CN1749267A - Protein separating and purifying method - Google Patents
Protein separating and purifying method Download PDFInfo
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- CN1749267A CN1749267A CN 200410051472 CN200410051472A CN1749267A CN 1749267 A CN1749267 A CN 1749267A CN 200410051472 CN200410051472 CN 200410051472 CN 200410051472 A CN200410051472 A CN 200410051472A CN 1749267 A CN1749267 A CN 1749267A
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Abstract
The protein separating and purifying method includes twice extraction in polymer/salt double water phase system, and chromatographic separation with anionic exchange resin to obtain protein of molecular weight 10,000-30,000. The method can eliminate great amount of impurity and cell segment simultaneously, has the features of maintaining protein activity, short phase separating time, no residue of organic solvent, easy engineering amplification and control operation, and can eliminate endotoxin to affect product quality. The method of the present invention is especially suitable for the separation and purification of hepatocytokine.
Description
Technical field:
The present invention relates to biological technical field, be specifically related to the separation and the purifying of the separation purification method of small protein, particularly hepatocyte growth-promoting factors (pHGF).
Background technology:
Aqueous two-phase extraction technology is the extracting process that obtains a kind of biologically active substance of developing rapidly in recent years.Its ultimate principle is to utilize superpolymer, salt etc. under certain composition and concentration, can form two mutual exclusive waters at same aqueous phase, be called double water-phase, utilize such double water-phase, come extraction purpose product from cell homogenates liquid or fermented liquid, realize extraction, the separation of purpose product, that its maximum advantage is to separate is easy, running cost is low, can keep the biological activity of purpose product effectively.At present, there has been multiple double-aqueous phase system to be applied, for example polyoxyethylene glycol (PEG)/dextran, PEG/ inorganic salt etc.CN 1195028A (" the aqueous two-phase extraction method is extracted the method for erythromycin ") proposes to extract erythromycin with the aqueous two-phase extraction method from the fermented liquid erythromycin that contains 3000～10000u/mL, adopt oxyethane-propylene oxide random copolymers and K2HPO4 to constitute double-aqueous phase system, leave standstill after the extraction or centrifugal phase-splitting, reach the purpose of extraction.Document (" Chinese microbiotic magazine ", 1998,23 (2), 144) has been introduced the method that the aqueous two-phase system that adopts PEG/ phosphoric acid salt to form extracts propionylspiramycin, and its research is to carry out in the propionyl spiral enzyme solution of 1000 μ g/mL.And document (" Guizhou University of Technology's journal (natural science) ", 2000,29 (4), 83) has also been reported employing PEG/ phosphoric acid salt aqueous two-phase system, separates the research of α-Dian Fenmei, and the concentration of enzyme reaches 1.05～5.5ku/mL, is up to 11.2ku/mL.Though these methods are keeping having obtained very big improvement aspect purpose its lytic activity, the raising product yield, the prerequisite of its application is that the kind of object is more single, the original concentration of object is very high, and does not have the issues of purification of object.For the aqueous two-phase extraction of the purpose product of lower concentration, still there not be the report of being correlated with at present
Hepatocyte growth-promoting factors (pHGF) is the higher bioactive treatment hepatitis medicament that has that extracts from the sucking pig liver.Classical preparation method is by the ethanol precipitation of La Breque in report in 1975.
PHGF is a molecule amount polypeptide distributed more widely, and its molecular weight distribution is in following scope: greater than 110,000,2～60,000 and less than 20,000.People such as the old light of China air hospital are at first on the traditional method of La Breque, improve, develop the technology (patent 89107217 that the negative pressure dialysis method prepares pHGF, " adopt the negative pressure dialysis method to prepare the method for hepatocyte growth factor "), promptly after the tissue of repeated multiple times is smashed to pieces, the centrifuging and taking supernatant liquor is dialysed with molecular weight cut-off 12000 daltonian dialysis tubings, obtains pHGF.Though the extraction process of this method is simple, and comparatively obvious defects is also arranged: the pHGF yield is low, protein concentration is low, production environment requires height, the production cycle is long.
