination protein SDS-PAGE electrophoresis eluted after renaturation.M is molecule
Marker (1-4 indicates 45kD, 27kD, 16kD, 10kD respectively) is measured, after B1 and B2 are respectively affinity chromatography renaturation and recycle renaturation
The destination protein eluted, 5 be the rhBMP-2 dimers after renaturation, and 6 be the rhBMP-2 monomers of renaturation.
Fig. 4 is rhBMP-2 dimer purity SDS-PAGE electrophoresis after dilution refolding.M is (1-4 points of molecular weight marker
45kD, 27kD, 16kD, 10kD are not indicated), F is recombinant protein solution, and 5 be the rhBMP-2 dimers after renaturation, and 6 be renaturation
RhBMP-2 monomers.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Recombinant human bone morphogenesis protein-2 (rhBMP-2) inclusion body is in patent in following embodiments
It discloses in CN200510132792.9, is specifically obtained according to the method included the following steps：
By recombinant bacterium BL21/pET-28a-BMP2-h, 37 DEG C of culture to OD600 values reach 0.8 in LB culture mediums, are added
The IPTG of final concentration of 1mM, Fiber differentiation 4h under the same terms.After culture, thalline were collected by centrifugation, as BL21/pET-
28a-BMP2-h inclusion bodys, 15%SDS-PAGE electrophoresis detections, size 13.4KD.
The amino acid sequence of recombinant human bone morphogenesis protein-2 (rhBMP-2) is sequence 2 in sequence table, encoding gene
Nucleotides sequence be classified as sequence 1 in sequence table.
Recombinant bacterium is the recombinant vector pET-28a-BMP2-h that will express recombinant human bone morphogenesis protein-2 (rhBMP-2)
It is transferred in e. coli bl21 (DE3), obtains recombinant bacterium BL21/pET-28a-BMP2-h.
The recombinant vector pET-28a-BMP2-h for expressing recombinant human bone morphogenesis protein-2 (rhBMP-2) is by sequence table
RhBMP-2 encoding genes shown in middle sequence 1 are inserted into the NdeI and XhoI of expression vector pET-28a and obtain recombinant vector.
The affinity chromatography renaturation of embodiment 1, recombinant human bone morphogenesis protein-2 rhBMP-2 inclusion bodys
One, rhBMP-2 inclusion bodys are denaturalized
Solubilization of inclusion bodies liquid：0.1mol/L Tris-HCl, 8mol/L urea, 0.1mol/L DTT, 5mmol/L EDTA；
pH8.5.Final concentration of each a concentration of respective components in lysate, solvent is water.
Denaturation：With 1 gram of recombinant human bone morphogenesis protein-2 rhBMP-2 inclusion body：The ratio of 10mL solubilization of inclusion bodies liquid
By solubilization of inclusion bodies, after 25 DEG C, 100r/min vibrate 6 small time variations, 12000g high speed centrifugation 30min, collecting supernatant is
To be denaturalized rhBMP-2 protein solutions.
Two, rhBMP-2 affinity chromatography renaturation after being denaturalized
1, prepurification
In order to improve the purity of albumen, reduce the content of the impurity such as nucleic acid, rhBMP-2 is passed through after the denaturation that above-mentioned one is prepared
Cation-exchange chromatography carries out prepurification, to remove foreign protein.
The chromatographic column specification of use：Bed volume 1.8L, column internal diameter 100mm.
Cation-exchange chromatography medium：SPSepharose (GE companies, 17-0729-04).
Equilibration buffer is SP-A, by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea and
Water forms, pH5.5.
Elution buffer is SP-B, by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea, end
A concentration of 1mol/L NaCl and water composition；pH5.5.
Concrete operations are as follows：
(1) column equilibration：Chromatographic column, balance to efflux are balanced with the flow velocity of 80 ± 20ml/min with equilibration buffer SP-A
Baseline it is steady.
