CN1789276A - One-step purification method of recombined human osteogenesis protein -1 - Google Patents
One-step purification method of recombined human osteogenesis protein -1 Download PDFInfo
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- CN1789276A CN1789276A CN 200510045370 CN200510045370A CN1789276A CN 1789276 A CN1789276 A CN 1789276A CN 200510045370 CN200510045370 CN 200510045370 CN 200510045370 A CN200510045370 A CN 200510045370A CN 1789276 A CN1789276 A CN 1789276A
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Abstract
The invention discloses a one-step purification method for the recombinant human osteogenic protein-1 expressed by genetic engineering bacterial, employing the mature fragments of peptides of the got human osteogenic protein-1, constructing expressing plasmid through DNA recombination technology, conversing engineering bacterial, fermenting and gathering, breaking the engineering bacterial, washing the inclusion body, denaturizating the recombinant protein, through one-step laminarization and purification to get the recombinant human osteogenic protein-1 with purity above 95%. The ont-step laminarization and purification method provided by this invention is fast and effective, and suited for mass production by company.
Description
Technical field
The present invention relates to a kind of purification process of human osteogenic protein-1, relate in particular to " single step purification " method of expressed recombinant human Osteogenic Protein-1 (rhOP-1) mature peptide of a kind of genetic engineering bacterium, belong to the biotechnological pharmaceutics field.
Background technology
(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: can be used for treating the damaged of bone and cartilage and be widely used in orthopedic and plastic surgery.Its 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.The treatment periodontopathy aspect OP-1 also have effects such as can promoting periodontal reconstruction, alveolar bone increase in density, this plantation for tooth have great application prospect (Giannobile WV et al., J Periodontol., 1998,69:129).
Utilize the genetically engineered prokaryotic expression system can obtain a large amount of target proteins, still, the purifying of OP-1, renaturation but are challenging difficult problems always, and common purifying needs the multistep chromatography to finish, and length consuming time is lost many.Express successful report though have at present, still do not have purifying, the refolding method of a cover high-level efficiency recombinant human Osteogenic Protein-1 (rhOP-1).
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides " single step purification " method of a kind of recombinant human Osteogenic Protein-1 (rhOP-1) mature peptide, utilize method provided by the invention, the crude product extracting solution of rhOP-1 is passed through the ion-exchange chromatography single step purification, can obtain highly purified rhOP-1 albumen, realize the purpose of purifying production rhOP-1, and method of the present invention also has efficiently advantage fast, the production amplification of suitable enterprise.
Expressed recombinant human Osteogenic Protein-1 (rhOP-1) mature peptide of genetic engineering bacterium of the present invention utilizes 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 recombinant human Osteogenic Protein-1 (rhOP-1) and efficiently expressed 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).Utilize conventional thermal induction to express, can obtain the high expression level of target protein matter, the expression amount of target protein accounts for about 30% of bacterial protein, and rhOP-1 of the present invention form with inclusion body in intestinal bacteria exists.
" single step purification " method of the recombinant human Osteogenic Protein-1 that genetic engineering bacterium of the present invention is expressed, it is characterized in that: with the engineering bacteria fragmentation, the inclusion body washing, the recombinant protein sex change, one step chromatography purification, the SDS-PAGE electrophoretic analysis can obtain the recombinant human Osteogenic Protein-1 of 95% above purity.
Wherein, described engineering bacteria fragmentation can be that the employing ice-bath ultrasonic is split bacterium or other routines are split the bacterium method, and engineering bacteria is crushed to microscopy bacteria breaking rate greater than 98%, promptly gets the inclusion body mixed solution.
Wherein, the washing of described inclusion body be with the inclusion body mixed solution in 4 ℃, 6000g, centrifugal 15～20min collects its throw out; Successively respectively with 4 ℃, the 8000g condition is washed centrifugal 20～30min with inclusion body washings 1 and inclusion body washings 2, the inclusion body throw out;
Above-mentioned inclusion body washings 1 composition is the 20mmol/L phosphate buffered saline buffer, and volume percent is 1% Triton 100, and pH 6.0; Above-mentioned inclusion body washings 2 compositions are 20mmol/L phosphate buffered saline buffers, 2mol/L Urea, and pH 6.0.
Wherein, described recombinant protein sex change, a step chromatography purification is meant that the inclusion body throw out that will obtain after the washing is level pad A dissolving with solubilization of inclusion bodies liquid, the centrifugal 20～30min of 12000g collects supernatant, promptly gets the rhOP-1 extracting solution; With described extracting solution with 0.45 μ m filtering with microporous membrane or the centrifugal 15～30min of 12000g after, get on filtrate or the supernatant liquor through level pad equilibrated strong cat ion exchange column and carry out chromatography purification; During wash-out, being earlier 10% elution buffer B wash-out part foreign protein with volume percent, is 20% elution buffer B wash-out target protein matter with volume percent again, and the fraction collection volume percent is the elution peak of 20% elution buffer B.
