CN100336825C - Renaturation of reconstituted human bone protein-1 and making method of its preparation - Google Patents
Renaturation of reconstituted human bone protein-1 and making method of its preparation Download PDFInfo
- Publication number
- CN100336825C CN100336825C CNB2005100456000A CN200510045600A CN100336825C CN 100336825 C CN100336825 C CN 100336825C CN B2005100456000 A CNB2005100456000 A CN B2005100456000A CN 200510045600 A CN200510045600 A CN 200510045600A CN 100336825 C CN100336825 C CN 100336825C
- Authority
- CN
- China
- Prior art keywords
- renaturation
- protein
- chromatography
- preparation
- mol
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention discloses the renaturation of a recombined human osteogenesis protein-1 and a preparation method for a preparation agent of the recombined human osteogenesis protein-1. A rhOP-1 monomer protein whose purity is more than 95 percent is obtained by chromatography is dialyzed in 2M to 6M of urea or 1M to 4M of a guanidine hydrochloride buffer solution in order to carry out the renaturation; the pH value of the renaturation buffer solution is from 8.0 to 10; the renaturation temperature is a room temperature or 4 DEG C.; the renaturation time is from 24 to 96 hours. Then, the purity of a target protein dimer reaches more than 90% by further chromatography and purification; freeze-drying powder of a genetic engineering product of a rhOP-1 mature peptide is obtained by a normal mode of dialyzing, degerming, subpackaging and freeze drying, and the freeze-drying powder or target protein liquid purified by chromatography is directly combined and sterilized with a collagen protein, an absorbable gelatin sponge, chitosan or materials of coral powder classes or materials of bone tissue engineering of synthetic macromolecules to prepare the renaturation preparation agent of the recombined human osteogenesis protein-1; the obtained preparation agent can be used for repairing bone defects, rectifying shapes, maintaining and restoring tooth extraction wound tooth space bones in mouth cavities, etc., and also be used for curing apoplexy, kidney diseases, etc.
Description
Technical field:
The present invention relates to a kind of formulation preparation method of Osteogenic Protein-1, relate in particular to the formulation preparation method of a kind of recombinant human Osteogenic Protein-1 (rhOP-1).Be to utilize genetic engineering means, utilize the method for escherichia coli expression and mass production recombinant human Osteogenic Protein-1 mature peptide, the reorganization human osteogenic protein-1 is carried out renaturation and formulation preparation thereof.Can be used for the damaged reparation of bone, orthopedic and be used for keeping and recovery etc. of oral cavity tooth extraction wound alveolar bone after the preparation sterilization of the present invention preparation clinically.Belong to the biotechnological pharmaceutics field.
Background technology:
Along with the fast development of genetic engineering technique, people provide some protein and peptide products from the heterogenous expression of matter for clinical and industrial production by egg more and more.Up to now, E.coli with its easy handling, genetic background is clear, fermentation costs is low and the protein expression level advantages of higher, still is the first-selected expression system of producing recombinant protein.But expressed proteins usually is deposited in the cell with the form of inclusion body in E.coli, show as the insoluble aggregate of non-activity, how efficiently recombinant protein is a most critical and the most difficult step in the engineered protein production process, has become one of bottleneck of industrialization
(Osteogenic protein-1, OP-1), (Bonemorphogenetic protein-7 BMP-7), is a member in the TGF-beta superfamily to Osteogenic Protein-1 to have another name called bone morphogenetic protein-7.OP-1 is a kind of multiduty albumen: the damaged of treatment bone and cartilage reaches in orthopedic and orthopedic application; Therapeutic action aspect fracture is proved very effective by a large amount of clinical trials, and it is damaged that it can cure nonunion and bone that existing all technology can't cure; Aspect the treatment periodontopathy: rhOP-1 can also promote that periodontal is rebuild, the alveolar ridge bone density increases, promote Dentinal formation and cemental formation, this plantation to artificial tooth has great application prospect (Giannobile MV et al., J Periodontol., 1998,69:129; ).Research finds that also OP-1 also has broad application prospects in the treatment of the difficult diseases such as regeneration of osteoporosis, renal failure and nervous tissue.