(2) loading and column is washed：The denaturation rhBMP-2 protein solution equilibration buffers SP-A dilutions 15 that above-mentioned one is prepared
It is loaded to after times on (1) processing rear pillar, applied sample amount is 500 ± 50ml, then with 90% (volume fraction) equilibration buffer SP-A+
The mixed liquor (wherein the content of NaCl is 0.1M) of 10% (volume fraction) elution buffer SP-B is with the stream of 80 ± 20ml/min
Speed rinses 1.5 column volumes to chromatographic column, with removal and the untight most of nucleic acid of filling adsorption and thalline foreign protein.
(3) it elutes：With 25% (volume fraction) equilibration buffer SP-A+75% (volume fraction) elution buffers SP-B's
Mixed liquor (wherein the content of NaCl be 0.75M) is to (2) treated pillar carries out one-step elution, 80 ± 20ml/min of flow velocity,
Elution time is 45 minutes, and elution volume is 3.3L, and ultraviolet detection starts to collect when playing peak, and peak to be detected is fallen after rise to baseline
When stop collect (eluting peak P3 shown in Fig. 1), collect 1 column volume eluent, as prepurification be denaturalized rhBMP-2.
2, affinity chromatography renaturation
Chromatograph renaturation solution：By final concentration of 0.1mol/L Tris-HCl, final concentration of 0.5mol/L urea, final concentration of
0.1mol/L 2- Cyclohexylaminos ethanesulfonic acid, final concentration of 0.2mol/L arginine, final concentration of 0.5mmol/L oxidized forms paddy Guang
Sweet peptide, final concentration of 1mmol/L reduced glutathiones and water composition, pH8.5-9.0.
Affinity column loads specification：Bed volume 1L, column internal diameter 100mm.
Chromatography media：Heparin Sepharose (GE companies, 17-0998-03).
Equilibration buffer H-A：It is made of final concentration of 50mmol/L Tris-HCl, final concentration of 8mol/L urea and water；
pH8.5。
Elution buffer FH-A：By final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea and water group
At；pH8.5.
Elution buffer is FH-B-3：By final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea, end
A concentration of 1mol/L NaCl and water composition；pH8.5；
Elution buffer is that FH-B-1 is that the NaCl final concentrations in elution buffer FH-B-3 are adjusted to 0.25mol/L to obtain
The solution arrived；
Elution buffer is that FH-B-2 is that the NaCl final concentrations in elution buffer FH-B-3 are adjusted to 0.45mol/L to obtain
The solution arrived；
Concrete operations are as follows：
(1) column equilibration：Above-mentioned 1 (3) treated layer is balanced with the flow velocity of 80 ± 20ml/min with equilibration buffer H-A
Column is analysed, the baseline of balance to efflux is steady.
(2) loading：It is loaded to (1) after the prepurification denaturation rhBMP-2 that step 1 obtains is diluted 5 times with weighing apparatus buffer solution H-A
On treated column, applied sample amount is 1000 ± 100ml.
(3) on-column refolding：With chromatography renaturation solution, with the flow velocity of 80 ± 20ml/min, to (2) treated, chromatographic column rinses 1
Then chromatographic column is placed in 4 degrees Celsius of refrigerators by a column volume, stand 4 days and carry out renaturation.
(4) recycling of renaturation solution is chromatographed
With elution buffer FH-A, with the flow velocity of 80 ± 20ml/min, to (3) treated, chromatographic column rinses 1 column volume
And efflux is collected, elution time is 25 minutes, and the eluent for collecting 0.8 column volume is to recycle chromatography renaturation solution, can be used
In chromatography renaturation next time；
(5) recycling of rhBMP-2 monomeric proteins：
With the mixed liquor of 75% (volume fraction) equilibration buffer FH-A+25% (volume fraction) elution buffers FH-B-1
(wherein the content of NaCl be 0.25M) with the flow velocity of 80 ± 20ml/min to (4) treated chromatographic column rinses 7 column volumes with
The rhBMP-2 monomeric proteins not yet folded are removed, elution time is 70 minutes, and the eluent for collecting 3 column volumes is rhBMP-2
Monomeric protein；
(6) elution of rhBMP-2 dimers：
With the mixed liquor of 55% (volume fraction) equilibration buffer FH-A+45% (volume fraction) elution buffers FH-B-2
(wherein the content of NaCl be 0.45M) pillar carries out one-step elution, flow velocity 80 ± 20ml/min elution times to (5) treated
It it is 38 minutes, elution volume is 3.8L, and ultraviolet detection starts to collect when playing peak, stops receiving when peak to be detected is fallen after rise to baseline
Collection collects the eluent of 1.5 column volumes.Eluate sample is through SDS-PAGE electrophoresis detections, and (size is rhBMP-2 dimers
26.8KD) content is 86%, and monomer (size 13.4KD) content is 14% (in Fig. 3 shown in B1).