Above-mentioned inclusion body throw out and solubilization of inclusion bodies liquid are the ratio of mixture of level pad A, in gram than milliliter, preferably 1: 10-50.
The composition that above-mentioned solubilization of inclusion bodies liquid is level pad A is 8mol/L Urea, 20mmol/L phosphate buffered saline buffer, pH 6.0 or 8mol/L urea, 20mmol/L Tris-HCI, pH6.0;
The composition of above-mentioned elution buffer B is 8mol/L Urea, 20mmol/L phosphate buffered saline buffer, 1mol/L NaCl, pH6.0.
In " single step purification " method of the expressed recombinant human Osteogenic Protein-1 of above-mentioned genetic engineering bacterium, described solubilization of inclusion bodies liquid is the EDTA (disodium ethylene diamine tetraacetate) that can also add 1-10mmol/L among the level pad A; The 3-mercaptoethanol of the DDT of 1-20mmol/L (dithiothreitol (DTT)) or 1-20mmol/L.
In " single step purification " method of the expressed recombinant human Osteogenic Protein-1 of above-mentioned genetic engineering bacterium, described strong cat ion exchange column is SP-Sepharose series (Amersham company), SP Resin HP series (Beijing Zhuo Guan), UNOsphere
TMOne of S series (Bio-Rad company).
Wherein, preferably SP-Sepharose Fast Flow (SP-FF) or UNOsphere of described strong cat ion exchange column
TMS Exchange Media strong cat ion exchange column.
In " single step purification " method of the expressed recombinant human Osteogenic Protein-1 of above-mentioned genetic engineering bacterium, it is that the target protein that the elution peak of 20% elution buffer B is collected carries out the SDS-PAGE electrophoretic analysis that described " single step purification " method is meant volume percent, utilize the purity of the target protein of gray scale scanning and software analysis collection, can obtain the recombinant human Osteogenic Protein-1 of 95% above purity.
The main points that " a step chromatography purification method " provided by the present invention purifying is produced the rhOP-1 method are: elder generation is 10% elution buffer B wash-out part foreign protein with volume percent, be 20% elution buffer B wash-out target protein matter (see figure 1) with volume percent again, wherein 20% B liquid elution peak 2 is target protein rhOP-1 substantially, part is respectively collected in the SDS-PAGE electrophoretic analysis, can obtain the target protein matter (see figure 2) of 95% above purity.
Being engaged in those skilled in the art can find out at an easy rate, can add the EDTA (disodium ethylene diamine tetraacetate) of 1-10mmol/L among the level pad A of the present invention; The 3-mercaptoethanol of the DDT of 1-20mmol/L (dithiothreitol (DTT)) or 1-20mmol/L; 1mol/L NaCl in the B liquid also can be the NaCl of 0.5-1mol/L concentration, calculates relative concentration, also can obtain the wash-out concentration of corresponding B liquid gradient concentration, but 1mol/L NaCl is after obtaining target protein, and the chromatography column of can directly regenerating has the advantage that improves purification efficiency.
With all the other conjugated protein and pillars of regenerating simultaneously of 100% B liquid wash-out; After the balance liquid A liquid balance, can repeat the sample purification of samples.Gradient concentration elution method of the present invention has improved efficient greatly, has reduced cost, has efficiently advantage fast, especially is fit to enterprise and produces amplification.
Description of drawings
The typical rhOP-1 purifying of Fig. 1 the present invention collection of illustrative plates
Wherein: peak 1 is for penetrating the peak; Peak 2 is the elution peak of 10%B liquid; Peak 3 is the 1st elution peak of 20%B liquid; Peak 4 is second elution peak of 20%B liquid.
The SDS-PAGE electrophorogram that the albumen that Fig. 2 the inventive method is collected carries out
Wherein: swimming lane 1 is the sample after inclusion body dissolves for the first time; Swimming lane 1 is the sample after inclusion body dissolves for the second time; Liquid swimming lane 3 is the target protein of second elution peak collection of 20% elution buffer B liquid; Swimming lane 4 is standard protein molecular weight marker.