Studies show that the BMP of natural extract has the activity that lures bone and repair cartilage, but has cost height, acquisition amount low (1 μ g/kg), activity to be difficult to shortcomings such as maintenance; Utilize the recombinant human bone morphogenetic protein of genetic engineering technique preparation to overcome the BMP extraction of natural origin and the limitation of purifying, its clinical value more and more receives publicity.The expression system that is used for genetically engineered drug production at present mainly contains eukaryotic expression system and prokaryotic expression system, though the former has active high advantage, its expression vector establishment complexity, cell transfection rate are low, cell clone select loaded down with trivial details, expressing quantity is extremely low and purifying difficulty.Above-mentioned deficiency has limited applying clinically.That prokaryotic expression system has is easy and simple to handle, stable gene, mass producible advantage.The OP-1 mature peptide has three glycosylation sites, and BMP-3 has two glycosylation sites, has the author to report and successfully uses escherichia coli expression BMP-2, BMP-3 mature peptide, has good bone-inducing activity after renaturation.
Utilize genetic engineering technique can obtain a large amount of target proteins in the prokaryotic expression living things system, still, the renaturation of prokaryotic expression OP-1 but is a challenging difficult problem always.The prokaryotic gene engineering product has production cost low, the expression amount height, and inclusion body protein is easy to advantages such as purifying.But the renaturation work of inclusion body is a difficult point and key link, if this problem can not get fine solution, the protein active of institute's renaturation is bad, renaturation yield is not high, and the prokaryotic gene engineering product economical and effective that just is far from being so is even expression amount is high more also meaningless.
Be cloned into the mature protein gene of OP-1 and set up rhOP-1 the host bacterium that to efficiently express system be present routine techniques, at home and abroad BMP-2, BMP-3 are in the success of intestinal bacteria system expression.OP-1 is a kind of more special protein, successful expression renaturation in eukaryotic cell CHO once, but yield poorly the cost height.Utilize the product of intestinal bacteria system expression people OP-1, though a large amount of evaluation articles is arranged, but all because of its renaturation that is bound to arouse fear (David CRueger, Biochenistry of bone morphogeneti proteins, in Bone MorphogeneticProteins From Laboratory To Clinical Practice/Slobodan Vukicevic, KuberT.Sampath ed Basel.Bosdton.Berlin:Birkhauser.2002 1-18) is restricted its use.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides the formulation preparation method of a kind of rhOP-1.This method can be at the rhOP-1 that can obtain to have clinical value.RhOP-1 after the renaturation through behind the purifying directly and carrier compound, can be prepared into can be used for the damaged reparation of bone, orthopedic and be used for oral cavity tooth extraction wound alveolar bone keep and preparation such as recovery is applied to clinical.
The present invention utilizes genetically engineered routine techniques design primer, has obtained goal gene from people's embryonic tissue; Utilize genetic engineering technique construction expression plasmid, by host bacterium transformation experiment screening, set up rhOP-1 and efficiently express system, made up and contained target protein OP-1 mature peptide gene (be people OP-1 mature peptide gene order, its nucleotides sequence is classified as
Http:// www.ncbi.nlm.nih.gov/, disclosed SEQ ID NO.1:
http：//www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nucleotide&val＝4502426
Open sequence: mat_peptide 999---1415/gene=" BMP7 ";
Is described nucleotide sequence SEQ ID NO.1 expressed protein sequence that (139 amino acid, its sequence are http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi to SEQ ID NO.2? val=4502427﹠amp; ItemID=7﹠amp; The open sequence of view=gpwithparts) expression system; The host bacterium is the BL21 series used of routine techniques, DH5 α intestinal bacteria (1. Li Bangyin; Zhuan Yuhui: the cloning and expression biotechnology communication 1999.03.30 of human osteogenic protein-1 mature peptide gene in intestinal bacteria; 10 (1): 17-19; 2. Chen Wen; Shi Junnan; Fu Shihong; Sun Yefang; Liang Guodong; Hou Yunde: the modification of human osteogenic protein-1 mature peptide gene 5 ' end and the expression tooth body dental pulp periodontology magazine 1998.03.30 in intestinal bacteria thereof; 8 (1): 3-6).