(7) recycling of rhBMP-2 polymers：
With elution buffer FH-B-3 to (6) treated pillar carries out one-step elution, 80 ± 20ml/min of flow velocity, elution
Volume is 2L, and ultraviolet detection starts to collect when playing peak, stops collecting when peak to be detected is fallen after rise to baseline, elution time 25
Minute, the eluent for collecting 1 column volume is rhBMP-2 polymers.
Three, affinity chromatography renaturation is recycled
In order to reduce waste and the reagent consumption of protein sample, to the rhBMP-2 monomeric proteins and step recycled in step 2
The polymer recycled in rapid two carries out recycling chromatography renaturation, and chromatography renaturation solution used is the chromatography renaturation of (3) in the 2 of step 2
Liquid, other reagents and equipment are identical as the 2 of step 2.Concrete operations are as follows：
1, rhBMP-2 polymers are denaturalized：
Solubilization of inclusion bodies liquid used in this step and the solubilization of inclusion bodies liquid phase described in step 1 are same.
Denaturation：With the polymer of above-mentioned two recycling of 1ml：The ratio of 5mL solubilization of inclusion bodies liquid dilutes polymer, in 25
DEG C, 100r/min vibrate 6 hours after, 12000g high speed centrifugation 30min, collect supernatant be denaturalized multimeric protein solution.
2, affinity chromatography renaturation
Affinity column loads specification：Bed volume 1L, column internal diameter 100mm.
Chromatography media：Heparin Sepharose (GE companies, 17-0998-03).
Concrete operations are as follows：
(1) column equilibration：With equilibration buffer H-A with the flow velocity of 80 ± 20ml/min balance above-mentioned two 2 (7) processing after
The baseline of chromatographic column, balance to efflux is steady.
(2) loading：The rhBMP-2 monomeric proteins that 2 (5) of above-mentioned denaturation multimeric protein solution and above-mentioned two are obtained
It is loaded to (1) together after diluting 5 times with weighing apparatus buffer solution H-A treated on column, 80 ± 20ml/min of loading flow velocity.
(3) on-column refolding：Chromatography renaturation solution is recycled with the flow velocity of 80 ± 20ml/min with (4) of 2 with above-mentioned two
To (2) treated, chromatographic column rinses 1 column volume, and then chromatographic column is placed in 4 degrees Celsius of refrigerators, stands 4 days and is answered
Property.
(4) removal of rhBMP-2 monomeric proteins
With the mixed liquor of 75% (volume fraction) elution buffer FH-A+25% (volume fraction) elution buffers FH-B
(wherein the content of NaCl be 0.25M) to (3) treated, chromatographic column rinses 6 column volumes with the flow velocity of 80 ± 20ml/min,
To remove the rhBMP-2 monomeric proteins not yet folded；
(5) elution of rhBMP-2 dimers：
With the mixed liquor of 55% (volume fraction) elution buffer FH-A+45% (volume fraction) elution buffers FH-B
(wherein the content of NaCl be 0.45M) to (4) treated pillar carries out one-step elution, 80 ± 20ml/min of flow velocity, when elution
Between be 20 minutes, elution volume is 2L, and ultraviolet detection starts to collect when playing peak, stops receiving when peak to be detected is fallen after rise to baseline
Collection, the eluent for collecting 0.8 column volume are the rhBMP-2 dimers (B2 of Fig. 2 and Fig. 3) that recycling chromatography renaturation obtains.
