CN100402646C - Method of producing recombination human bone morphopoiesis protein - Google Patents
Method of producing recombination human bone morphopoiesis protein Download PDFInfo
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- CN100402646C CN100402646C CNB2005100506103A CN200510050610A CN100402646C CN 100402646 C CN100402646 C CN 100402646C CN B2005100506103 A CNB2005100506103 A CN B2005100506103A CN 200510050610 A CN200510050610 A CN 200510050610A CN 100402646 C CN100402646 C CN 100402646C
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Abstract
The present invention relates to a method for producing recombination human bone morphopoiesis protein. The method comprises the following steps: a fermented bacterium is crushed, and inclusion bodies are separated and cracked by a denaturant; the denatured and cracked recombination human bone morphopoiesis protein is renatured and correctly folded to form a soluble conformer with bioactivity; the protein is purified by a chromatographic method to obtain the recombination human bone morphopoiesis protein with high purity. The recombination human bone morphopoiesis protein produced by the method of the present invention has the advantages of high yield, bioactivity and purify, simple process, low production cost, etc.
Description
Technical field
The present invention relates to the method for production recombinant human bone morphogenetic protein, belong to biotechnology and pharmacy field.
Background technology
The repairing and treating that the bulk bone is damaged is the difficult problem of clinical orthopaedics always.Because its possibility that heals voluntarily is minimum, so main for a long time clinically the employing carried out repairing and treating from methods such as body, allogenic bone transplantation or the implantation of artificial bio-membrane's material.But all there is some unsurmountable shortcoming in all these methods, thereby make their clinical application be subjected to very big restriction.Studies show that in recent years, some somatomedins such as transforming growth factor-beta, insulin like growth factor-1 I etc. are to the specific effect that grown of bone and cartilage, especially the discovery of Delicious peptide has brought new hope for clinicist's repairing bone defect, has opened up a new approach for the clinical treatment bone is damaged.
Delicious peptide is the class protein of being discovered in the sclerotin extract of decalcification by the U.S. scientist Urist MR nineteen sixty-five.Now verified this proteinoid belongs to the transforming growth factor-beta superfamily member, had been found that ten surplus a family member.In to multiple animal experiment studies such as monkey, dog, rats, confirm, Delicious peptide can not only promote the reparation that bone is damaged, and good result of treatment is all arranged in the treatment fields such as reparation, periodontopathy and acute renal failure of nerve injury, be a kind of cytokine with extensive clinical use value.
The human bone morphogenesis protein-2 bioactive molecule respectively contains 114 amino acid whose peptide chains by identical 2 and forms homodimer.The about 30kd of molecular weight, iso-electric point is 7.9.The common feature that other members of human bone morphogenesis protein-2 and transforming growth factor-beta superfamily have is: each subunit all contains 7 very conservative cysteine residues, and wherein 6 form three pairs of intrachain disulfide bonds, and 1 forms interchain disulfide bond in addition.Also have a heparin binding site and a glycosylation site in the human bone morphogenesis protein-2 molecular structure.Research has proved that glycosylation does not have obvious influence to the physiologically active of bone morphogenesis protein-2.The people's bone morphogenetic protein that adopts genetic engineering technique to produce reorganization in intestinal bacteria is the field that the various countries scientist explores always.But the trace that people's bone morphogenetic protein protokaryon technology of preparing of the reorganization of home and abroad report is finished active ingredient in all can only the laboratory reclaims, and can't accomplish scale production.
Summary of the invention
The objective of the invention is to be implemented in the proteic deficiency of mass production recombinant human bone form in the colibacillus engineering for overcoming existing genetic engineering technique.The method that a kind of technology is simple, with low cost, energy scale operation has bioactive recombinant human bone morphogenetic protein is provided.
