CN101302504B - Method for purifying lysostaphin by antibody affinity chromatography - Google Patents
Method for purifying lysostaphin by antibody affinity chromatography Download PDFInfo
- Publication number
- CN101302504B CN101302504B CN2007100405614A CN200710040561A CN101302504B CN 101302504 B CN101302504 B CN 101302504B CN 2007100405614 A CN2007100405614 A CN 2007100405614A CN 200710040561 A CN200710040561 A CN 200710040561A CN 101302504 B CN101302504 B CN 101302504B
- Authority
- CN
- China
- Prior art keywords
- buffer
- chromatography column
- chromatography
- sample
- damping fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying lysostaphin by antibody affinity chromatography, comprising the following steps of: (1) cleaning and eluting an antibody sample containing lysostaphin by passing the antibody sample through a chromatographic column which is filled with protein A resin and is balanced by buffer liquid A; collecting and drying by freezing eluting peak; (2) mixing the freeze-dried product with an affinity chromatography medium to form an immunized affinity chromatography medium; (3) making the lysostaphin sample pass through the chromatography column which is filled with immunoaffinity chromatography medium and is previously balanced with the buffer liquid A; then cleaning the sampled immunoaffinity chromatography column with the buffer liquid A; and eluting the lysostaphin from the chromatography column with a buffer liquid C; and collecting the eluting peak. The method has the advantages of rapid processing speed, high recycling rate and good repeatability, long column efficiency and basically invariable joint efficiency, along with a recycling rate of an active protein of between 85 and 95 percent, a specific activity of more than 1000 U/mg, a multiple of protein purification of more than between 3 and 5 times, and a purity of between 95 and 98 percent.
Description
Technical field
The present invention relates to a kind of purification process of enzyme, relate to the chromatography purification method of staphylococcus lysozyme specifically.
Background technology
Staphylococcus lysozyme (Lysostaphin) is that Staphylococus Simulans staphylococcus secretes the protein product outside born of the same parents, and molecular weight is 27kDa, contains 246 amino acid, is a kind of endopeptidase.This enzyme can cut off the pentaglycine peptide bond bridge construction in the aureus cell wall peptidoglycan, thereby reaches the purpose of rapid dissolving and killing bacteria.Even the resistance streptococcus aureus, MRSA and " superbacteria ", staphylococcus lysozyme has very strong germicidal action too, and is not easy to induce the generation Resistant strain, is expected to become the novel biological agent of treatment drug resistant bacterial infections.
Fudan University in 1987 expresses staphylococcus lysozyme and succeeds in Bacillus sphaericus, and set up the separation purification method of this enzyme, this method comprises uses cellulosic anion chromatography of DEAE and the cellulosic positively charged ion chromatography of CM, because the chromatography media rigidity is weak, the albumen carrying capacity is low, cause chromatography process flow velocity can not be too fast, can only handle small samples, handling 10 liters of samples needs 4 days time, and all undesirable at aspects such as yield, purity.
The applicant made up a kind of from the Bacillus sphaericus culture supernatant that contains the staphylococcus lysozyme gene technology (patent publication No.: CN1743453 of purifying lysostaphin; Open day: 2006.3.8), this method comprises ion-exchange and hydrophobic chromatography, handle 45 liters of samples and only need 2 working dayss, total yield is 50%, specific activity reaches 1000U/mg protein, but this method only is applicable to industrialized big production, and the staphylococcus lysozyme purity of producing gained reaches about 90%, purity requirement that still can not fulfilling medicinal.
In addition, the applicant has made up a kind of technology (patent publication No.: CN1900290 with E, coli to express lysostaphin in high efficiency via external secretion; Open day: 2007.1.24), meet the requirement of the medicinal engineering bacteria that new drug declares, expression product was present in the substratum steps such as no renaturing inclusion bodies with final ripe staphylococcus lysozyme activity form; Purifying is simpler, and host's mycoprotein pollutes few; Rate of recovery height, expression amount can reach 200-400mg/L, and the rate of recovery is greater than 60%, but the staphylococcus lysozyme purity of gained also can't reach medicinal requirement, needs further purifying.