The rhBMP-2 dimers that this experiment obtains are mixed with the rhBMP-2 dimers that the 2 of step 2 (6) obtain, are obtained
Dimer protein rhBMP-2 after to renaturation.
Four, the calculating of renaturation yield
It will as follows be detected by the inclusion body for carrying out renaturation the step of above-mentioned one-three：
Dimer purity passes through SDS-PAGE electrophoresis after dimer purity and recycling chromatography renaturation after inclusion body chromatography renaturation
Analysis, the results are shown in Figure 4, and the purity of the two is that 86% and 89% (electrophoretic image system is soft after the photo of shooting glue respectively
Part can account for the percentage of entire swimming lane total amount by the way that each component is calculated)；
Dimer purity albumen concentration can pass through after dimer purity and recycling chromatography renaturation after inclusion body chromatography renaturation
Bradford methods measure, and the two is respectively 0.62mg/ml and 0.42mg/ml；The protein sample volume of the two is 3200ml, multiple
Inclusion body protein concentration is measured as 2.1mg/ml by Bradford methods before property, waits for renaturation inclusion body sample volume 1000ml.
Inclusion body protein amount before dimer protein amount/renaturation after total renaturation yield=renaturation
Dimer protein amount=inclusion body chromatography renaturation dimer protein amount+recycling chromatography renaturation dimer protein after renaturation
Amount
Dimer purity × inclusion body chromatography is multiple after inclusion body chromatographs renaturation dimer protein amount=inclusion body chromatography renaturation
Property after albumen concentration × inclusion body chromatograph refolded protein sample volume
Egg after dimer purity × recycling chromatography renaturation after recycling chromatography renaturation dimer protein amount=recycling chromatography renaturation
White concentration × recycling chromatography refolded protein sample volume
Inclusion body protein amount before renaturation=inclusion body protein concentration before renaturation × waits for renaturation inclusion body sample volume
The total renaturation yield of this technique is calculated up to 60.3% by above-mentioned formula.
Comparative example 1, dilution refolding
It is pre- to have carried out inclusion body in the renaturation process for carrying out recombinant human bone morphogenesis protein-2 by the present inventor
After purification, it additionally uses direct dilution refolding technique and renaturation is carried out to inclusion body, the results show that the renaturation yield of dilution refolding technique
Not as good as the renaturation yield of column chromatography renaturation technique, and reagent consumption is chromatograph renaturation technique 10 times, of high cost.Concrete operations
It is as follows：
One, inclusion body is denaturalized
With the step of embodiment 1 one.
Two, rhBMP-2 dilution refoldings after being denaturalized
1, prepurification
With the step of embodiment 1 two 1, obtain prepurification denaturation rhBMP-2.
2, dilution refolding
Renaturing inclusion bodies liquid：0.1mol/L Tris-HCl, 0.5mol/L urea, 0.1mol/L 2- Cyclohexylamino ethanesulfonic acids
(CHES), 0.2mol/L arginine, 0.5mmol/L oxidizeds form of glutathione, 1mmol/L reduced glutathiones, pH8.5-
9.0。
Using a step direct dilution method renaturation, first to waiting for recombinant protein (prepurification that step 1 obtains be denaturalized rhBMP-2)
The renaturing inclusion bodies liquid that 9 times of volumes are added carries out it ten times of dilutions, is subsequently placed on constant-temperature table and vibrates, 4 DEG C of set temperature,
150 revs/min of rotating speed (shaking table used is the ZWY-211B models shaking table of Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.'s production),
It carries out renaturation 4 days, obtains rhBMP-2 dimers.