Embodiment
Embodiment 1:
Ratio in 1g thalline 10ml adds lysate suspension engineering bacteria, and ice-bath ultrasonic is split bacterium.Ultrasonic power 400～1200w, ultrasonic 10s, 15s repeats 45 times (19 minutes) totally at interval.Detect cleavage rate with phase microscope, the suspension liquid after the centrifugal cracking (4 ℃, 6000g, centrifugal 15min) supernatant discarded keeps precipitation.Microscopic examination is if reach 98% above bacterium and do not break, and will precipitate to have hanged the above bacterium of repetition cracking to 98% with lysate again and break, and with above-mentioned condition centrifugal collecting precipitate, is the inclusion body mixture.
With inclusion body washings 1 (20mmol/L phosphate buffered saline buffer, pH 6.0,1% Triton 100) and inclusion body washings 2 (the 20mmol/L phosphate buffered saline buffer, pH 6.0,2mol/L Urea) washs centrifugal (8000g, 25min) collection inclusion body throw out respectively successively.
With the inclusion body of above-mentioned collection level pad A (8mol/L Urea, the 20mmol/L phosphate buffered saline buffer, pH6.0) in (gram than milliliter) 1g wet thallus: 2ml or inclusion body throw out and solubilization of inclusion bodies liquid be the ratio of mixture of level pad A with 1: the mixed of 10-20, more than 4 ℃ of conditions dissolving inclusion body 30min or spend the night, the centrifugal 30min of 12000g collects supernatant liquor, precipitation is again with above-mentioned steps dissolving, centrifugal, repeat 1-2 time, carry out the SDS-PAGE electrophoretic analysis, look target protein matter content and determine to collect supernatant liquor.Merge the supernatant liquor of collecting, be the rhOP-1 extracting solution.
Behind the filtering with microporous membrane or 12000g centrifugal 20min of rhOP-1 extracting solution, get ion exchange column SP-Sepharose Fast Flow (SP-FF) strong cat ion exchange column chromatography purification on filtrate or the supernatant liquor with 0.45 μ m.Concrete steps are:
1, with level pad A (pH 6.0 for 8mol/L Urea, a 20mmol/L phosphate buffered saline buffer) balance 2-3 column volume, flow velocity is 20cm-100cm/ hour linear rate of flow.
2, after post effluent liquid baseline is steady, will be by SP-FF post on the filtering with microporous membrane of 0.45 μ m or the rhOP-1 extracting solution sample constant speed behind the centrifugal 30min of 12000g.
3, upward wash a post 1-2 column volume with level pad A behind the sample.
4, (wash-out 1-2 column volume of A liquid+1mol/LNaCl) removed the part foreign protein with 90% balance liquid A and 10% elution buffer B.
5, with balance liquid A liquid and 3-5 column volume of 20% buffer B wash-out of 80%, the elution peak of fraction collection 20% buffer B.
6, with all the other conjugated protein and pillars of regenerating simultaneously of 100% buffer B wash-out; After the balance liquid A liquid balance, can repeat above step, sample purification of samples in the continuation.
In the step 5, the elution peak of 80% A liquid and 20% B liquid has 2 (see figure 1)s, and wherein elution peak 2 is target protein rhOP-1 substantially, and part is respectively collected in the SDS-PAGE electrophoretic analysis, can obtain the target protein matter (see figure 2) of 95% above purity.
Embodiment 2:
Prepare solubilization of inclusion bodies liquid (8mol/L urea, pH 6.0, the 20mmol/L phosphate buffered saline buffer contains the EDTA (disodium ethylene diamine tetraacetate) of 1mmol/L; The DDT of 10mmol/L (dithiothreitol (DTT)).
Split bacterium, washing, centrifugal acquisition inclusion body in embodiment 1 mode, the inclusion body of collection dissolves with above-mentioned solubilization of inclusion bodies liquid, and with the mixed of 1g wet thallus: 8ml, 4 ℃ of dissolving inclusion body 30min are above or spend the night.Liquid in the centrifugal 25min collection of 12000g, precipitation are again with the above-mentioned steps dissolving, and is centrifugal, repeats 2 times, carries out the SDS-PAGE electrophoretic analysis, looks target protein matter content and determine to collect supernatant liquor.Merge the supernatant liquor of collecting, be the rhOP-1 extracting solution.
The rhOP-1 extracting solution of above-mentioned collection is gone up ion exchange column UNOsphereTM S Exchange Media (Bio-Rad company) strong cat ion exchange column chromatography gradient purifying after with the filtering with microporous membrane of 0.45 μ m.Concrete steps are:
1. with level pad A (20mmol/L phosphate buffered saline buffer, pH 6.0, a 8mol/L Urea) balance 2-3 column volume, flow velocity is 40cm-120cm/ hour linear rate of flow.
