CN105017412A - Method for separating high-purity bovine serum albumin from bovine serum - Google Patents
Method for separating high-purity bovine serum albumin from bovine serum Download PDFInfo
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Abstract
The invention belongs to the technical field of protein purification, and concretely discloses a method for separating high-purity bovine serum albumin from bovine serum. Diluted bovine serum is successively subjected to ammonium sulfate salting out, molecular sieve chromatography, anion exchange chromatography, pathogenic organism inactivation, molecular sieve chromatography, ultrafiltration condensation and filtering sterilization, so that high-purity bovine serum albumin is finally obtained. The purity of bovine serum albumin is higher than 99.5%, also bovine serum albumin does not contain micromolecule substances such as sodium chloride, ammonium sulfate, amino acids and the like, and additionally, bovine serum albumin does not contain live virus.
Description
Technical field
The present invention relates to Protein purification techniques field, particularly, relate to a kind of method of separating high-purity bovine serum albumin from bovine serum.
Background technology
Bovine serum albumin (BSA), also known as BSA (Cohn Fraction V), be also the main component of bovine blood, comprise 583 amino-acid residues, molecular weight is 66.430 kDa, and iso-electric point is about 4.7.This name (BSA) rule comes from the Cohn serum albumin partition method of original adoption cold ethanol precipitation method.According to CohnShi method, in the 5th ethanol fraction, have found serum albumin.So-called bovine serum albumin BSA is a kind of main mixture containing bovine serum albumin in fact, instead of pure bovine serum albumin.Albumin can be combined with multiple positively charged ion, negatively charged ion and other small-molecule substances.Albumin in blood mainly plays the effect of maintenance osmotic pressure, pH shock absorption, carrier function and trophism.In animal cell non-serum is cultivated, add albumin and can play physiology and mechanical protection effect and carrier function.Bovine serum albumin is widely used in biochemical test, such as, at western blot and the closed reagent prepared in elisa plate.In the preservation solution that BSA is generally used to restriction enzyme or modifying enzyme as stablizer and reaction solution, because some enzyme is unstable or active low at low concentrations.After adding BSA, it may play " protection " or " carrier " effect, and many enzymes can make its activity increase substantially after adding BSA.Bovine serum albumin is a kind of protein with multi-functional feature such as very strong-hydrophobicity and wetting ability etc., and other oroteins does not have such feature, if bovine serum albumin is impure, such as, containing other oroteins or small-molecule substance, will affect its function.
Be separated the pure sero-abluminous object of acquisition and be mainly separated other more than 200 kinds of foreign proteins and the pollution substance that may exist in removing blood plasma, obtain pyrogen-free, without antigen antibody reaction, free of contamination albumin etc.At present, the report majority of serum albumin purifying is about the sero-abluminous separation and purification in people source: early stage method as, the chlorination nanofarad (1847) of Denis, the sodium sulphate method (1890) of Hofmeister and the grade ammonium sulfate salting-out crystallization process (1894) of Hermmarsten.China early application ammonium sulfate salting-out process produces human serum albumin, but due to ammonium sulfate production process loaded down with trivial details, also adopt cold ethanol precipitation method afterwards.So far the main method of producing human serum albumin is industrially still.Utilize cold ethanol precipitation method to have some shortcomings: ethanol is a kind of non-specific precipitation agent, although remarkable to human serum albumin production effect, product purity is not very high yet; Ethanol as organic solvent also may make protein aggregates or sex change; In non-enclosed environment, organic solvent is also comparatively large to staff's harm, residues in ethanol in product simultaneously and is difficult to remove.Also someone uses the PEG precipitator method instead for this reason, and to avoid protein aggregation or sex change, and not easily remove from albumin solution, industrial production has certain difficulty.For solving the problem that the small-molecule chemical reagent such as ammonium sulfate, ethanol, PEG remain in serum albumin and serum albumin purity is not high, just adopt the albumin that chromatography is produced.But the simple chromatography that adopts is difficult to effectively suppress or except pyrogen removal or exogenous virus etc., have a strong impact on the security of product.For making up chromatographic deficiency, the method that immediate development chromatogram combines with cold ethanol precipitation, the two is had complementary advantages, application produce after certain effect.