The present invention utilizes thermal induction to express, and has obtained the high expression level of target protein matter, and the expression amount of target protein accounts for about 30% of bacterial protein.RhOP-1 form with inclusion body in intestinal bacteria exists, through splitting bacterium, washing, centrifugal acquisition inclusion body, the guanidine hydrochloride dissolution inclusion body of 8 moles of 1/L urea or 6 mol, behind the centrifugal collection supernatant at first to the metaprotein solution purification.The renaturation of rhOP-1 is that (more than 95%) carries out renaturation again obtain highly purified rhOP-1 monomeric protein by column chromatography after, and the renaturation of rhOP-1 can be carried out in 6M urea or 4M Guanidinium hydrochloride damping fluid, renaturation temperature room temperature or 4 degree, and the pH value of rhOP-1 renaturation buffer is between 8.0～10.The advantage of present method is: recombinant protein matter concentration can be higher protein concentration, 100～1000ug/ml.This can improve annealing efficiency greatly, makes things convenient for subsequent purification.
The renaturation of rhOP-1 also can be carried out in 2M urea or 1M Guanidinium hydrochloride damping fluid, but this moment, the renaturation buffer of rhOP-1 must add the L-arginine, and will be with concentration dilution to 10～100 μ g/ml of rhOP-1, its pH value is between 8.0～10, otherwise the renaturation poor effect, even the protein aggregation precipitation appears.
The formulation preparation method of the rhOP-1 that the present invention relates to, it is characterized in that obtaining the rhOP-1 monomeric protein of 95% above purity by chromatography, carry out renaturation at 2M～6M urea or/and dialyse in 1M～4M Guanidinium hydrochloride damping fluid, renaturation temperature room temperature or 4 degree, the pH value of renaturation buffer is 8.0～10, and sampling in 6～12 hours at interval, use the SDS-PAGE of irreducibility to detect the situation that dimer forms, 24～96 hours renaturation time; Through further chromatography purification the dimeric purity of target protein is reached more than 90% more afterwards, after dialysis, degerming, packing and lyophilize, obtain freeze-drying part of genetic engineered product of rhOP-1 mature peptide or in a usual manner that target protein and collagen protein, absorbable gelatin sponge, chitosan or coral powder class material or synthetic macromolecule engineering material of bone tissue is compound in a usual manner, sterilize, make the application preparation of rhOP-1.
In above-mentioned method, described renaturation adopts at lower concentration denaturing agent 2M urea or/and when renaturation was carried out in dialysis in the 1M Guanidinium hydrochloride damping fluid, the arginine of 0.2～1.0 mol was necessary.
Above-mentioned chromatography is an ion exchange chromatography or/and hydrophobic chromatography, reversed phase chromatography, affinity chromatography, molecular exclusion chromatography.
In the above-mentioned renaturation step, can add tensio-active agent, redox system, renaturation additive.
In above-mentioned method, described tensio-active agent is one of Triton 100, Triton 114, polysorbas20, tween 80, sarcosyl, hexadecyl brometo de amonio, NP40, and its consumption concentration is volume percent 0.05%～1%.
In above-mentioned method, described redox system be 0.1 micromoles per liter～0.1 mmole/liter cupric ion or/and ratio is a mol ratio from reductive agent/oxidizer system of 10: 1 to 1: 2, wherein the concentration range of reductive agent be 0.05～10 mmole/liter.
Wherein, described reductive agent is: one of reduced glutathion, halfcystine and hydrochloride thereof, mercaptoethanol, dithiothreitol (DTT), the red tinea sugar alcohol of two sulphur.
Wherein, described oxygenant is: one of the red tinea sugar alcohol of the dithiothreitol (DTT) of Sleep-promoting factor B, oxidized form and two sulphur, Gelucystine and hydrochloride thereof.
In above-mentioned method, the Macrogol 4000 that described renaturation additive is 0.05～10 grams per liter or poly-second two ferment 6000 or polyoxyethylene glycol 8000 or cetomacrogol 1000 0; 0.02 the arginine of～1.0 mol or its hydrochloride or other basic aminoacids or its hydrochloride or three (methylol) aminomethane, volume percent is 1%～30% glycerine.
Wherein, described renaturation additive is the Macrogol 4000 of 0.05～10 grams per liter or the arginine of poly-second two ferment 6000,0.4～1.0 mol preferably, or three (methylol) aminomethane, and volume percent is 1%～20% glycerine.
In above-mentioned method, the renaturation preparation of the rhOP-1 of described system is to be used for the damaged reparation of bone, orthopedic and be used for the renaturation preparation of the rhOP-1 that keeps and recover of oral cavity tooth extraction wound alveolar bone clinically.