After renaturation, the calculating of protein renaturation rate is carried out by following formula：
Albumen concentration before rhBMP-2 dimers purity × refolded protein concentration/renaturation after renaturation yield=renaturation
RhBMP-2 dimers purity is by SDS-PAGE electrophoretic analysis wherein after renaturation, SDS electrophoresis results as shown in figure 4,
The purity of rhBMP-2 dimers is 39%, and refolded protein concentration is measured as 0.19mg/ml by Bradford methods, before renaturation
Albumen concentration is measured as 0.20mg/ml by Bradford methods.RhBMP-2 protein renaturations rate is calculated up to 37%.
Claims (4)
1. a kind of refolding method of recombinant human bone morphogenesis protein-2 inclusion body, includes the following steps：
1）It is denaturalized recombinant human bone morphogenesis protein-2 inclusion body, albumen after being denaturalized；
2）Affinity chromatography renaturation is carried out to albumen after the denaturation, collects eluent successively, renaturation solution, again is chromatographed after being recycled
Group human bone morphogenesis protein-2 monomer, recombinant human bone morphogenesis protein-2 dimer and recombinant human bone morphogenesis protein-2
Polymer；
3）It is denaturalized the recombinant human bone morphogenesis protein-2 polymer, obtains the change of recombinant human bone morphogenesis protein-2 polymer
Albumen after property；
4）Albumen and the recombinant human bone morphogenesis protein-after being denaturalized to the recombinant human bone morphogenesis protein-2 polymer
2 monomers carry out affinity chromatography renaturation again, collect eluent, obtain the recombinant human bone morphogenesis protein-2 of recycling chromatography renaturation
Dimer；
5）Merge step 2）The obtained recombinant human bone morphogenesis protein-2 dimer and step 4）Obtained recycling chromatography
The recombinant human bone morphogenesis protein-2 dimer of renaturation realizes the renaturation of recombinant human bone morphogenesis protein-2 inclusion body；
In step 1）With step 2）Between, further include the steps that prepurification：By albumen after the denaturation through cation-exchange chromatography
Purifying collects elution buffer, obtains albuminate after prepurification；
Step 2）Include the following steps：
2）- 1, albumen after the denaturation is loaded to affinity column, obtains affinity column after loading, then with chromatography renaturation solution
Affinity column after the loading is flowed through, renaturation is stood；
The chromatography renaturation solution is by final concentration of 0.1mol/L Tris-HCl, final concentration of 0.5mol/L urea, final concentration of
0.1mol/L 2- Cyclohexylaminos ethanesulfonic acid, final concentration of 0.2mol/L arginine, final concentration of 0.5mmol/L oxidized forms paddy
The sweet peptide of Guang, final concentration of 1mmol/L reduced glutathiones and water composition；
2）- 2, with A pairs 2 of elution buffer）- 1 treated that chromatographic column is eluted, and collects eluent, is chromatographed after being recycled
Renaturation solution；The elution buffer A is elution buffer FH-A；
2）- 3, with B pairs 2 of elution buffer）- 2 treated that chromatographic column is eluted, and collects eluent, obtains recombinant human bone shape
- 2 monomer of albumen occurs for state；The elution buffer B is the equilibration buffer FH-A and volume fraction 25% by volume fraction 75%
Elution buffer FH-B-1 composition mixed liquor；
2）- 4, with C pairs 2 of elution buffer）- 3 treated that chromatographic column is eluted, and collects eluent, obtains recombinant human bone shape
- 2 dimer of albumen occurs for state；The elution buffer C is to be by 55% equilibration buffer FH-A of volume fraction and volume fraction
The mixed liquor of 45% elution buffer FH-B-2 compositions；
2）- 5, with D pairs 2 of elution buffer）- 4 treated that chromatographic column is eluted, and collects eluent, obtains recombinant human bone shape
- 2 polymer of albumen occurs for state；The elution buffer D is elution buffer FH-B-3；
The elution buffer FH-A is made of final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea and water；
The elution buffer FH-B-3 is by final concentration of 50mmol/L Tris-HCl, final concentration of 3mol/L urea, final concentration