Such as, but following shortcoming can be there is in the bovine serum albumin of these method purifying: 1) residual a large amount of small-molecule substance or chemical reagent, sodium-chlor, ammonium sulfate, PEG, ethanol, amino acid, VITAMIN, trace element etc. in serum albumin; 2) serum albumin purity is not high enough; 3) in serum, the microorganism such as exogenous virus has not had removal, affects sero-abluminous security.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of method of separating high-purity bovine serum albumin from bovine serum is provided.
To achieve these goals, the present invention is achieved by the following technical programs:
A method for separating high-purity bovine serum albumin from bovine serum, comprises the following steps:
S1. in bovine serum, physiological saline is added or bovine serum dilutes by PBS solution; Moderate amount of sulfuric acid ammonium is added, 2 ~ 8 DEG C of standing more than 30min, collected by centrifugation supernatant liquor in the bovine serum after dilution;
S2. in supernatant liquor, add the PBS solution of 2 ~ 3 times of volumes, after mixing, carry out hydrophobic chromatography, use PBS solution wash-out, collect elutriant;
S3. elutriant is carried out sieve chromatography, collect not saliferous and other micromolecular protein peak; Described chromatographic stuffing is G10-G75 dextran;
S4. use anion-exchange chromatography to be further purified bovine serum albumin, carry out gradient elution by 0.1 ~ 1.5M concentration sodium-chlor, collect consummate bovine serum albumin;
S5. the inactivator adding nonprotein sex change class carries out deactivation to pathogenic micro-organism, carries out sieve chromatography afterwards, uses ultrapure water wash-out to collect bovine serum albumin; Described sieve chromatography filler is the dextran of G25 or G50;
S6. use the film bag that aperture is 30K to carry out ultrafiltration and concentration, make bovine serum albumin ultimate density reach more than 5mg/ml; The filter membrane of 0.2 micron is finally used to carry out filtration sterilization.
In bovine serum, kinds of protein composition is complicated, therefore want by bovine serum albumin therefrom high purity separation out, the doping of other foreign proteins must be got rid of.Conventional art is adopt blanket chlorination nanofarad, sodium sulphate method and grade ammonium sulfate salting-out crystallization process mostly, although these blanket methods also can be separated to bovine serum albumin, but cannot get rid of the doping of other foreign proteins, the purity of the bovine serum albumin namely obtained is not high at all.The present invention is by for many years to the analysis and research that the one-tenth of bovine serum is grouped into, and the biological activity according to serum albumin and other foreign proteins or miscellaneous material is distinguished, and develops a kind of albuminous method of high purity separation.Although the hydrophobic chromatography that the present invention uses, sieve chromatography, ion exchange chromatography are all the routine techniques means for separation and purification of protein, but, the sequencing of various chromatography but arbitrarily can not adjust change, and we study discovery, the resolving power of hydrophobic chromatography is all good than ion-exchange when being separated albumin with current capacity, and therefore this combination is optimum combination.
Preferably, the add-on of physiological saline described in S1 or PBS solution is 1 ~ 3 times of bovine serum volume.
The effect of ammonium sulfate makes protein denaturation, and expose the hydrophobic grouping in albumen, to be separated according to hydrophobic property difference, preferably, the consumption of ammonium sulfate described in S1 is add 10 ~ 30g ammonium sulfate in every 100 milliliters of dilute serums.
More preferably, ammonium sulfate described in S1 adds by the amount adding 15 ~ 25g ammonium sulfate in every 100 milliliters of dilute serums.
Most preferably, ammonium sulfate described in S1 adds by the amount adding 20 ~ 25g ammonium sulfate in every 100 milliliters of dilute serums.