Launch to further specify with regard to foregoing below:
The present invention obtains the target product with the inclusion body formal representation after engineering bacteria ferments, splits bacterium, washing through routine, inclusion body dissolves through the urea of 7～8 mol or the guanidine hydrochloride denaturation of 5～6 mol; Ion exchange chromatography (or/and hydrophobic chromatography again, reversed phase chromatography) after purifying reaches purity more than 95%, at the urea that contains relative lower concentration denaturing agent 4～6 mol or/and 2～4 mol Guanidinium hydrochlorides, or the tensio-active agent of adding lower concentration (volume percent 0.05-1%) is (as Triton 100, Triton 114, polysorbas20, tween 80, sarcosyl, the hexadecyl brometo de amonio, one of NP40), suitable redox system (as 0.1 micromoles per liter～0.1 mmole/liter cupric ion or/and reductive agent/oxidant ratio is that mol ratio was from 10: 1 to 1: 2, wherein the concentration range of reductive agent 0.05～10 mmole/liter, employed reductive agent can be: reduced glutathion, halfcystine and hydrochloride thereof, mercaptoethanol, dithiothreitol (DTT), one of red tinea sugar alcohol of two sulphur, employed oxygenant can be: Sleep-promoting factor B, the red tinea sugar alcohol of the dithiothreitol (DTT) of oxidized form and two sulphur, one of Gelucystine and hydrochloride thereof); And other renaturation additive (as: Macrogol 4000 of 0.05-10 grams per liter or poly-second two ferment 6000 or polyoxyethylene glycol 8000 or cetomacrogol 1000 0,0.02 the arginine of～1.0 mol and hydrochloride thereof or other basic aminoacids and hydrochloride or three (methylol) aminomethane thereof, 1～30% glycerine and a certain amount of heparin) meta-alkalescence (pH8.0～10.0) renaturation solution in, renaturation temperature room temperature or 4 degree, renaturation 24～96 hours (6～12 hours SDS-PAGE with irreducibility detect the situation that dimer forms at interval), pass through further chromatography purification (ion-exchange again, anti-phase, hydrophobic, non-special affine and size-exclusion) purity of target protein is reached more than 90%, then 1. through dialysis, degerming, obtain freeze-drying part of the genetic engineered product of rhOP-1 mature peptide after packing and the lyophilize.2. target protein and collagen protein, absorbable gelatin sponge, chitosan or coral powder class material, other artificial engineering materialss are compound, can be used for the damaged reparation of bone, orthopedic and be used for keeping and recovery etc. of oral cavity tooth extraction wound alveolar bone.
Perhaps will be through ion exchange chromatography (or/and hydrophobic chromatography, reversed phase chromatography) purifying reaches the rhOP-1 monomer of 95% above purity, its urea or 1 mol Guanidinium hydrochloride with 2 mol is diluted to 10～100 μ g/ml, and add the arginine of 0.2～1.0 mol therein, preferred 0.3～0.8 mol arginine, and its pH value should be between 8.0～10,24～60 hours (see figure 3)s of renaturation, pass through further chromatography purification (ion-exchange again, anti-phase, hydrophobic, non-special affine and size-exclusion) the dimeric purity of target protein is reached more than 90%, then 1. through dialysis, degerming, obtain freeze-drying part of the genetic engineered product of rhOP-1 mature peptide after packing and the lyophilize.2. target protein and collagen protein, absorbable gelatin sponge, chitosan or coral powder class material, decalcified bone matrix and other people worker's tissue engineering material are compound, can be used for the damaged reparation of bone, orthopedic and be used for keeping and recovery etc. of oral cavity tooth extraction wound alveolar bone.
The invention provides the refolding method of a kind of rhOP-1, this method can be at the rhOP-1 that can obtain to have clinical value.RhOP-1 after the renaturation through behind the purifying directly and carrier compound, can be used for the damaged reparation of bone, orthopedic and be used for keeping and recovery etc. of oral cavity tooth extraction wound alveolar bone after the goods sterilization of the present invention preparation clinically, also can prepare becomes the treatment that other preparations are used for apoplexy, kidney disease etc.
The purifying of this experiment, renaturation technology are simple and easy to do, have obtained rhOP-1 separation and purification, the renaturation process program of laboratory level, for the scale operation of rhOP-1, the further research and the rhOP-1 of 26S Proteasome Structure and Function are applied to clinical laying a good foundation early.
Description of drawings
Fig. 1: be the figure of the SDS-PAGE before the rhOP-1 renaturation.
