CN101029077A - Method for purifying gene-recombinant insulin precursor - Google Patents
Method for purifying gene-recombinant insulin precursor Download PDFInfo
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- CN101029077A CN101029077A CN 200710026682 CN200710026682A CN101029077A CN 101029077 A CN101029077 A CN 101029077A CN 200710026682 CN200710026682 CN 200710026682 CN 200710026682 A CN200710026682 A CN 200710026682A CN 101029077 A CN101029077 A CN 101029077A
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Abstract
A process for purifying the genetically recombined insulin precursor features that the activated carbon as adsorbent and cationic chromatography are used to remove the pigment from fermented liquid and purify the active protein. Its recovery rate is more than 85% and its purity is more than 95%.
Description
Technical field
The invention belongs to a kind of production method of Regular Insulin, relate generally to the expression of insulin precursor protein in pichia pastoris phaff of gene recombination.
Background technology
From animals and plants, extract natural insulin and do not satisfy clinical demand, utilize intestinal bacteria, yeast saccharomyces cerevisiae, host expresses Recombulins such as debaryomyces hansenii have appeared in the newspapers, but output lower (＜0.5mg/mL).The external source pork insulin gene of synthetic is expressed among the hosts such as yeast saccharomyces cerevisiae intestinal bacteria; There is easy formation inclusion body, it is high and produce deficiency such as more foreign protein to express productive rate.
Overcome above-mentioned deficiency with pichia spp as expression system.With pichia spp as the host can improve greatly Recombulin output (〉=1.0mg/mL), have a good application prospect.But can produce a large amount of pigments and some foreign proteins in the pichia spp fermentative production Recombulin process, the existence of pigment has a strong impact on the separation and purification in downstream.Thereby decolouring is the main task of the Recombulin initial gross separation of pichia spp production.The decolouring and the separation method of bibliographical information generally adopt HPLC at present, are unfavorable for scale operation.And the method that adopts cation-exchange chromatography can not be removed the pigment in the pichia spp fermented liquid fully, though hydrophobic chromatography can be removed most of pigment, it is less to exist exchange capacity, or bonding force too a little less than, or the low deficiency that waits of activity recovery, its application is restricted.Gel-filtration also can be removed pigment well, is subjected to the restriction of applied sample amount and can not be applied to large-scale production but exist the medium that is used for fractional separation.
Summary of the invention
The objective of the invention is to:, propose a kind of insulin precurosor purification process of gene recombination at the deficiencies in the prior art.
The insulin precurosor purification process of gene recombination of the present invention is mainly concerned with the expression of reorganization pork insulin precursor protein in pichia pastoris phaff, and as expression system, fermented liquid contains a large amount of pigments for pichia spp.These pigments not only kind are many, and amount is big.Only the several steps decolouring technology does not reach decolorizing effect.And for this biological activity protein of insulin precurosor, owing to will protect biological activity, many envrionment conditionss are restricted on technology, and as pH value, temperature, salt concn, organic solvent concentration or the like cause that easily protein-denatured factor all wants strictness assurance.
Gene-recombinant insulin precursor gene recombination purification process of the present invention, be the novel technique that adopts gac and cation-exchange chromatography to separate and decolour at gene-recombinant insulin precursor, at first utilize gac that the fermentation supernatant is carried out pre-treatment, separate and decolour at gene-recombinant insulin precursor through active carbon adsorption column and cation-exchange chromatography then, its operational path and technical scheme are as follows:
1 operational path
Gene recombined Pichia pastoris → shake bottle a kinds → fermentor tank → fermented liquid → adding gac → stirring → centrifugal → supernatant liquor → activated carbon column → through liquid → cation seperation column chromatography → contain gene-recombinant insulin precursor component elutriant → ultrafiltration desalination → lyophilize → gene-recombinant insulin precursor
2, technical scheme
1) gac that the present invention relates to is all through pre-treatment.At room temperature soak＞12h with≤0.5mol/L HCl, clear water is washed till neutrality, dries standby behind the suction filtration.