Afterwards, there was multiple improved pHGF preparation technology to occur again.
For example, ethanol sedimentation coupled ion exchange filtration method (CN 1068036A, " a kind of preparation technology of excited growth factor from newborn pig's liver "), can not be directly used in clinical at pHGF raw product in original ethanol precipitation, and the refining defective that needs the chemical reagent of complicated technical process and costliness, improve, simplified original technology, through 95 ℃ of heating in water bath, 95% ethanol sedimentation, leave standstill after the filtration, phosphate buffered saline buffer dissolving ethanol sedimentation thing with 0.02M pH 7.2, carrying out DEAE ion-exchange filters, carry out wash-out twice with different concentrations of sodium chloride liquid again, promptly obtain the highly finished product of pHGF.
And for example two go on foot ultrafiltration process (CN 1038512C, " medicine for improving growth of liver cell and its method for production "), through 95 ℃ heating in water bath, cryosel cooling, centrifugal, clarify with 0.3 micron membrane filtration after, through two times of ultrafiltration, molecular weight cut-off is the pHGF product at 8000～10000 daltonian protein ingredients.
These improved methods are simplified on technology, and yield, the purity of product all increase.But they all have a common characteristic, place water-bath to be heated to 95 ℃ cell homogenates liquid earlier exactly, isolate clear liquid with centrifugal or filtering way then, further handle with technologies such as precipitation, ultrafiltration and ion-exchange filtrations again.So just bring a problem, i.e. the filtration of the cell homogenates liquid after the heating and centrifugal very difficult.In the filtration, filtering membrane is very easily blocked, causes filter pressure to increase, and power consumption increases, and filtering membrane also damages easily.And in centrifugal, also exist and be difficult to clear liquid and turbid liquid are separated fully, make troubles for the technology of back, and increase centrifugal force or centrifugal rotational speed, the problem of also bringing power consumption to increase.Owing to filter or centrifugal difficulty, make above-mentioned technology be easier to go wrong at ratio aspect product yield, product activity and the quality product, phenomenons such as intracellular toxin or antigen exceed standard appear inevitably.
PHGF is in 18000～24000 molecular weight ranges, and its activity degree is higher, clinical effectiveness good (document " clinical liver and bladder disease magazine " " extracting and the experimental study of the liver stimulating growth factor ", 1990,6 (3), 41).And to carry out the separation of the object of different molecular weight, rely on above-mentioned two aqueous phase extraction method all to be difficult to accomplish.
Research has also shown (document " Northeast China Normal University's journal natural science edition " " progress of aqueous two phase extraction technique " simultaneously, 2000,32 (3), 34), can carry out proteinic separation and Extraction though the aqueous two-phase extraction method is found the earliest, in recent years, find that also this method can also be used for extracting microbiotic, but, do not find that also this method can also extract other impurity that comprise intracellular toxin simultaneously.
Summary of the invention:
Technical problem to be solved by this invention be to provide a kind of from the higher cell slurries of foreign protein content separation and purification have the method for the activated protein of certain molecular weight scope, this method can be removed a large amount of impurity and cell debris simultaneously, have maintain protein active, the phase-splitting time is short, organic solvent-free is residual, be easy to that engineering is amplified and the characteristics of operate continuously, can remove the intracellular toxin that influences quality product simultaneously.
Method of purifying protein disclosed by the invention adopts the aqueous two-phase system of being made up of superpolymer/salt to extract, and through twice aqueous two-phase extraction, analyses separation in conjunction with anion exchange resin layer, extracts molecular weight and be 10000～30000 protein.
Superpolymer of the present invention is selected from molecular weight and is respectively in 400,1000,2000,4000,6000 or 20000 the polyoxyethylene glycol (PEG) one or several.