Add ammonium sulfate in the bovine serum after dilution after, preferably leave standstill at 2 ~ 8 DEG C and carry out centrifugal again in more than 1 hour, collect supernatant liquor; Described centrifugal optimum condition is 5000 ~ 8000 revs/min, centrifugal 30 minutes to 1 hour.
Hydrophobic chromatography utilizes the hydrophobic ligands of coupling on stationary phase carrier combine with some the hydrophobic molecule generation reversibilitys in moving phase and be separated.The method based on be the hydrophobic difference of protein, in high level salt solution, protein can combine with hydrophobic aglucon, and other foreign protein does not then have this kind of character, utilizes this kind of character, can by separation preliminary for protein.Preferably, the filler of hydrophobic chromatography described in S2 is phenyl-dextran or other dewatering filling of butyl-dextran.During hydrophobic chromatography, foreign protein is present in and penetrates in liquid, uses PBS solution wash-out, collects elutriant, main containing serum albumin in elutriant.
The object that the present invention carries out sieve chromatography is desalination and removes small-molecule substance, such as, and the materials such as ammonium sulfate, sodium-chlor, amino acid, VITAMIN, small protein.
Preferably, the filler of anion-exchange chromatography described in S4 is DEAE-dextran.
Preferably, described in S5, the inactivator of nonprotein sex change class is beta-propiolactone.The optimum condition using beta-propiolactone to carry out deactivation to pathogenic micro-organism is 37 DEG C of deactivations 24 hours.The add-on that beta-propiolactone is 5 ‰ according to its final concentration in the solution adds.
Compared with prior art, the present invention has following beneficial effect:
In the bovine serum albumin prepared by the present invention, the purity of bovine serum albumin is higher than 99.5%, and, not such as, containing small-molecule substance, sodium-chlor, ammonium sulfate, amino acid etc. in bovine serum albumin; In addition, live virus is not contained in bovine serum albumin.
Accompanying drawing explanation
Fig. 1 is bovine serum phenyl in embodiment 1-albuminous design sketch of dextran hydrophobic chromatography separation of serum; Wherein, blue curve is the separation variation situation of Proteins in Serum in hydrophobic chromatography process; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak.
Fig. 2 is the design sketch that in embodiment 1, bovine serum albumin solution screens out salt and small-molecule substance by G25 dextran molecule; Wherein, blue curve is albumen change in protein situation in sieve chromatography process in bovine serum albumin solution; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak; Red arrow is small-molecule substance protein peak.
Fig. 3 be in embodiment 1 bovine serum albumin by the consummate further design sketch of DEAE-dextran ion exchange chromatography; Wherein, blue curve is that the thick pure sample product of bovine serum albumin are by ion exchange chromatography protein separation situation; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak.
Fig. 4 be in embodiment 1 consummate bovine serum albumin solution by the design sketch of G50 dextran molecule sieve chromatography desalination and small-molecule substance; Wherein, blue curve is protein separation situation in consummate rear bovine serum albumin solution; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak; Red arrow is small-molecule substance protein peak.
Fig. 5 is that bovine serum albumin in embodiment 1 after consummateization and bovine serum carry out SDS-PAGE electrophoretic effects comparison chart; M: standard protein Marker; 2: the bovine serum albumin after consummate; 3: the molecular weight of the protein that arrow is corresponding.
Fig. 6 is bovine serum butyl in embodiment 2-albuminous design sketch of dextran hydrophobic chromatography separation of serum; Wherein, blue curve is the separation variation situation of Proteins in Serum in hydrophobic chromatography process; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak.
Fig. 7 is the design sketch that in embodiment 2, bovine serum albumin solution screens out salt and small-molecule substance by G50 dextran molecule; Wherein, blue curve is albumen change in protein situation in sieve chromatography process in bovine serum albumin solution; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak; Red arrow is small-molecule substance protein peak.