2) pigment that relates among the present invention and proteic molecular weight are higher, also contain impurity such as small molecules pigment, so adopt the gac of mesopore and micropore mixed type.The gac fines that produces in the decolorization is removed with the millipore filtration suction filtration, so not only make the light absorption value of mensuration more can embody actual decolorizing effect, and has eliminated its influence to subsequent purification.
3) the present invention is adjusted to the pH value under≤5 acidic conditions with fermented liquid, and the proteic iso-electric point of wide makes albumen be difficult for being adsorbed.
4) gac that adds for the first time adopts powdered carbon, and add-on is the 1%-3% (weight) of treatment solution; Generally be no more than 3% of treatment solution.Stir, treatment time 5-10min generally is no more than 10min.After high speed centrifugation, 20-30min removes somatic cells and gac, collects supernatant liquor.The gac that adsorbs a large amount of pigments is discarded, does not reclaim.
5) the present invention is at room temperature, and the gac that adds adopts the ADSORPTION IN A FIXED BED mode for the second time.The activated carbon that adds is a granular carbon, and the amount of adding is no more than 5% (weight) of treatment solution.After the first step gac is removed a large amount of pigments, be equipped with in the round shape adsorption column of gac in process, treatment solution passes through from top to down continuously, contacts with big carbon content active, reaches very high percent of decolourization at the outlet at bottom place.Be no more than 30 minutes the duration of contact of gac and treatment solution.This method is beneficial to the usefulness of giving full play to gac.Activated carbon after the use reclaims through acid-base method regeneration.
6) combine the column chromatography method of cationic exchange among the present invention.Allow uncharged pigment directly penetrate, charged pigment is adsorbed with target protein, by changing elution requirement, pigment and target protein is separated, and obtains highly purified target protein.Can remove most of pigment with cation-exchange chromatography (SP) a step, simultaneously concentrating sample.
7) ultra-filtration technique of the present invention's employing.Target protein solution desalination in molecular weight cut-off≤5KDa tubular fibre or hyperfiltration membrane assembly with the cation-exchange chromatography collection.Collect concentrated solution.When reaching desalination and exchange buffering liquid purpose, further remove remaining pigment, solved the decolouring problem.
8) the concentrated solution vacuum lyophilization of the present invention after with the ultrafiltration of collecting.
The present invention compared with prior art has following advantage and effect:
The target protein rate of recovery of the present invention reaches more than 85%, and purity is more than 95%, can reach to decolour fully and keep the higher rate of recovery and the dual purpose of purity.Compare with other purifying decoloring method, the present invention has that processing unit is simple, activated protein purity and rate of recovery height, and exchange capacity is big, and speed is fast, low cost and other advantages, can satisfy needs of scale production.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment
1 with pretreated powdered active carbon, cross 60 mesh sieves after, take by weighing 20 grams, be added in 2 liters the fermented liquid.Glass stick stirred 10 minutes, and is centrifugal through 8000 supercentrifuges that change then, 20 minutes time, collects supernatant liquor 1L.
2 with pretreated granular carbon (20mm * 40mm) claims 30 grams, is contained on 20cm * 2.6cm glass column.After the cleaning of 1% aqueous acetic acid, with pump the supernatant liquor of collecting is imported in the post, the control flow velocity is at 3mL/min.With 1% aqueous acetic acid sample is also ejected from activated carbon column again after last sample is intact.Collection penetrates liquid 1.2L.
3 positively charged ion filler sp550EC get 100mL and are loaded on 20cm * 2.6cm glass column.