When post effluent liquid baseline steadily after. UNOsphereTM S Exchange Media post on will be by the rhOP-1 extracting solution sample constant speed behind the filtering with microporous membrane of 0.45 μ m.
3. wash a post 1-2 column volume (reequilibrate) with level pad after going up sample.
4. (wash-out 1-2 column volume of A liquid+1mol/LNaCl) removed the part foreign protein with 90% balance liquid A and 10% buffer B.
5. with balance liquid A liquid and 3-5 column volume of 20% buffer B wash-out of 80%, the elution peak of fraction collection 20% buffer B.Part is respectively collected in the SDS-PAGE electrophoretic analysis, can obtain the target protein matter of 95% above purity.
6. with all the other conjugated protein and pillars of regenerating simultaneously of 100% buffer B wash-out; After the balance liquid A liquid balance, can repeat above step, sample purification of samples in the continuation.
Claims (10)
1. " single step purification " method of the expressed recombinant human Osteogenic Protein-1 of a genetic engineering bacterium, it is characterized in that with the engineering bacteria fragmentation inclusion body washing, recombinant protein sex change, one step chromatography purification, the SDS-PAGE electrophoretic analysis can obtain the recombinant human Osteogenic Protein-1 of 95% above purity.
2. by the described method of claim 1, it is characterized in that described engineering bacteria fragmentation is that engineering bacteria is crushed to microscopy bacteria breaking rate greater than 98%, promptly gets the inclusion body mixed solution.
3. by the described method of claim 1, it is characterized in that described inclusion body washing be with the inclusion body mixed solution in 4 ℃, the centrifugal 15～20min of 6000g collects its throw out; With inclusion body washings 1 and inclusion body washings 2 successively respectively with 4 ℃, the 8000g condition, washing, centrifugal 20～30min, the inclusion body throw out.
4. by the described method of claim 1, it is characterized in that described recombinant protein sex change, a step chromatography purification is meant that the inclusion body throw out that will obtain after the washing is level pad A dissolving with solubilization of inclusion bodies liquid, the centrifugal 20～30min of 12000g, collect supernatant, promptly get the rhOP-1 extracting solution; With described extracting solution with 0.45 μ m filtering with microporous membrane or the centrifugal 15～30min of 12000g after, get on filtrate or the supernatant liquor through level pad equilibrated strong cat ion exchange column and carry out chromatography purification; During wash-out, being earlier 10% elution buffer B wash-out part foreign protein with volume percent, is 20% elution buffer B wash-out target protein matter with volume percent again, and the fraction collection volume percent is the elution peak of 20% elution buffer B.
5. by the described method of claim 4, it is characterized in that described inclusion body throw out and solubilization of inclusion bodies liquid are the ratio of mixture of level pad A, is 1 in gram than milliliter: 10-50.
6. by the described method of claim 4, it is characterized in that the composition that described solubilization of inclusion bodies liquid is level pad A is 8mol/L Urea, 20mmol/L phosphate buffered saline buffer, pH 6.0 or 8mol/L urea, 20mmol/L Tris-HCI, pH6.0; The composition of described elution buffer B is 8mol/L Urea, the 20mmol/L phosphate buffered saline buffer, and 1mol/L NaCl, pH 6.0.
7. by the described method of claim 6, it is characterized in that described solubilization of inclusion bodies liquid is the disodium ethylene diamine tetraacetate that can add 1-10mmol/L among the level pad A; The dithiothreitol (DTT) of 1-20mmol/L or the 3-mercaptoethanol of 1-20mmol/L.
8. by the described method of claim 4, it is characterized in that described strong cat ion exchange column is SP-Sepharose series, SP Resin HP series, UNOsphere
TMOne of S series.
9. by the described method of claim 8, it is characterized in that described strong cat ion exchange column is SP-Sepharose FastFlow or UNOsphere
TMS Exchange Media strong cat ion exchange column.
10. by the described method of claim 4, it is characterized in that 20% B liquid elution peak the 2nd peak is target protein recombinant human Osteogenic Protein-1 peak, with the volume percent of collecting is that the elution peak of 20% elution buffer B carries out the SDS-PAGE electrophoretic analysis, can obtain the recombinant human Osteogenic Protein-1 of 95% above purity.
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| CN112812969A (en) * | 2021-01-12 | 2021-05-18 | 福建基诺厚普生物科技有限公司 | System purification method for recombinant expression polypeptide in genetic engineering |
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| CN112812969A (en) * | 2021-01-12 | 2021-05-18 | 福建基诺厚普生物科技有限公司 | System purification method for recombinant expression polypeptide in genetic engineering |
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