Salt of the present invention is selected from one or several in ammonium sulfate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate or the potassiumphosphate.
The present invention adopts twice aqueous two-phase extraction method of separating protein specifically to comprise the following steps
(1) form aqueous two-phase system with the ratio of superpolymer/salt=4～18%/6～20% (w/v), the pair cell homogenate extracts, and by leaving standstill or centrifugation method, isolate the last phase that is rich in target protein matter, obtains crude extract for the first time;
(2) form aqueous two-phase system with the ratio of superpolymer/salt=4～18%/6～20% (w/v), and add the NaCL solution of 0.1%～1.0% (w/v), to the first time crude extract extract, by leaving standstill or centrifugation method, isolate the following phase that is rich in target protein matter, as protein crude extract;
(3) protein crude extract reinstalls with in DEAE-dextran or the chromatography column of Mierocrystalline cellulose as anionite-exchange resin, with containing the wash-out that the aqueous solution of mixture more than 2 kinds or 2 kinds in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate or the sodium-chlor carries out chromatography column, collect the elutriant under 0.42%～0.86% (w/v) concentration, be needed molecular weight and be 10000～30000 protein.
In the aqueous two-phase extraction method of separating protein of the present invention, the salt preferably sulfuric acid ammonium of aqueous two-phase extraction for the first time.
When the aqueous two-phase extraction system selects two kinds of superpolymer for use, the weight ratio of two superpolymer preferred (1.0～3.0): (1.0～4.0).
When the aqueous two-phase extraction system selects two kinds of salt for use, the weight ratio of two kinds of salt preferred (1.0～8.5): (1.0～8.5).
Twice aqueous two-phase extraction separation method of the present invention is applicable to the separation and purification of small protein, is specially adapted to the preparation of hepatocyte growth-promoting factors.
Twice aqueous two-phase extraction separation method of the present invention also is applicable to antibiotic extraction.
Adopt aqueous two-phase extraction technology, avoided the filtration and the centrifugal very difficult problem of the cell homogenates liquid after the heating in the traditional technology.Owing in the composition of aqueous two-phase system, adopted the combination of the PEG of different molecular weight, and successively constitute aqueous two-phase system with two class salt, slightly carry by primary, to contain intracellular protein and other impurity is separated, again by slightly carrying for the second time, and add NaCL solution, and the purpose proteinoid can be extracted, have the selectivity of extraction target protein matter.After obtaining target protein phase crude extract, adopt anionite-exchange resin to carry out chromatography again, can isolate needed molecular weight and be 10000～30000 protein.Whole technological operation is easy, does not have complicated filter plant, only can reach the purpose of roughing out by the liquid-liquid phase centrifugation that realizes easily, has reduced the input and the running cost of equipment.Simultaneously, because entire operation aqueous phase at normal temperatures carries out, can keep the biological activity of target protein effectively.
Specific embodiment:
Embodiment 1
With mixture and PEG2000 and ammonium sulfate and the phosphoric acid salt composition double water-phase of PEG400 and PEG6000, in conjunction with DEAE-dextran chromatography, preparation pHGF.
Get fresh sucking pig liver,, carry out behind the high-pressure homogenization stand-by through wiping out adventitia, rubbing the back and add purified water.Get PEG400 and PEG6000, according to the quality ratio of components is 1.0: 2.8 (w/w), mix with cell homogenates liquid with a little purified water dissolving back, add ammonium sulfate again, comprise that the PEG of cell homogenates liquid and ammonium sulfate ratio are 9%/11% (w/v) after feasible the mixing, carry out the adjusting of concentration with purified water, vibrate so that its thorough mixing evenly after, leave standstill 1.2h.Isolate phase this moment, discards down phase, obtains crude extract for the first time.