The method of production recombinant human bone morphogenetic protein of the present invention may further comprise the steps:
1) after the plasmid transform bacteria and abduction delivering that contain coding recombinant human bone morphogenetic protein mature peptide sequence, centrifugal collection bacterium;
2) with bacteria breaking, centrifugal collection inclusion body with lavation buffer solution repetitive scrubbing inclusion body, is removed impurity;
3) with lysis buffer continuously stirring cracking inclusion body 2-8 hour that contains denaturing agent and reductive agent;
4) inclusion body solution centrifugal that cracking is good is got supernatant and is added lentamente continuously and do dilution refolding in the renaturation buffer, makes it progressively to recover biological activity in 0-28 ℃ environment, and the proteinic final concentration of renaturation solution is adjusted in the scope of 0.02-0.3mg/ml;
5) with the chromatography separation and purification bioactive dimer is arranged.
Among the present invention, broken bacterium can be adopted the whole bag of tricks, as broken thalline such as ultrasonication, high pressure dispersion and grindings, makes it to discharge inclusion body, because inclusion body density height ratio is great, can obtain purer inclusion body through centrifugal.
Among the present invention, the damping fluid of washing inclusion body is made up of denaturing agent, tensio-active agent, metal ion chelation agent and buffering salt, said denaturing agent is that Guanidinium hydrochloride, the tensio-active agent of 1-3M urea or 0.5～1M is that 0.01-1% triton 100, metal ion chelation agent are the 1-5mM disodium ethylene diamine tetraacetate, and buffering salt is 20mM-50mM hydrochloric acid Tutofusin tris pH 7.5-9.5.
Among the present invention, the used lysis buffer of cracking inclusion body is made up of 50mM Tutofusin tris pH7.5-9.5,6-7M Guanidinium hydrochloride, 5-20mM dithiothreitol (DTT) and 1-5mM disodium ethylene diamine tetraacetate.
Among the present invention, used renaturation buffer is grouped into by 25～100mM Tutofusin tris pH 7.5-9.5,0.5-3M urea, 0.1-2M arginine, 0.5-2M sodium-chlor, 1～5mM disodium ethylene diamine tetraacetate and oxidized form Triptide/reduced glutathione peer-group, and wherein the concentration ratio of oxidized form Triptide/reduced glutathione is 1: 1-1: 10.
The temperature of renaturation should be constant in 0-28 ℃ of scope, makes the recombinant human bone morphogenetic protein peptide chain of sex change progressively recover its natural structure picture.In order to guarantee best renaturation effect, the proteinic final concentration of renaturation solution should be adjusted in the scope of 0.02-0.3mg/ml.How much time of renaturation can be judged or judge according to the alkaline phosphatase activities of its stimulation l cell according to dimeric in the non-reduced SDS-PAGE running gel.
After renaturation process of the present invention finished, the polypeptide chain, the multimeric protein that use dimer active ingredient that anion-exchange chromatography or hydrophobic chromatography will correctly fold and various foreign proteins, do not have correctly to fold separated.
The chromatography media that anion exchange chromatography is selected is Q Sepharose FF, to go up sample behind the level pad balance pillar of forming with 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1-3M urea, 10mM NaCl and 5-20% dimethyl formamide; The gradient elution that increases progressively as salt concn of the elution buffer of forming with 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1-3M urea, 0.1-1M sodium-chlor and 5-20% dimethyl formamide again.
Hydrophobic chromatography selects the phenyl sepharose gel to make medium, go up sample behind the level pad balance pillar with 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1-3M urea, 5-20% dimethyl formamide, 1-4M sodium-chlor composition, do the gradient elution that salt concn is successively decreased with the elution buffer that 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1-3M urea and 5-20% dimethyl formamide are formed.
The present invention compares the useful effect that has with background technology:
The inventive method overcome existing genetic engineering technique can not be in intestinal bacteria the proteic defective of mass production recombinant human bone form, realized with colibacillus engineering production recombinant human bone morphogenetic protein on a large scale.This method technology is simple, low production cost, and the recombinant human bone morphogenetic protein of producing has yield height (＞15%), good (alkaline phosphatase activities＞10 of biological activity
5IU/ml), purity height advantages such as (＞95%).Be applicable to the production preparation of the recombinant conversion growth factor-beta superfamily member that the engineering bacterium is expressed.
