CN101575373A - Preparation method of hemoglobin extract - Google Patents
Preparation method of hemoglobin extract Download PDFInfo
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- CN101575373A CN101575373A CNA2009101040700A CN200910104070A CN101575373A CN 101575373 A CN101575373 A CN 101575373A CN A2009101040700 A CNA2009101040700 A CN A2009101040700A CN 200910104070 A CN200910104070 A CN 200910104070A CN 101575373 A CN101575373 A CN 101575373A
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Abstract
The invention provides a preparation method of hemoglobin extract, which implements the following operations in sequence of: breaking red blood cells in a dialysis bag through hypotonicity to obtain the breaking liquid of hematid; using ammonium sulphate with an amount corresponding to the final saturation degree of 5-40 percent and the final saturation degree of 40-80 percent at the temperature of 0 DEG C to implement two steps of deposition and then heavily suspending the deposited protein and transferring the protein into the dialysis bag to dialyze and remove the ammonium sulphate, and finally using buffer solution to dilute the dialyzed protein liquid and centrifugating to remove particulate matters. When the method is used for preparing the hemoglobin extract, the hemoglobin recycling rate is more than 85 percent; the lipoprotein and phospholipid residual quantity are less than 2 percent; and the hemoglobin extract is easy to penetrate a 0.45 Mum filterable membrane and is very suitable for the requirement of massive production of anion-exchange column chromatography.
Description
Technical field
The invention belongs to the separation purifying technique technical field of oxyphorase, specifically, relate to the method for preparing hemoglobin extract in the oxyphorase separation purifying technique.
Background technology
The separation purifying technique of oxyphorase is an important process step of taking the oxygen material development.Existing is the taking in the oxygen material of carrier with the stroma-free hemoglobin, except that the recombinant expressed oxyphorase of genetically engineered, most of oxyphorase all derives from animal, comprise ox, pig, people etc., utilize these animal blood haemoglobins, at first need a kind of low cost, easy standardized oxyphorase purification process.
Existing oxyphorase purification process mainly contains heating method, ultrafiltration process, extraction process, anion-exchange column chromatography etc.Heating method and extraction process are inevitable to the influence of the space structure generation of oxyphorase, and the organic solvent that extraction process is introduced also is a kind of potential Hazard Factor; Though ultrafiltration process is convenient to carry out airtight streamline production, ultra-filtration membrane needs often to change, and is with high costs; The anion-exchange column chromatography cost is low, to the not influence of space structure of oxyphorase, and does not introduce organic solvent, thereby is the most promising oxyphorase purification process.
Anion-exchange column chromatography has the requirement of following two aspects to the protein extract that is used for upper prop: (1) protein extract must not contain the macrobead thing, and general requirement is through 0.45 μ m membrane filtration; (2) protein ingredient in the protein extract should be the least possible, and reduce the content with anion exchange chromatography medium effect intensive composition as far as possible.
The preparation method of traditional hemoglobin extract that is used for anion-exchange chromatography generally comprises following steps:
(1) with packed red cells and hypotonic solution (normally water) according to certain volume than mixing to prepare hypotonic environment, standing over night makes erythroclasis;
(2) centrifugal erythroclasis liquid is transferred to new centrifuge tube with supernatant;
(3) with supernatant with 0.45 μ m membrane filtration, obtain hemoglobin extract;
(4) hemoglobin extract is replaced to anion-exchange chromatography level pad system.
Prepare hemoglobin extract by above-mentioned traditional method, long-pending big except erythroclasis liquid, be unfavorable for that subsequent step is carried out beyond, the more important thing is the problem that has following two aspects:
(1) hemoglobin extract that obtains is difficult to by 0.45 μ m filter membrane, therefore need change filter membrane continually, and the process of changing filter membrane has improved the content of methemoglobin in the extracting solution;
(2) contain more phosphatide and lipoprotein in the hemoglobin extract that obtains, its wash-out must can not be carried out cleaning procedure with the elute soln of the tolerant maximum strength of filler.Cleaning procedure need expend a large amount of ethanol or Virahol, and the time is longer, is not suitable for scale operation.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method who is applicable to the hemoglobin extract of anion-exchange column chromatography mass production requirement, adopt the hemoglobin extract of this method preparation to be easy to by 0.45 μ m filter membrane, lipoprotein and phospholipids content are few.