Fig. 8 be in embodiment 2 bovine serum albumin by the consummate further design sketch of DEAE-dextran ion exchange chromatography; Wherein, blue curve is that the thick pure sample product of bovine serum albumin are by ion exchange chromatography protein separation situation; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak.
Fig. 9 be in embodiment 2 consummate bovine serum albumin solution by the design sketch of G25 dextran molecule sieve chromatography desalination and small-molecule substance; Wherein, blue curve is protein separation situation in consummate rear bovine serum albumin solution; Red curve is conductance in solution (various ionic concn) changing conditions; Black arrow is bovine serum albumin white peak; Red arrow is small-molecule substance protein peak.
Figure 10 is that bovine serum albumin in embodiment 2 after consummateization and bovine serum carry out SDS-PAGE electrophoretic effects comparison chart; M: standard protein Marker; 2: the bovine serum albumin after consummate; 3: the molecular weight of the protein that arrow is corresponding.
Embodiment
To make the present invention below in conjunction with Figure of description and specific embodiment and elaborating further, described embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.The test method used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc. if no special instructions, are the reagent that can obtain from commercial channels and material.
embodiment 1
A method for separating high-purity bovine serum albumin from bovine serum, concrete steps are as follows:
S1. in 50mL bovine serum, add the mixing of 50mL physiological saline, then add the mixing of 20g ammonium sulfate, put into 2 ~ 8 DEG C of refrigerators static 2 hours, then 8000 revs/min, centrifugal 30 minutes, collect supernatant liquor.
S2. in supernatant liquor, add the PBS solution of 300 mL, after mixing, direct loading carries out hydrophobic chromatography, and chromatographic stuffing is phenyl-dextran (GE company), uses normal saline solution wash-out filler, collects physiological saline elution peak.The albuminous design sketch of hydrophobic chromatography separation of serum is shown in Fig. 1.
S3. then use molecular sieve to carry out removal salt and small-molecule substance, chromatographic stuffing is G25 dextran (GE company), collects not saliferous and other micromolecular protein peak.The design sketch being screened out salt and small-molecule substance by G25 dextran molecule is shown in Fig. 2.
S4. then carry out anion-exchange chromatography, chromatographic stuffing is DEAE-dextran (GE company), uses 0.5M sodium-chlor to carry out gradient elution after the direct loading of sample, collects bovine serum albumin white peak.The consummate further design sketch of DEAE-dextran ion exchange chromatography is shown in Fig. 3.
S5. add beta-propiolactone, its final concentration is in the solution 5 ‰, 37 DEG C of deactivations 24 hours; Then carry out sieve chromatography, use G50 dextran (GE company) chromatographic stuffing carry out desalination and remove small-molecule substance, this process uses ultrapure water wash-out, collects bovine serum albumin.The design sketch that G50 dextran molecule screens out salt and small-molecule substance is shown in Fig. 4.
S6. then use the film bag that aperture is 30K to carry out ultrafiltration and concentration, make final bovine serum albumin concentration reach more than 5mg/mL; Finally use the filter membrane of 0.2 micron to carry out filtration sterilization and namely obtain high-purity bovine serum albumin; In bovine serum albumin, the purity of bovine serum albumin is 99.7%.Bovine serum albumin after consummate and bovine serum are carried out the comparison of SDS-PAGE electrophoretic effects, the results are shown in Figure 5.
embodiment 2
s1. in 50mL bovine serum, add the mixing of 100mL physiological saline, then add the mixing of 40g ammonium sulfate, put into 2 ~ 8 DEG C of refrigerators static 2 hours, then 8000 revs/min, centrifugal 30 minutes, collect supernatant liquor.
S2. in supernatant liquor, add 300mL physiological saline, after mixing, direct loading carries out hydrophobic chromatography, and chromatographic stuffing is butyl-dextran (GE company), uses the PBS solution wash-out filler of 0.1M, collects the PBS elution peak of 0.1M.The albuminous design sketch of hydrophobic chromatography separation of serum is shown in Fig. 6.