Embodiment
Further specify the present invention below in conjunction with embodiment:
Example 1
Recombinant human bone morphogenesis protein-2 with the production escherichia coli expression is an example, may further comprise the steps:
1) after the plasmid transform bacteria and abduction delivering that contain coding recombinant human bone morphogenetic protein mature peptide sequence, bacterium is collected in centrifugal and filtration;
2) ultrasonication thalline makes it to discharge inclusion body, centrifugal collection inclusion body, wash with the ratio of 100ml lavation buffer solution with every gram inclusion body, lavation buffer solution is by 2M urea, 1% triton 100, and 2mM disodium ethylene diamine tetraacetate and 50mM hydrochloric acid Tutofusin tris pH 8.0 form.The washing process repeated multiple times is removed and irrelevant cell protein, nucleic acid and the mycoderm composition of recombinant human bone morphogenesis protein-2 as far as possible;
3) lysis buffer is made up of 50mM Tutofusin tris pH8.0,7M Guanidinium hydrochloride, 10mM dithiothreitol (DTT) and 2mM disodium ethylene diamine tetraacetate.The lysis buffer continuously stirring cracking of per 1.5 gram inclusion bodys usefulness 20ml 2-8 hour, 25000xg is centrifugal 20 minutes then, abandons precipitation.Measure the protein concentration of supernatant liquor with Kao Mashi light blue method, and protein concentration is adjusted to 5-20mg/ml.
4) renaturation buffer is formed: 100mM Tutofusin tris pH 8.5,0.5M arginine, 2mM disodium ethylene diamine tetraacetate, 1M sodium-chlor, 3M urea, 1mM oxidized form Triptide and 5mM prototype Triptide.
The inclusion body that cracking is good adds in the renaturation buffer continuously and slowly, and making proteic final concentration is 0.02-0.3mg/ml, in 0-28 ℃ of isoperibol renaturation 1-24 days then.Check the effect of renaturation with non-reducing SDS-PAGE electrophoresis.
5) after renaturation process finishes, use the anion exchange chromatography separation and purification that bioactive dimer is arranged:
Chromatography media adopts the Q Sepharose FF of AM General (GE) company.
Level pad: 100mM 2-cyclohexylamino ethyl sulfonic acid pH 8.5,10mM NaCl, 10% dimethyl formamide, 3M urea.
Elution buffer: 100mM 2-cyclohexylamino ethyl sulfonic acid pH 8.5,3M urea, 1M sodium-chlor, 10% dimethyl formamide.
With going up sample behind the good pillar of level pad balance, continue no longer to fluctuate with level pad washing pillar to baseline, carry out the gradient elution that salt concn is risen progressively with elution buffer then, collect main peak.
Example 2
Produce recombinant human bone morphogenesis protein-2, step is as follows:
1) after the plasmid transform bacteria and abduction delivering that contain coding recombinant human bone morphogenetic protein mature peptide sequence, bacterium is collected in centrifugal and filtration;
2) ultrasonication thalline, make it to discharge inclusion body, centrifugal collection inclusion body, wash with the ratio of 100ml lavation buffer solution with every gram inclusion body, lavation buffer solution is made up of Guanidinium hydrochloride, 1% triton 100,2mM disodium ethylene diamine tetraacetate and the 20mM hydrochloric acid Tutofusin tris pH 7.5 of 0.5M.The washing process repeated multiple times is removed and irrelevant cell protein, nucleic acid and the mycoderm composition of recombinant human bone morphogenesis protein-2 as far as possible;
3) lysis buffer is made up of 50mM Tutofusin tris pH 8.0,7M Guanidinium hydrochloride, 10mM dithiothreitol (DTT) and 2mM disodium ethylene diamine tetraacetate.The lysis buffer continuously stirring cracking of every 1.5g inclusion body usefulness 20ml 2-8 hour, 25000xg is centrifugal 20 minutes then, abandons precipitation.With the protein concentration of Kao Mashi light blue method mensuration supernatant liquor, and the protein concentration of adjusting lysate is 5-20mg/ml.