For achieving the above object, the preparation method of hemoglobin extract of the present invention may further comprise the steps, and each step is all carried out under 0～10 ℃ ambient temperature conditions:
(1) packed red cells is placed dialysis tubing, through the Hyposmolality fragmentation, obtain erythroclasis liquid with damping fluid;
(2) ammonium sulfate that whole saturation ratio is 5～40% corresponding amounts in the time of will be with 0 ℃ adds in the erythroclasis liquid, and it is centrifugal with 4000～16000G to treat that ammonium sulfate fully dissolves the back;
Whole saturation ratio is the ammonium sulfate of 40～80% corresponding amounts when (3) adding with 0 ℃ in the supernatant liquor of the centrifugal gained of step (2), treats that ammonium sulfate is centrifugal with 2000～10000G after fully dissolving;
(4) albumen precipitation of the centrifugal gained of step (3) is changed in the dialysis tubing after resuspended, remove ammonium sulfate with the damping fluid dialysis;
(5) protein liquid through dialysis is to dilute in 1: 1～1: 10 with damping fluid by volume, and is centrifugal with 8000～16000G then, removes particulate matter.
Adopt the mode of dialysing to make the Hyposmolality environment in the step of the present invention (1) and make erythroclasis, effectively reduced the volume of erythroclasis liquid, increased the density of solution, thereby created condition for follow-up ammonium sulfate fractional separation; And, saved the consumption of ammonium sulfate greatly because erythroclasis liquid is long-pending little.Adopt the two steps ammonium sulfate precipitation operation of step (2) and step (3), prepare different solution densities, make lipoprotein and phosphatide under corresponding density and centrifugal force condition, be in different positions in the centrifuge tube, thereby the lipoprotein and the part foreign protein of the overwhelming majority are removed with oxyphorase.
Practice shows, through the hemoglobin extract of the inventive method preparation, compares with the traditional hemoglobin extract preparation method who is used for anion-exchange chromatography, and its lipoprotein and phospholipids content residual rate are easy to by 0.45 μ m filter membrane less than 2%; And the ammonium sulfate consumption is few, and the purifying cost is low, and the oxyphorase rate of recovery is higher, has good application prospects.
Description of drawings
To be the present invention compare the SDS-PAGE analytical results contrast figure of the hemoglobin extract of preparation with the traditional hemoglobin extract preparation method who is used for anion-exchange chromatography with accompanying drawing;
Among the figure: M represents the molecular weight of albumen standard;
C represents the hemoglobin extract of traditional method preparation;
D represents the hemoglobin extract of the inventive method preparation.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
Embodiment 1:
Under 0～10 ℃ ambient temperature conditions, carry out following operation successively:
(1) packed red cells being placed average molecular weight cut-off is that the dialysis tubing of 5～14kDa obtains erythroclasis liquid through the Hyposmolality fragmentation, the used damping fluid of dialysing is the Tris-HCl damping fluid through deoxidation treatment, the Tris base concentration of damping fluid is 50mM, and the pH value is 9.0;
(2) ammonium sulfate that whole saturation ratio is 40% corresponding amount in the time of will be with 0 ℃ adds in the erythroclasis liquid, and it is centrifugal with 12000G to treat that ammonium sulfate fully dissolves the back;
Whole saturation ratio is the ammonium sulfate of 80% corresponding amount when (3) adding with 0 ℃ in step (2) in the supernatant liquor of centrifugal gained, treats that ammonium sulfate is centrifugal with 16000G after fully dissolving;
(4) protein precipitation of step (3) gained is changed in the dialysis tubing that average molecular weight cut-off is 5～14kDa over to dialysis after resuspended to remove ammonium sulfate, the used damping fluid of dialysing is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 100mM, NaCl concentration is 50mM, and the pH value of damping fluid is 9.0;
(5) protein liquid through dialysis is that 1: 10 ratio is diluted with damping fluid by volume, then with the centrifugal removal particulate matter of 8000G, used damping fluid is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 100mM, NaCl concentration is 50mM, and the pH value of damping fluid is 9.0.