Balance liquid is pH4.0,50mmolNaAc-HAC
Washings is pH4.0,50mmolNaAc-HAC+0.1mol/L NaCl+40% ethanol
Elutriant is pH4.0,50mmolNaAc-HAC+0.5mol/L NaCl+40% ethanol
Go up all product and collect liquid 1.2L for gene-recombinant insulin precursor through activated carbon column.Flow rate control is 10ml/min.Pillar is gone up sample behind 3 column volumes of balance liquid balance (BV), wash 10BV with washings again, crosses post with elutriant again, and collecting elution peak is the insulin precurosor of gene recombination, collects 220ml altogether.Regeneration condition is: 1mol/LNaCl, 5BV → 0.2mol/LNaOH, 5BV → pure water, 5BV → balance liquid, 5BV
4 target protein solution hyperfiltration membrane assemblies in molecular weight cut-off 3KDa with the cation-exchange chromatography collection.
Micromolecular foreign protein and pigment are leached.With the pH value is that 3 acetate buffer solution exchanges, and obtains the gene-recombinant insulin precursor acidic solution 100mL of spissated desalination.
5 obtain the gene-recombinant insulin precursor sample with ultrafiltration and concentration liquid vacuum lyophilization.
Detected result:
Solution with each step of the foregoing description obtains detects light absorption value at wavelength 420nm place.And on the analytical instrument of the HPLC of waters test sample concentration and purity, the purifying decolorizing effect of its more whole processing step sees following table for details.
| Sample | Light absorption value A | Purity/% | Concentration/mg/ml | Liquor capacity/ml | Gene-recombinant insulin precursor amount/g | The rate of recovery/% |
| The collection liquid that the centrifuged supernatant that the centrifuged supernatant that does not add active carbon adds active carbon is crossed activated-charcoal column is crossed the collection liquid ultrafiltration concentration liquid of cation chromatography post | 1.452 0.940 0.341 0.083 0.012 | 63.85 75.88 88.17 96.30 97.03 | 1.20 1.17 0.91 4.90 10.7 | 1000 1000 1200 220 100 | 1.20. 1.17 1.10 1.08 1.07 | / 97.75 93.64 98.65 98.92 |
| Final purity 97.03% total yield 89.16% | ||||||
Claims (6)
1, a kind of method for purifying gene-recombinant insulin precursor, its technological process is as follows:
Gene recombined Pichia pastoris → shake bottle a kinds → fermentor tank → fermented liquid → adding gac → stirring → centrifugal → supernatant liquor → activated carbon column → through the component elutriant → ultrafiltration desalination → lyophilize → insulin precurosor of liquid → cation seperation column chromatography → insulin-containing precursor.
2, method for purifying gene-recombinant insulin precursor according to claim 1 is characterized in that: its gac is at room temperature to soak＞12h with≤0.5mol/L HCl, and clear water is washed till neutrality, and the air dried method is carried out pre-treatment behind the suction filtration.
3, method for purifying gene-recombinant insulin precursor according to claim 1 is characterized in that: a kind of albumen-insulin precurosor of Pichia anomala expression, fermented liquid are regulated the pH value at≤5 acidic conditions.
4, method for purifying gene-recombinant insulin precursor according to claim 1 is characterized in that: fermentation liquor treatment, adding amounts of activated carbon by weight is 1-3%.
5, method for purifying gene-recombinant insulin precursor according to claim 1, it is characterized in that: supernatant liquor is by in the round shape adsorption column that gac is housed, the amount of gac is no less than 3% of pending supernatant by weight, treatment solution passes through from top to down continuously, contact its duration of contact≤30 minute with gac.
6, method for purifying gene-recombinant insulin precursor according to claim 1 is characterized in that: the ultrafiltration desalination is target protein solution desalination in molecular weight cut-off≤5KDa tubular fibre or hyperfiltration membrane assembly that cation-exchange chromatography is collected.