Get PEG2000, after a little purified water dissolving, add in the crude extract first time, behind the thorough mixing, add again with purified water preparation, be the phosphate solution that mixes at 1.0: 3.8 by Sodium phosphate dibasic and potassium primary phosphate mass ratio, the NaCL solution that adds 0.76% (w/v) again, make PEG2000 and the phosphoric acid salt proportion of composing in total system reach 8.5%/11.2% at last, form aqueous two-phase system, to the first time crude extract extract, with the centrifugal method below the 4000rpm, isolate the following phase that is rich in target protein matter, be protein crude extract.
Again protein crude extract is packed in order in the chromatography column of DEAE-dextran as anionite-exchange resin, carry out the wash-out of chromatography column with the aqueous solution that contains potassium primary phosphate and sodium-chlor (it is 1.0: 2.3 that quality is formed (w/w)), the elutriant total concn is 0.8%～1.2% (w/v), carry out the wash-out of chromatography column again with the aqueous solution that contains Sodium phosphate dibasic and sodium-chlor (it is 1.0: 1.5 that quality is formed (w/w)), the elutriant total concn is 0.42%～0.86% (w/v), collect the elutriant under this concentration, can obtain needed molecular weight and be 18000～24000 hepatocyte growth-promoting factors, purity is 78.0%～84.0% (W/W) (18000～24000 molecular weight accounts for the per-cent of total protein content), and yield is 0.24%～0.82% (W/W) (Tot Prot accounts for the per-cent of cell homogenates liquid).
Embodiment 2
With mixture and PEG4000 and ammonium sulfate and the phosphoric acid salt composition double water-phase of PEG20000 and PEG1000, in conjunction with DEAE-dextran chromatography, preparation pHGF.
Get fresh sucking pig liver, wipe out behind adventitia, rubbing, the high-pressure homogenization stand-by.Get PEG20000 and PEG1000, according to the quality ratio of components is 2.7: 1.3 (w/w), mix with homogenate the dissolving back, add ammonium sulfate, making PEG and ammonium sulfate reach ratio in homogenate is 8.2%/12.3% (w/v), and water carries out the adjusting of concentration, after fully mixed, after leaving standstill 1.3h, isolate phase, obtain crude extract for the first time.
Get PEG4000 again, after the dissolving, add in the crude extract first time, the NaCL solution that adds 0.76% (w/v) again, after this, add SODIUM PHOSPHATE, MONOBASIC, form aqueous two-phase system, make PEG4000 and the phosphoric acid salt proportion of composing in total system reach 10.3%/12.7%, behind the thorough mixing, centrifugal (≤4000rpm), introduce in the chromatography column mutually down with isolated, anionite-exchange resin is the DEAE-dextran, the aqueous solution with dipotassium hydrogen phosphate and sodium-chlor (it is 1.0: 2.8 that quality is formed (w/w)) carries out wash-out earlier, the elutriant total concn is 0.8%～1.2% (w/v), use the aqueous solution of Sodium phosphate dibasic and sodium-chlor (it is 1.0: 1.5 that quality is formed (w/w)) to carry out wash-out again, the elutriant total concn is 0.42%～0.86% (w/v), collect the elutriant under this concentration, can obtain needed product, purity is 81.0%～86.0% (W/W) (18000～24000 molecular weight accounts for the per-cent of total protein content), and yield is 0.16%～0.67% (W/W) (Tot Prot accounts for the per-cent of cell homogenates liquid).
Claims (8)
1. the separation purification method of a small protein, it is characterized in that this method adopts the aqueous two-phase system extraction of being made up of superpolymer/salt, through twice aqueous two-phase extraction, analyse separation in conjunction with anion exchange resin layer, extract molecular weight and be 10000～30000 protein or microbiotic.
2. separation purification method according to claim 1 is characterized in that wherein said protein is hepatocyte growth-promoting factors.
3. separation purification method according to claim 1 is characterized in that it is in 400,1000,2000,4000,6000 or 20000 the polyoxyethylene glycol one or several that wherein said superpolymer is selected from molecular weight.
4. separation purification method according to claim 1 is characterized in that wherein said salt is selected from one or several in ammonium sulfate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate or the potassiumphosphate.