Embodiment 2:
Under 0～10 ℃ ambient temperature conditions, carry out following operation successively:
(1) packed red cells is obtained erythroclasis liquid through the Hyposmolality fragmentation in the dialysis tubing that average molecular weight cut-off is 32kDa, used dialyzate is the Tris-HCl damping fluid through deoxidation treatment, and the Tris base concentration of damping fluid is 5mM, and the pH value is 7.0;
(2) ammonium sulfate that whole saturation ratio is 5% corresponding amount in the time of will be with 0 ℃ adds in the erythroclasis liquid, and it is centrifugal with 8000G to treat that ammonium sulfate fully dissolves the back;
Whole saturation ratio is the ammonium sulfate of 40% corresponding amount when (3) adding with 0 ℃ in step (2) in the supernatant liquor of centrifugal gained, treats that ammonium sulfate is centrifugal with 2000G after fully dissolving;
(4) protein precipitation of step (3) gained is changed in the dialysis tubing that average molecular weight cut-off is 32kDa over to dialysis after resuspended to remove ammonium sulfate, the used damping fluid of dialysing is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 10mM, NaCl concentration is 10mM, and the pH value of damping fluid is 8.0;
(5) protein liquid through dialysis is that 1: 1 ratio is diluted with damping fluid by volume, then with the centrifugal removal particulate matter of 16000G, used damping fluid is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 10mM, NaCl concentration is 10mM, and the pH value of damping fluid is 8.0;
Embodiment 3:
Under 0～10 ℃ ambient temperature conditions, carry out following operation successively:
(1) packed red cells is obtained erythroclasis liquid through the Hyposmolality fragmentation in the dialysis tubing that average molecular weight cut-off is 60kDa, the used damping fluid of dialysing is the Tris-HCl damping fluid through deoxidation treatment, the Tris base concentration of damping fluid is 100mM, and the pH value is 8.0;
(2) ammonium sulfate that whole saturation ratio is 25% corresponding amount in the time of will be with 0 ℃ adds in the erythroclasis liquid, and it is centrifugal with 4000G to treat that ammonium sulfate fully dissolves the back;
Whole saturation ratio is the ammonium sulfate of 60% corresponding amount when (3) adding with 0 ℃ in step (2) in the supernatant liquor of centrifugal gained, treats that ammonium sulfate is centrifugal with 6000G after fully dissolving;
(4) protein precipitation of step (3) gained is changed in the dialysis tubing that average molecular weight cut-off is 60kDa over to dialysis after resuspended to remove ammonium sulfate, the used damping fluid of dialysing is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 55mM, NaCl concentration is 30mM, and the pH value of damping fluid is 8.5;
(5) protein liquid through dialysis is that 1: 5 ratio is diluted with damping fluid by volume, then with the centrifugal removal particulate matter of 10000G, used damping fluid is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, the Tris base concentration of damping fluid is 55mM, NaCl concentration is 30mM, and the pH value of damping fluid is 8.5.
The parameter such as the following table of above-mentioned three embodiment gained hemoglobin extracts:
As can be seen, utilize the inventive method to prepare hemoglobin extract, the oxyphorase rate of recovery is greater than 80%, and lipoprotein and phosphatide residual quantity are easy to be very suitable for the mass-produced requirement of anion-exchange column chromatography by 0.45 μ m filter membrane less than 2%.
Figure of description is the SDS-PAGE check analysis figure of the hemoglobin extract of the inventive method and traditional method preparation.As can be seen from the figure, protein ingredient is effectively reduced in the hemoglobin extract of employing the inventive method preparation, and content of hemoglobin improves.
Claims (4)
1. the preparation method of a hemoglobin extract is characterized in that, may further comprise the steps, and each step is all carried out under 0～10 ℃ ambient temperature conditions:
(1) packed red cells is placed dialysis tubing, through the Hyposmolality fragmentation, obtain erythroclasis liquid with damping fluid;
(2) ammonium sulfate that whole saturation ratio is 5～40% corresponding amounts in the time of will be with 0 ℃ adds in the erythroclasis liquid, and it is centrifugal with 4000～16000G to treat that ammonium sulfate fully dissolves the back;
Whole saturation ratio is the ammonium sulfate of 40～80% corresponding amounts when (3) adding with 0 ℃ in the supernatant liquor of the centrifugal gained of step (2), treats that ammonium sulfate is centrifugal with 2000～10000G after fully dissolving;
(4) albumen precipitation of the centrifugal gained of step (3) is changed in the dialysis tubing after resuspended, remove ammonium sulfate with the damping fluid dialysis;
(5) protein liquid through dialysis is to dilute in 1: 1～1: 10 with damping fluid by volume, and is centrifugal with 8000～16000G then, removes particulate matter.