4) renaturation buffer is formed: 100mM Tutofusin tris pH 8.5,0.5M arginine, 2mM disodium ethylene diamine tetraacetate, 0.2M sodium-chlor, 1M urea, 1mM oxidized form Triptide and 5mM prototype Triptide.
The inclusion body that cracking is good adds in the renaturation buffer continuously and slowly, and making proteic final concentration is 0.02-0.3mg/ml, is placed in the 0-28 ℃ of isoperibol renaturation 1-24 days then.Check the effect of renaturation with non-reducing SDS-PAGE electrophoresis.
5) after renaturation process finished, re-using the hydrophobic chromatography separation and purification had bioactive dimer:
Chromatography media adopts the phenyl sepharose gel.
Level pad: 100mM 2-cyclohexylamino ethyl sulfonic acid pH 8.5,1M urea, 3M sodium-chlor and 10% dimethyl formamide.
Elution buffer: 100mM 2-cyclohexylamino ethyl sulfonic acid pH8.5,1M urea and 10% dimethyl formamide.
Chromatography column is with going up sample after the level pad balance, till stream occurring and wearing the peak, continue no longer to fluctuate with level pad washing pillar to baseline, uses elution buffer again instead and carries out the gradient elution that salt concn is successively decreased.
The peak sample of collecting carries out the SDS-PAGE electrophoretic analysis, the cell alkaline phosphatase activities detects and the interior dystopy skeletonization experiment of the flesh bag of mouse quadriceps muscle of thigh.
The biological activity determination of recombinant human bone morphogenetic protein of the present invention:
1, cultured cell in vitro is surveyed and is lived:
Measure alkaline phosphatase activities with the strain of mouse 2T3 interstital stem cell.The alkaline phosphatase activities unit definition: the alkaline phosphatase specific activity of enzyme refers to the enzyme activity unit number that contains in every milligram of protein, be expressed as U/mg, an enzyme activity unit is defined as: at 37 ℃, per minute transforms the required enzyme amount of 1umol p-nitrophenyl phosphoric acid under pH 10.5 conditions.Detect proof: alkaline phosphatase activities and recombinant human bone morphogenesis protein-2 dosage are dependency, and promptly alkaline phosphatase activities also increases with the dosage increase of recombinant human bone morphogenesis protein-2, illustrates that this product has clear and definite biological activity.
2, dystopy skeletonization experiment in the body:
Prepare mouse quadriceps muscle of thigh bag model according to universal method in the world.Give 0.5mg recombinant human bone morphogenesis protein-2 in each mouse quadriceps muscle of thigh lacuna, the type i collagen that adds equivalent is as excipient; Control group is implanted the 1mg type i collagen.After three weeks, carry out the X-ray detection, to show the activity of skeletonization.Control group should not have dystopy skeletonization phenomenon, and as seen the laboratory animal of implantation recombinant human bone morphogenesis protein-2 should have tangible dystopy skeletonization.
Though the present invention is to be that example is set forth with the recombinant human bone morphogenesis protein-2.Because other members' of transforming growth factor-beta superfamily structural similitude.Therefore, can adapt to other members' of transforming growth factor-beta superfamily production.So application the inventive method all should be included in the claims scope of the present invention other members' of transforming growth factor-beta superfamily production, is subjected to the protection and the restriction of claim of the present invention.
Claims (6)
1. method of producing recombinant human bone morphogenetic protein is characterized in that may further comprise the steps:
1) after the plasmid transformation escherichia coli and abduction delivering that contain coding recombinant human bone morphogenetic protein mature peptide sequence, centrifugal collection intestinal bacteria;
2) with the intestinal bacteria fragmentation, centrifugal collection inclusion body with lavation buffer solution repetitive scrubbing inclusion body, is removed impurity;
3) with lysis buffer continuously stirring cracking inclusion body 2-8 hour that contains denaturing agent and reductive agent;
4) inclusion body solution centrifugal that cracking is good is got supernatant and is added lentamente continuously and do dilution refolding in the renaturation buffer, makes it progressively to recover biological activity in 0-28 ℃ environment, and the proteinic final concentration of renaturation solution is adjusted in the scope of 0.02-0.3mg/ml;
5) with the chromatography separation and purification bioactive dimer is arranged.