It is formed for 1 mol/L NaCl and water；
The elution buffer FH-B-2 is that the NaCl final concentrations in the elution buffer FH-B-3 are adjusted to 0.45mol/L
Obtained solution；
The elution buffer FH-B-1 is that the NaCl final concentrations in the elution buffer FH-B-3 are adjusted to 0.25mol/L
Obtained solution；
Described 2）- 2, the eluent of collecting is the eluent for collecting 0.8 column volume；The time of the elution is 25 minutes
Described 2）- 3, the eluent of collecting is the eluent for collecting 3 column volumes；The time of the elution is 70 minutes,
Described 2）- 4, the eluent of collecting is the eluent for collecting 1.5 column volumes；The time of the elution is 38 minutes,
Described 2）- 5, the eluent of collecting is the eluent for collecting 1.5 column volumes；The time of the elution is 25 minutes；
Step 4）Include the following steps：
4）- 1, albumen and the recombinant human bone morphogenetic protein after the recombinant human bone morphogenesis protein-2 polymer is denaturalized
- 2 monomer loading affinity column in vain obtains affinity column after loading, then recycling chromatography renaturation solution is flowed through on described
Chromatographic column after sample stands renaturation；
4）- 2, wash 4 with the elution buffer B）- 1 treated chromatographic column；The washing flow velocity is 80 ± 20ml/min；
4）- 3, elute 4 with the elution buffer C）- 2 treated chromatographic columns collect eluent, obtain recycling chromatography renaturation
Recombinant human bone morphogenesis protein-2 dimer；
4）In -3, the eluent of collecting is the eluent for collecting 0.8 column volume, and the time of the elution is 20 minutes；
The chromatography media of the cation-exchange chromatography is SPSepharose；
The chromatography media of the affinity chromatography is Heparin Sepharose；
The amino acid sequence of the recombinant human bone morphogenesis protein-2 is sequence 2.
2. according to the method described in claim 1, it is characterized in that：
The temperature for standing renaturation is 4 degrees Celsius, the time is 4 days；
The flow velocity that elution is often walked in the affinity chromatography is 80 ± 20ml/min.
3. method according to claim 1 or 2, it is characterised in that：
In the prepurification, it is described by albumen after the denaturation through cation-exchange chromatography purifying be protein stream after by the denaturation
It is eluted through cation-exchange chromatography post, then with elution buffer, collects the eluent of 1 column volume, obtain prepurification denaturation egg
In vain；
The elution buffer is by the elution buffer of the equilibration buffer SP-A and volume fraction 75% of volume fraction 25%
The mixed liquor of SP-B compositions；
The equilibration buffer SP-A is by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea and water group
At；
The elution buffer SP-B is by final concentration of 50mmol/L sodium dihydrogen phosphates, final concentration of 8mol/L urea, final concentration
It is formed for 1 mol/L NaCl and water；
The elution time of the prepurification is 45 minutes；
The elution flow rate of the prepurification is 80 ± 20ml/min.
4. method according to claim 1 or 2, it is characterised in that：The denaturing soln that the denaturation uses is by 0.1mol/L
Tris-HCl, 8mol/L urea, 0.1mol/L DTT, 5mmol/L EDTA and water composition；
The condition of the denaturation is 25 DEG C, 100r/min vibrates 6 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002044203A2 (en) * | 2000-11-29 | 2002-06-06 | Scil Proteins Gmbh | Production of recombinant bmp-2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002044203A2 (en) * | 2000-11-29 | 2002-06-06 | Scil Proteins Gmbh | Production of recombinant bmp-2 |
| CN104447977A (en) * | 2013-09-22 | 2015-03-25 | 烟台正海生物技术有限公司 | Purification production method for recombinant human bone morphogenetic protein-2 |
Non-Patent Citations (2)
| Title |
|---|
| On-column refolding of bone morphogenetic protein-2 using cation exchange resin;Anuja M. Rane,et al.;《Protein Expression and Purification》;20130831;第90卷(第2期);摘要，第136页全部 * |
| 蛋白质层析柱复性及工艺评价;高文等;《中国生物工程杂志》;20150331;第35卷(第3期);第84-91页 * |
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