2. the preparation method of a kind of hemoglobin extract as claimed in claim 1 is characterized in that, the average molecular weight cut-off of used dialysis tubing is less than 64kDa in described step (1) and the step (4).
3. the preparation method of a kind of hemoglobin extract as claimed in claim 1, it is characterized in that, used damping fluid is the Tris-HCl damping fluid through deoxidation treatment in the described step (1), and the Tri s base concentration of damping fluid is 5～100mM, and the pH value is 7.0～9.0.
4. the preparation method of a kind of hemoglobin extract as claimed in claim 1, it is characterized in that, damping fluid used in step (4) and the step (5) is the Tris-HCl through deoxidation treatment, the NaCl damping fluid, Tris base concentration in the damping fluid is 10～100mM, NaCl concentration is 10～50mM, and the pH value of damping fluid is 8.0～9.0.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200910104070 CN101575373B (en) | 2009-06-12 | 2009-06-12 | Preparation method of hemoglobin extract |
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| CN 200910104070 CN101575373B (en) | 2009-06-12 | 2009-06-12 | Preparation method of hemoglobin extract |
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| CN101575373A true CN101575373A (en) | 2009-11-11 |
| CN101575373B CN101575373B (en) | 2013-03-20 |
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| CN 200910104070 Expired - Fee Related CN101575373B (en) | 2009-06-12 | 2009-06-12 | Preparation method of hemoglobin extract |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100183A (en) * | 2018-10-26 | 2020-05-05 | 中国科学院青岛生物能源与过程研究所 | Method for preparing solution-permeable target protein precipitate and separating and purifying target protein from host cell |
| CN113150120A (en) * | 2021-05-21 | 2021-07-23 | 江南大学 | Method for separating and purifying porcine myoglobin in fermentation liquor |
| CN113959807A (en) * | 2021-10-26 | 2022-01-21 | 上海瀚诺威生物科技有限公司 | Preparation method of glycosylated hemoglobin calibration quality control product |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602639B (en) * | 2013-08-16 | 2016-04-27 | 科兴（大连）疫苗技术有限公司 | A kind of method adopting Hyposmolality harvest liquid to gather in the crops virus |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE2449885C3 (en) * | 1974-10-21 | 1980-04-30 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | Process for the production of chemically modified, long-life hemoglobin preparations as well as the modified hemoglobin preparation produced by this process |
| CN1169836C (en) * | 2002-04-11 | 2004-10-06 | 中南民族大学 | Process for separating and purifying haematoglobin by liquid-solid extracting system |
| CN1786018B (en) * | 2005-12-12 | 2011-06-22 | 天津协和生物科技发展有限公司 | Separation and purification of high purity hemoglobin and virus inactivation technology |
| CN101289493B (en) * | 2008-04-22 | 2010-11-10 | 中国人民解放军第三军医大学野战外科研究所 | Process for abstracting high-purity hemoglobin from pig blood |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100183A (en) * | 2018-10-26 | 2020-05-05 | 中国科学院青岛生物能源与过程研究所 | Method for preparing solution-permeable target protein precipitate and separating and purifying target protein from host cell |
| CN111100183B (en) * | 2018-10-26 | 2022-07-15 | 中国科学院青岛生物能源与过程研究所 | Method for preparing solution-permeable target protein precipitate and separating and purifying target protein from host cell |
| CN113150120A (en) * | 2021-05-21 | 2021-07-23 | 江南大学 | Method for separating and purifying porcine myoglobin in fermentation liquor |
| CN113959807A (en) * | 2021-10-26 | 2022-01-21 | 上海瀚诺威生物科技有限公司 | Preparation method of glycosylated hemoglobin calibration quality control product |
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