Sample before the renaturation is monomer, and wherein: 2 swimming lanes are standard protein Marker (molecular weight standard is followed successively by 97.4,66,43,31 from top to bottom, 20.1 and 14.4Kd).
Fig. 2: be the reductibility SDS-PAGE figure of the renaturation effect under the different renaturation conditions of rhOP-1.
Wherein: the 1-7 swimming lane is the different condition renaturation of same sample, and 3 swimming lanes are best practice of the present invention, and renaturation yield has 40% approximately.10 swimming lanes are standard protein Marker, and molecular weight standard is followed successively by 97.4,66,43,31 from top to bottom, 20.1 and 14.4Kd.
Fig. 3: the renaturation result under the lower concentration denaturing agent different condition.
Wherein: the 1-6 swimming lane is the renaturation result of pH＜8 o'clock; 7 swimming lane .pH8.0 renaturation, 8 swimming lane .pH8.0 renaturation (containing L-Arg), 9 swimming lane .pH9.0 renaturation, 10 swimming lane .pH9.0 renaturation (containing L-Arg).
Embodiment:
Embodiment 1:
The rhOP-1 engineering bacteria by fermentation, centrifugal collection thalline, split bacterium, washing obtains inclusion body, inclusion body is through the urea-denatured dissolving of 8 mol, after ion-exchange again (or/and hydrophobic, affine and size-exclusion) chromatography is collected purifying and reached purity more than 95%, it is diluted to 200～600 μ g/ml.After pack into through in processed conventionally dialysis tubing (molecular weight cut-off is 10000), (pH8.0～9.5) air at room temperature oxidizing and refolding is 24～96 hours in the renaturation solution of the arginine of the Macrogol 4000 of the urea of 6 mol, 0.05 grams per liter, 0.4～0.6 mol, 20 mmoles/rise phosphate buffered saline buffer, through further ion exchange chromatography, size-exclusion purifying the dimer purity of target protein is reached more than 90% again.The urea of the urea of the urea of the urea of 5 mol, 4 mol, 2 mol, 1 mol dialysis is to 20 mmoles/rise phosphate buffered saline buffer again, and lyophilize obtains the rhOP-1 lyophilized powder.-20 ℃ of preservations are standby behind the ethylene oxide sterilizing.
Perhaps (one): dimer purity is reached the ratio of 90% the above object albumen (urea of 6 mol) with 1mg: 5～20mg, directly compound with a certain amount of absorbable gelatin sponge material.Compound tense is taken out repeatedly in vacuum system, is exitted, to the bubble of almost completely driving away in the gelfoam material, compound finishing.Again through the urea dialysis of the urea of the urea of the urea of 5 mol, 4 mol, 2 mol, 1 mol to 20 mmoles/rise phosphate buffered saline buffer.Be sub-packed in after the lyophilize in polyethylene bag or other suitable containers, ethylene oxide sterilizing promptly gets and can be used for the damaged reparation of bone, orthopedic and be used for keeping and the preparation of recovery etc. of oral cavity tooth extraction wound alveolar bone.
(2), with the rhOP-1 lyophilized powder after the above-mentioned lyophilize, the urea of 6 mol dissolving, with the ratio of 1mg: 1～20mg, compound with the commercially available absorbable gelatin sponge material that becomes about diameter 5mm with the punch tool preparation.Take out repeatedly, exit, to almost completely driving away gelfoam material bubble, compound finishing.Again step by step urea dialysis to 20 mmoles/rise phosphate buffered saline buffer.Be sub-packed in after the lyophilize in the appropriate vessel, the preparation of ethylene oxide sterilizing after 50 minutes can be used for keeping of oral cavity tooth extraction wound alveolar bone and recovery etc.
Embodiment 2:
The rhOp-1 engineering bacteria by fermentation, centrifugal collection thalline, split bacterium, washing obtains inclusion body, inclusion body is through the guanidine hydrochloride denaturation dissolving of 6 mol; After ion-exchange again (or/and hydrophobic, affine and size-exclusion) chromatography is collected purifying and is reached purity more than 95%, the arginine of the Guanidinium hydrochloride of 4 mol and 0.2～0.5 mol, 0.1 micromoles per liter～0.1 mmole/liter cupric ion (cupric chloride or copper sulfate); The room temperature renaturation is 24～96 hours in 20 mmoles/liter phosphatic renaturation solution (pH8.0～9.0), passes through further ion exchange chromatography purifying (or/and anti-phase, hydrophobic, non-special affine and size-exclusion) again the purity of target protein is reached more than 90%.The sample that obtains is direct and solid support material (one of collagen protein, absorbable gelatin sponge, chitosan or coral powder class, synthetic macromolecule engineering material of bone tissue) is compound, compound tense vacuum is repeatedly taken out, is exitted, and then dialysis is to 20 mmoles/rise phosphate buffered saline buffer, place the freeze drier freeze-drying ,-20 ℃ of preservations are standby behind the ethylene oxide sterilizing.