5. separation purification method according to claim 1 is characterized in that this method specifically comprises the following steps:
(1) form aqueous two-phase system with the ratio of superpolymer/salt=4～18%/6～20% (w/v), the pair cell homogenate extracts, and by leaving standstill or centrifugation method, isolate the last phase that is rich in target protein matter, obtains crude extract for the first time;
(2) form aqueous two-phase system with the ratio of superpolymer/salt=4～18%/6～20% (w/v), and add the NaCL solution of 0.1%～1.0% (w/v), to the first time crude extract extract, by leaving standstill or centrifugation method, isolate the following phase that is rich in target protein matter, as protein crude extract;
(3) protein crude extract reinstalls with in DEAE-dextran or the chromatography column of Mierocrystalline cellulose as anionite-exchange resin, with containing the wash-out that the aqueous solution of mixture more than 2 kinds or 2 kinds in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate or the sodium-chlor carries out chromatography column, collect the elutriant under 0.42%～0.86% (w/v) concentration, be needed molecular weight and be 10000～30000 protein.
6. separation purification method according to claim 4 is characterized in that the salt of wherein said step (1) the aqueous two-phase extraction first time is ammonium sulfate.
7. separation purification method according to claim 2, when it is characterized in that wherein said superpolymer is selected two kinds for use, weight ratio is (1.0～3.0): (1.0～4.0).
8. separation purification method according to claim 3, when it is characterized in that wherein said salt is selected two kinds for use, weight ratio is (1.0～8.5): (1.0～8.5).
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100453642C (en) * | 2006-10-10 | 2009-01-21 | 山西职工医学院 | Extraction method of peroxidase |
| CN100471542C (en) * | 2006-12-15 | 2009-03-25 | 中国科学院长春应用化学研究所 | Process for separating superfine particles |
| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
| CN103275941A (en) * | 2013-06-14 | 2013-09-04 | 齐鲁工业大学 | Method for preparing pyruvic oxidase |
| CN104263706A (en) * | 2014-10-15 | 2015-01-07 | 广西大学 | Purification method for tannase |
| CN105732797A (en) * | 2016-04-18 | 2016-07-06 | 山东众山生物科技有限公司 | Purification method for urinary gonadotropin |
| CN106432410A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Protein separation and purification device and protein separation and purification method |
| CN114112612A (en) * | 2021-10-28 | 2022-03-01 | 丽珠集团福州福兴医药有限公司 | Separation and purification method of teicoplanin I5 impurity and application thereof |
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2004
- 2004-09-15 CN CN 200410051472 patent/CN1749267A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100453642C (en) * | 2006-10-10 | 2009-01-21 | 山西职工医学院 | Extraction method of peroxidase |
| CN100471542C (en) * | 2006-12-15 | 2009-03-25 | 中国科学院长春应用化学研究所 | Process for separating superfine particles |
| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
| CN103275941A (en) * | 2013-06-14 | 2013-09-04 | 齐鲁工业大学 | Method for preparing pyruvic oxidase |
| CN103275941B (en) * | 2013-06-14 | 2014-12-31 | 齐鲁工业大学 | Method for preparing pyruvic oxidase |
| CN104263706A (en) * | 2014-10-15 | 2015-01-07 | 广西大学 | Purification method for tannase |
| CN105732797A (en) * | 2016-04-18 | 2016-07-06 | 山东众山生物科技有限公司 | Purification method for urinary gonadotropin |
| CN105732797B (en) * | 2016-04-18 | 2019-07-02 | 山东众山生物科技有限公司 | A kind of purification process of urinary gonadotropin |
| CN106432410A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Protein separation and purification device and protein separation and purification method |
| CN114112612A (en) * | 2021-10-28 | 2022-03-01 | 丽珠集团福州福兴医药有限公司 | Separation and purification method of teicoplanin I5 impurity and application thereof |
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