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| CN2007100266823A CN101029077B (en) | 2007-02-02 | 2007-02-02 | Method for purifying gene-recombinant insulin precursor |
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| CN2007100266823A CN101029077B (en) | 2007-02-02 | 2007-02-02 | Method for purifying gene-recombinant insulin precursor |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532257A (en) * | 2010-12-13 | 2012-07-04 | 鲁南制药集团股份有限公司 | Method for purifying recombinant human proinsulin |
| US8440878B2 (en) | 2008-02-04 | 2013-05-14 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene in prawns and uses thereof |
| CN103145829A (en) * | 2013-03-29 | 2013-06-12 | 江苏诺泰制药有限公司 | Purification method of insulin detemir |
| CN101981046B (en) * | 2008-02-04 | 2015-01-07 | 内盖夫研究与发展部本-古里安大学 | Insulin-like gene in prawns and uses thereof |
| CN105153294A (en) * | 2015-08-31 | 2015-12-16 | 济南康和医药科技有限公司 | Recombinant insulin and insulin analogue precursor purification method |
| CN107383161A (en) * | 2012-06-29 | 2017-11-24 | Emd密理博公司 | The purifying of biomolecule |
| CN112341535A (en) * | 2019-08-07 | 2021-02-09 | 中国科学院大连化学物理研究所 | Method for preparing insulin by separation and purification through ion exchange chromatography |
| CN113563413A (en) * | 2021-08-17 | 2021-10-29 | 汉肽生物医药集团有限公司 | Method for removing pigment in high-density fermentation process of pichia pastoris |
| WO2023154467A1 (en) * | 2022-02-11 | 2023-08-17 | Clara Foods Co. | Protein compositions and consumable products thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100460508C (en) * | 2004-11-03 | 2009-02-11 | 马延高 | Secretory expression for human insulin gene in methyl alcohol yeast |
| CN1854299A (en) * | 2005-04-29 | 2006-11-01 | 上海新生源医药研究有限公司 | Production of recombinant insulinum primary C peptide |
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2007
- 2007-02-02 CN CN2007100266823A patent/CN101029077B/en active Active
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9049850B2 (en) | 2008-02-04 | 2015-06-09 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene of prawns and uses thereof |
| CN101981046B (en) * | 2008-02-04 | 2015-01-07 | 内盖夫研究与发展部本-古里安大学 | Insulin-like gene in prawns and uses thereof |
| US8440878B2 (en) | 2008-02-04 | 2013-05-14 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene in prawns and uses thereof |
| CN102532257A (en) * | 2010-12-13 | 2012-07-04 | 鲁南制药集团股份有限公司 | Method for purifying recombinant human proinsulin |
| CN102532257B (en) * | 2010-12-13 | 2015-03-18 | 鲁南制药集团股份有限公司 | Method for purifying recombinant human proinsulin |
| US10865224B2 (en) | 2012-06-29 | 2020-12-15 | Emd Millipore Corporation | Purification of biological molecules |
| CN107383161A (en) * | 2012-06-29 | 2017-11-24 | Emd密理博公司 | The purifying of biomolecule |
| CN107383161B (en) * | 2012-06-29 | 2021-08-17 | Emd密理博公司 | Purification of biomolecules |
| CN103145829B (en) * | 2013-03-29 | 2015-06-03 | 江苏诺泰制药有限公司 | Purification method of insulin detemir |
| CN103145829A (en) * | 2013-03-29 | 2013-06-12 | 江苏诺泰制药有限公司 | Purification method of insulin detemir |
| CN105153294A (en) * | 2015-08-31 | 2015-12-16 | 济南康和医药科技有限公司 | Recombinant insulin and insulin analogue precursor purification method |
| CN112341535A (en) * | 2019-08-07 | 2021-02-09 | 中国科学院大连化学物理研究所 | Method for preparing insulin by separation and purification through ion exchange chromatography |
| CN113563413A (en) * | 2021-08-17 | 2021-10-29 | 汉肽生物医药集团有限公司 | Method for removing pigment in high-density fermentation process of pichia pastoris |
| WO2023154467A1 (en) * | 2022-02-11 | 2023-08-17 | Clara Foods Co. | Protein compositions and consumable products thereof |
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