Embodiment 3:
With the urea-denatured dissolving of inclusion body, after ion exchange chromatography is collected purifying and reached purity more than 95% again, it is diluted to 100～600 μ g/ml through 8 mol.After pack into through in processed conventionally dialysis tubing (molecular weight cut-off is 10000), directly (pH9.0～9.5) air at room temperature oxidizing and refolding 24～96 hours in the renaturation solution of the urea of 6 mol, 20 mmoles/rise phosphate buffered saline buffer reaches more than 90% the dimer purity of target protein through further ion exchange chromatography, size-exclusion purifying again.The urea of the urea of the urea of the urea of 5 mol, 4 mol, 2 mol, 1 mol dialysis is to 20 mmoles/rise phosphate buffered saline buffer again, and lyophilize obtains the rhOP-1 lyophilized powder.-20 ℃ of preservations are standby behind the ethylene oxide sterilizing.Perhaps dimer purity is reached the ratio of 90% the above object albumen with 1mg: 5～10mg, directly compound with a certain amount of absorbable gelatin sponge material.Compound tense is taken out repeatedly in vacuum system, is exitted, to the bubble of almost completely driving away in the gelfoam material, compound finishing.Dialyse to distilled water through gradient urea again.Be sub-packed in after the lyophilize in polyethylene bag or other suitable containers, ethylene oxide sterilizing promptly gets and can be used for the damaged reparation of bone, orthopedic and be used for keeping and the preparation of recovery etc. of oral cavity tooth extraction wound alveolar bone.
Embodiment 4:
With the urea-denatured dissolving of inclusion body, after ion exchange chromatography is collected purifying and reached purity more than 95% again, it is diluted to 200～600 μ g/ml through 8 mol.After pack into through in processed conventionally dialysis tubing (molecular weight cut-off is 10000), urea in 6 mol, the tensio-active agent polysorbas20 that adds lower concentration (volume percent 0.1-1%), redox system (as 0.1 micromoles per liter～0.1 mmole/liter cupric ion or/and reductive agent/oxidant ratio is that mol ratio was from 10: 1 to 1: 2, wherein the concentration range of reductive agent 1～10 mmole/liter, employed reductive agent can be: reduced glutathion, halfcystine and hydrochloride thereof, mercaptoethanol, half Guang ammonia, dithiothreitol (DTT), the red tinea sugar alcohol of two sulphur, employed oxygenant can be: Sleep-promoting factor B, the red tinea sugar alcohol of the dithiothreitol (DTT) of oxidized form and two sulphur, Gelucystine and hydrochloride thereof); And other renaturation additive (as: Macrogol 4000 of 0.5～10 grams per liter or poly-second two ferment 6000 or polyoxyethylene glycol 8000 or cetomacrogol 1000 0,0.2 the arginine of～1.0 mol and hydrochloride thereof or other basic aminoacids and hydrochloride or three (methylol) aminomethane thereof, 1～20% glycerine and a certain amount of heparin) meta-alkalescence (pH8.0～9.5) renaturation solution in renaturation, reductibility SDS-PAGE detects the renaturation effect.Through further ion exchange chromatography, size-exclusion purifying the dimer purity of target protein is reached more than 90% again.The urea of the urea of the urea of the urea of 5 mol, 4 mol, 2 mol, 1 mol dialysis is to 20 mmoles/rise phosphate buffered saline buffer again, and lyophilize obtains the rhOP-1 lyophilized powder.-20 ℃ of preservations are standby behind the ethylene oxide sterilizing.