S3. then use molecular sieve to carry out removal salt and small-molecule substance, chromatographic stuffing is G50 dextran (GE company), collects not saliferous and other micromolecular protein peak.The design sketch that G50 dextran molecule screens out salt and small-molecule substance is shown in Fig. 7.
S4. then carry out ion exchange chromatography, chromatographic stuffing is DEAE-dextran (GE company), uses 0.5M sodium-chlor to carry out gradient elution after the direct loading of sample, collects bovine serum albumin white peak.The consummate further design sketch of DEAE-dextran ion exchange chromatography is shown in Fig. 8.
S5. add beta-propiolactone, its final concentration is in the solution 5 ‰, 37 DEG C of deactivations 24 hours.Then carry out sieve chromatography, use G25 dextran chromatography filler carry out desalination and remove small-molecule substance, this process uses ultrapure water wash-out, collects bovine serum albumin.The design sketch that G25 dextran molecule screens out salt and small-molecule substance is shown in Fig. 9.
S6. then use the film bag that aperture is 30K to carry out ultrafiltration and concentration, use final bovine serum albumin concentration to reach more than 5mg/ml.Finally use the filter membrane of 0.2 micron to carry out filtration sterilization and namely obtain high-purity bovine serum albumin.In bovine serum albumin, the purity of bovine serum albumin is 99.6%.Bovine serum albumin after consummate and bovine serum are carried out the comparison of SDS-PAGE electrophoretic effects, the results are shown in Figure 10.
embodiment 3
A method for separating high-purity bovine serum albumin from bovine serum, concrete steps are as follows:
S1. in 50mL bovine serum, add the mixing of 50mL physiological saline, then add the mixing of 30g ammonium sulfate, put into 2 ~ 8 DEG C of refrigerators static 3 hours, then 8000 revs/min, centrifugal 60 minutes, collect supernatant liquor.
S2. in supernatant liquor, add the PBS solution of 250 mL, after mixing, direct loading carries out hydrophobic chromatography, and chromatographic stuffing is phenyl-dextran (GE company), uses normal saline solution wash-out filler, collects physiological saline elution peak.
S3. then use molecular sieve to carry out removal salt and small-molecule substance, chromatographic stuffing is G25 dextran (GE company), collects not saliferous and other micromolecular protein peak.
S4. then carry out anion-exchange chromatography, chromatographic stuffing is DEAE-dextran (GE company), uses 0.2M sodium-chlor to carry out gradient elution after the direct loading of sample, collects bovine serum albumin white peak.
S5. add beta-propiolactone, its final concentration is in the solution 5 ‰, 37 DEG C of deactivations 24 hours; Then carry out sieve chromatography, use G50 dextran (GE company) chromatographic stuffing carry out desalination and remove small-molecule substance, this process uses ultrapure water wash-out, collects bovine serum albumin.
S6. then use the film bag that aperture is 30K to carry out ultrafiltration and concentration, make final bovine serum albumin concentration reach more than 5mg/mL; Finally use the filter membrane of 0.2 micron to carry out filtration sterilization and namely obtain high-purity bovine serum albumin.
comparative example 1
A method for separating high-purity bovine serum albumin from bovine serum, elementary operation, with embodiment 1, is uniquely removed unlike by S3 step, and in the bovine serum albumin finally obtained, the purity of bovine serum albumin is 80%.