2. production recombinant human bone morphogenetic protein method according to claim 1 is characterized in that the damping fluid that washs inclusion body is made up of denaturing agent, tensio-active agent, metal ion chelation agent and buffering salt.Said denaturing agent is that Guanidinium hydrochloride, the tensio-active agent of 1-3M urea or 0.5～1M is that 0.01-1% triton 100, metal ion chelation agent are the 1-5mM disodium ethylene diamine tetraacetate, and buffering salt is 20mM-50mM hydrochloric acid Tutofusin tris pH 7.5-9.5.
3. production recombinant human bone morphogenetic protein method according to claim 1 is characterized in that lysis buffer is made up of 50mM Tutofusin tris pH 7.5-9.5,6-7M Guanidinium hydrochloride, 5-20mM dithiothreitol (DTT) and 1-5mM disodium ethylene diamine tetraacetate.
4. production recombinant human bone morphogenetic protein method according to claim 1, it is characterized in that renaturation buffer is made up of component 25～100mM Tutofusin tris pH 7.5-9.5,0.5-3M urea, 0.1-2M arginine, 0.5-2M sodium-chlor, 1～5mM disodium ethylene diamine tetraacetate and oxidized form Triptide/reduced glutathione, wherein the concentration ratio of oxidized form Triptide/reduced glutathione is 1: 1-1: 10.
5. production recombinant human bone morphogenetic protein method according to claim 1, it is characterized in that separation and purification has bioactive dimer to adopt anion exchange chromatography, chromatography media is Q Sepharose FF, goes up sample behind the level pad balance pillar with 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1～3M urea, 10mM NaCl and 5-20% dimethyl formamide composition; The gradient elution that increases progressively as salt concn of the elution buffer of forming with 25～100mM2-cyclohexylamino ethyl sulfonic acid pH7.5～9.5,1-3M urea, 0.1-1M sodium-chlor and 5-20% dimethyl formamide again.
6. the method for production recombinant human bone morphogenetic protein according to claim 1, it is characterized in that separation and purification has bioactive dimer to adopt hydrophobic chromatography, chromatography media is the phenyl sepharose gel, with 25～100mM 2-cyclohexylamino ethyl sulfonic acid pH 7.5～9.5,1～3M urea, the 5-20% dimethyl formamide, go up sample behind the level pad balance pillar that 1～4M sodium-chlor is formed, use 25～100mM 2-cyclohexylamino ethyl sulfonic acid of pH 7.5～9.5 again, the elution buffer that 1～3M urea and 5-20% dimethyl formamide are formed is done the gradient elution that salt concn is successively decreased.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100410371C (en) * | 2006-05-13 | 2008-08-13 | 暨南大学 | Expression method of recombination human bone formation protein-2 |
| CN102336829B (en) * | 2010-09-09 | 2014-07-16 | 杭州九源基因工程有限公司 | Method for producing recombinant human bone morphogenetic protein-2 mature peptide |
| CN102776198A (en) * | 2011-05-09 | 2012-11-14 | 扬州泰达生物科技有限公司 | Method for expressing recombinant human bone morphogenetic protein in insect cell |
| WO2017098627A1 (en) * | 2015-12-10 | 2017-06-15 | 株式会社メニコン | Peptide composition |
| CN107098959B (en) * | 2017-05-27 | 2019-10-18 | 中国农业科学院蜜蜂研究所 | The preparation method of bumblebee peptidoglycan recognition protein |
| CN107759683A (en) * | 2017-11-23 | 2018-03-06 | 湖北昊龙生物科技有限公司 | A kind of large-scale preparation method of Human bone morphogenetic protein |
| CN117164696B (en) * | 2023-11-03 | 2024-03-22 | 北京市春立正达医疗器械股份有限公司 | Production method of recombinant human bone morphogenetic protein-2 mature peptide dimer |
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| CN1273976A (en) * | 2000-04-21 | 2000-11-22 | 西南药业股份有限公司 | Process for preparing recombined morphogenetic protein (rhOP-1) of human bone |
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