Embodiment 5:
To reach the rhOP-1 monomer of 95% above purity through ion exchange chromatography (or/and hydrophobic chromatography, reversed phase chromatography) purifying, its urea or 1 mol Guanidinium hydrochloride with 2 mol is diluted to 50～100 μ g/ml, and add 0.4～0.6 mol arginine therein, transfer its pH value between 8.0～10, (reductibility SDS-PAGE detected the renaturation effect to renaturation, Fig. 3) in 24～60 hours under the room temperature.The renaturation sample passes through further chromatography purification (ion-exchange, anti-phase, hydrophobic, non-special affine and size-exclusion) again reaches more than 90% the purity of dimer purity, 1. obtains freeze-drying part of the genetic engineered product of rhOP-1 mature peptide then after dialysis, degerming, packing and lyophilize.2. target protein and collagen protein, absorbable gelatin sponge, chitosan or coral powder class material, other artificial engineering materialss are compound, can be used for the damaged reparation of bone, orthopedic and be used for keeping and recovery etc. of oral cavity tooth extraction wound alveolar bone.
Claims (9)
1. the formulation preparation method of a recombinant human Osteogenic Protein-1, it is characterized in that obtaining the recombinant human Osteogenic Protein-1 monomeric protein of 95% above purity by chromatography, contain 2M～6M urea or/and in 1M～4M guanidine hydrochloride denaturation agent damping fluid dialysis carry out renaturation, the pH value of renaturation buffer is 8.0～10, renaturation temperature room temperature or 4 degree, 24～96 hours renaturation time; Through further chromatography purification the dimeric purity of target protein is reached more than 90% more afterwards, after dialysis, degerming, packing and lyophilize, obtain in a usual manner the rhOP-1 mature peptide genetic engineered product lyophilized powder or in a usual manner with the target protein liquid of chromatography purification directly and collagen protein, absorbable gelatin sponge, chitosan or coral powder class material or synthetic macromolecule engineering material of bone tissue compound, sterilize, make the renaturation preparation of recombinant human Osteogenic Protein-1;
Wherein: described renaturation adopts when renaturation is carried out in dialysis in lower concentration denaturing agent 2M urea or 1M Guanidinium hydrochloride damping fluid, and the arginine of 0.2～1.0 mol is necessary.
2. the method for claim 1 is characterized in that described chromatography is ion exchange chromatography or hydrophobic chromatography, reversed phase chromatography, affinity chromatography, molecular exclusion chromatography.
3. the method for claim 1 is characterized in that in the described renaturation step, adds tensio-active agent, redox system, renaturation additive.
4. method as claimed in claim 3, it is characterized in that described tensio-active agent is one of Triton 100, Triton 114, polysorbas20, tween 80, sarcosyl, hexadecyl brometo de amonio, NP40, its consumption concentration is volume percent 0.05%-1%.
5. method as claimed in claim 3, it is characterized in that described redox system be 0.1 micromoles per liter～0.1 mmole/liter cupric ion or/and ratio is a mol ratio from reductive agent/oxidizer system of 10: 1 to 1: 2, wherein the concentration range of reductive agent be 0.05～10 mmole/liter.
6. method as claimed in claim 5 is characterized in that described reductive agent is: one of reduced glutathion, halfcystine and hydrochloride thereof, mercaptoethanol, dithiothreitol (DTT), the red tinea sugar alcohol of two sulphur.
7. method as claimed in claim 5 is characterized in that described oxygenant is: one of the red tinea sugar alcohol of the dithiothreitol (DTT) of Sleep-promoting factor B, oxidized form and two sulphur, Gelucystine and hydrochloride thereof.
8. method as claimed in claim 3, it is characterized in that Macrogol 4000 or poly-second two ferment 6000 or polyoxyethylene glycol 8000 or cetomacrogol 1000 0 that described renaturation additive is 0.05～10 grams per liter, 0.02 the arginine of～1.0 mol or its hydrochloride or other basic aminoacids or its hydrochloride or three (methylol) aminomethane, volume percent is 1%～30% glycerine.