Claims (8)
1. the method for separating high-purity bovine serum albumin from bovine serum, is characterized in that, comprise the following steps:
S1. in bovine serum, add physiological saline or PBS solution, bovine serum is diluted; Moderate amount of sulfuric acid ammonium is added, 2 ~ 8 DEG C of standing more than 30min, collected by centrifugation supernatant liquor in the bovine serum after dilution;
S2. in supernatant liquor, add the PBS solution of 2 ~ 3 times of volumes, after mixing, carry out hydrophobic chromatography, use PBS solution wash-out, collect elutriant;
S3. elutriant is carried out sieve chromatography, collect not saliferous and other micromolecular protein peak; Described chromatographic stuffing is G10 ~ G75 dextran;
S4. use anion-exchange chromatography to be further purified bovine serum albumin, carry out gradient elution by 0.1 ~ 1.5M concentration sodium-chlor, collect consummate bovine serum albumin;
S5. the inactivator adding nonprotein sex change class carries out deactivation to pathogenic micro-organism, carries out sieve chromatography afterwards, uses ultrapure water wash-out to collect bovine serum albumin; Described sieve chromatography filler is the dextran of G25 or G50;
S6. use the film bag that aperture is 30K to carry out ultrafiltration and concentration, make bovine serum albumin ultimate density reach more than 5mg/ml; The filter membrane of 0.2 micron is finally used to carry out filtration sterilization.
2. method according to claim 1, is characterized in that, the add-on of physiological saline described in S1 or PBS solution is 1 ~ 3 times of bovine serum volume.
3. method according to claim 2, is characterized in that, ammonium sulfate described in S1 adds by the amount adding 10 ~ 30g ammonium sulfate in every 100 milliliters of dilute serums.
4. method according to claim 3, is characterized in that, ammonium sulfate described in S1 adds by the amount adding 15 ~ 25g ammonium sulfate in every 100 milliliters of dilute serums.
5. method according to claim 4, is characterized in that, ammonium sulfate described in S1 adds by the amount adding 20 ~ 25g ammonium sulfate in every 100 milliliters of dilute serums.
6. method according to claim 1, is characterized in that, the filler of hydrophobic chromatography described in S2 is phenyl-dextran or butyl-dextran.
7. method according to claim 1, is characterized in that, the filler of anion-exchange chromatography described in S4 is DEAE-dextran.
8. method according to claim 1, is characterized in that, described in S5, the inactivator of nonprotein sex change class is beta-propiolactone.
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| CN106749626A (en) * | 2017-01-24 | 2017-05-31 | 西藏康威科技发展有限公司 | A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum |
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| CN108191971B (en) * | 2018-01-02 | 2022-02-22 | 汇海(苏州)生物技术有限公司 | Preparation method of recombinant human metallothionein III alpha fragment pure product |
| CN108191971A (en) * | 2018-01-02 | 2018-06-22 | 冯永良 | The preparation method of III α segment sterlings of recombinant human metallothionein |
| CN108546294A (en) * | 2018-07-19 | 2018-09-18 | 北京博莱得利生物技术有限责任公司 | The preparation method and products thereof of dog albumin |
| CN109627322A (en) * | 2019-01-07 | 2019-04-16 | 中国科学院过程工程研究所 | A method of human serum albumins is isolated and purified from V supernatant of Cohn component |
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| CN109734796B (en) * | 2019-02-01 | 2022-04-15 | 广州蕊特生物科技有限公司 | Process for separating albumin from haemolytic serum |
| CN109776673A (en) * | 2019-02-20 | 2019-05-21 | 天津理工大学 | A kind of high-purity bovine serum albumin purifying process of suitable pilot scale volume production |
| CN109776673B (en) * | 2019-02-20 | 2022-03-29 | 天津理工大学 | High-purity bovine serum albumin purification process suitable for pilot scale production |
| CN110339209A (en) * | 2019-07-18 | 2019-10-18 | 上海泰坦科技股份有限公司 | A kind of preparation method for the calf serum going Thyroid binding protein |
| CN112083158A (en) * | 2020-08-29 | 2020-12-15 | 赵峻岭 | Preparation method of bovine serum for confining liquid |
| CN113968905A (en) * | 2021-10-27 | 2022-01-25 | 哈尔滨国生生物科技股份有限公司 | Bovine serum albumin and application thereof |
| CN114276437A (en) * | 2021-12-04 | 2022-04-05 | 南京岚煜生物科技有限公司 | Preparation method of compound high-value reference substance for five immune terms |
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