9. the method for claim 1, the renaturation preparation that it is characterized in that the recombinant human Osteogenic Protein-1 of described system are to be used for the damaged reparation of bone, orthopedic and be used for the preparation of the recombinant human Osteogenic Protein-1 of keeping and recovering of oral cavity tooth extraction wound alveolar bone clinically.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100456000A CN100336825C (en) | 2005-12-20 | 2005-12-20 | Renaturation of reconstituted human bone protein-1 and making method of its preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100456000A CN100336825C (en) | 2005-12-20 | 2005-12-20 | Renaturation of reconstituted human bone protein-1 and making method of its preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1786017A CN1786017A (en) | 2006-06-14 |
| CN100336825C true CN100336825C (en) | 2007-09-12 |
Family
ID=36783634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100456000A Expired - Fee Related CN100336825C (en) | 2005-12-20 | 2005-12-20 | Renaturation of reconstituted human bone protein-1 and making method of its preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100336825C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2281827B1 (en) | 2008-05-08 | 2013-08-21 | Ajinomoto Co., Inc. | Protein refolding method |
| FR2948573B1 (en) * | 2009-07-31 | 2011-11-18 | Adocia | NEW FORM OF ADMINISTRATION OF OSTEOGENIC PROTEIN COMPLEXES |
| WO2016201608A1 (en) | 2015-06-15 | 2016-12-22 | 张鹏 | Method capable of being independently used for protein renaturation or capable of being used for preceding operations of protein renaturation |
| CN105420209A (en) * | 2015-12-30 | 2016-03-23 | 海口奇力制药股份有限公司 | Method for preparing rhCNB dimer |
| CN109942668B (en) * | 2019-03-27 | 2021-04-06 | 安徽环球基因科技有限公司 | Method for dilution dialysis renaturation of inclusion body protein |
| CN112341517A (en) * | 2019-08-09 | 2021-02-09 | 无锡傲锐东源生物科技有限公司 | Renaturation method of recombinant protein |
| CN111574583B (en) * | 2020-04-10 | 2021-06-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
-
2005
- 2005-12-20 CN CNB2005100456000A patent/CN100336825C/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 人成骨蛋白-1成熟肽在大肠杆菌中的克隆、表达和诱骨活性测定 王琦 等，中国矫形外科杂志，第12卷第21.22期 2004 * |
| 人成骨蛋白成熟肽基因在毕氏酵母(Pichia Yeast)中的高效表达 王忠泽等，生命科学研究，第6卷第1期 2002 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1786017A (en) | 2006-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100336825C (en) | Renaturation of reconstituted human bone protein-1 and making method of its preparation | |
| CN112626074B (en) | Hydroxyproline-modified recombinant human III-type collagen mature peptide and preparation method and application thereof | |
| US11780905B2 (en) | Preparation method for collagen hydrogel | |
| CN113717290B (en) | Composite transdermal recombinant fibronectin and application thereof | |
| CN110511280B (en) | Transdermal recombinant fibronectin and application thereof | |
| CN100336824C (en) | Recombinant protein efficient renaturation method | |
| CN113621053A (en) | Recombinant human collagen and preparation method and application thereof | |
| RU2010107271A (en) | OPTIMIZED METHOD FOR CLEANING RECOMBINANT PROTEIN OF GROWTH FACTOR | |
| CN1793177A (en) | Recombined collagen and synthesizing and expressing purifying process thereof | |
| CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
| EP0240348A2 (en) | Agent for the removal of nucleic acids and/or endotoxin and method for the removal thereof | |
| CN113274486A (en) | Stable acidic fibroblast growth factor preparation and preparation method and application thereof | |
| RU2478706C1 (en) | Method of producing suspensions of hydrogel microparticles with given dimensions based on recombinant cobweb protein and use thereof | |
| CN1757723A (en) | Method of producing recombination human bone morphopoiesis protein | |
| CN111423516B (en) | Protein and application thereof in wound repair and bacteriostasis | |
| CN101514229B (en) | Human interferon alpha derivative and polyethylene glycol modified substance thereof | |
| JPH05176789A (en) | Preparation of gamma-interferon containing no human cystine by gene recombination containing no methionine on n-terminal | |
| CN101875699B (en) | Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof | |
| CN1807459A (en) | Activated collagen protein stent material and its special restoration factor for binding | |
| CN102167733A (en) | Construction, expression and application of acidly cleavable high-copy antihypertensive peptide tandem gene | |
| CN101875923A (en) | Fusion protein of human serum albumin and urate oxidase and preparation method thereof | |
| CN109836487B (en) | Human fibroblast growth factor 18, and soluble recombinant expression method, preparation and application thereof | |
| CN111748505B (en) | Genetically engineered bacterium for expressing carboxypeptidase G2 and preparation method and application thereof | |
| CN114133444A (en) | Preparation method of human BMP2 and analogues thereof | |
| CN112625117A (en) | Non-denaturing purification method and application of soluble recombinant teriparatide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070912 Termination date: 20100120 |