Affinity chromatography be according to biomacromolecule can be special with part, the characteristic that reversibly combines, from the biological sample of complexity, separate the separation purification method that obtains required target product.It is based on the biological specificity between biomacromolecule and the part, have that selectivity is strong, purification efficiency is high, step is simple, be the main direction of studying of all kinds of protein purifications in recent years, part Study person also attempts using the affinity chromatography purifying lysostaphin: as using Sepharose4B as affinity adsorbent, affinity chromatography (the Zhang Peide that carries out as part with dead somatic cells and the whole cell peptidoglycan of streptococcus aureus, horsepower, Chen Shigen. the affinity chromatography of staphylococcus enzyme. industrial microorganism, 1992,21 (5): 1-5).Have and adopt chitosan as carrier, with specific substrate (bacterium somatic cells) immobilization of enzyme effect, as the molten grape ball of adsorption medium purifying enzyme (Zhou Yibin, Zhang Peide. industrial microorganism .2002,332 (1): 19-22).Also have with aureus cell wall peptidoglycan (PG) to be coupled to BrCN activatory Sepharose4B be affine adsorption medium in order to purifying lysostaphin (Zhang Peide, horsepower. industrial microorganism .1992,5:1-5).But imitate very shortly with the affinity adsorption column post that somatic cells or whole cell peptidoglycan make as part, just lose affinity interaction substantially after using 2-3 times, and adsorptive capacity is lower, can't be applied to suitability for industrialized production.
Therefore seeking the dissolving staphylococcal bacteria enzyme purification process that a species specificity is good, product purity is high, the sample adsorptive capacity is big is the difficult problem that relevant field extremely presses for solution.
Summary of the invention
The technical issues that need to address of the present invention are the methods that disclose a kind of purifying lysostaphin by antibody affinity chromatography, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
(1) processing of dissolving staphylococcal bacteria enzyme antibody:
The sample that will contain the dissolving staphylococcal bacteria enzyme antibody with the good chromatography column that protein A resin is housed of the buffer A balance of 0.05～0.2mol/L pH7.0-9.0, cleans the protein A chromatography column of going up behind the sample with the buffer A of 0.05～0.2mol/LpH7.0-9.0 by in advance then; Use the flow velocity of the buffer B of 0.05～0.2mol/LpH2.0～4.0 with per minute 1～4ml again, with dissolving staphylococcal bacteria enzyme antibody wash-out and collect elution peak from the chromatography column, freeze-drying obtains dissolving staphylococcal bacteria enzyme antibody lyophilized products;
Contain in the sample of dissolving staphylococcal bacteria enzyme antibody, the content of dissolving staphylococcal bacteria enzyme antibody is 0.1～3%, the dissolving staphylococcal bacteria enzyme antibody sample that the protein A resin of every ml can purifying 1-50mg.
The preparation method of said dissolving staphylococcal bacteria enzyme antibody sample is with reference to public publication [(U.S.) J. Sa nurse Brooker, E.F. Ritchie not, T. Manny A Disi. molecular cloning experiment guide (second edition). the 18 chapter, first segment, the .1992 of PP853-858. Science Press];
Used staphylococcus lysozyme can adopt patent documentation (patent publication No.: CN1900290 in the antibody preparation process; Open day: 2007.1.24) disclosed method was prepared, or adopted the commercially available prod, as the staphylococcus lysozyme standard substance of SIGMA company production;
The said chromatography column of protein A resin that is equipped with is available from the Hightrap ProteinA of Pharmacia company prepacked column;
Said damping fluid balance is equipped with the method for the chromatography column of protein A resin, comprises the steps:
With the buffer A of the 0.05～0.2mol/L pH7.0-9.0 flow velocity with per minute 1～4ml, by chromatography column, consumption is 3～10 times of chromatography column volume, by ultraviolet detection guarantee chromatography column balance to baseline;
The method of the protein A resin chromatography column on the said buffer solution for cleaning behind the sample comprises the steps:
With the buffer A of 0.05～0.2mol/L pH7.0-9.0 flow velocity with per minute 1～4ml, protein A resin chromatography column in the cleaning behind the sample, consumption is 3～10 times of protein A resin chromatography column volume, guarantees that by ultraviolet detection chromatography column has cleaned to baseline;
The buffer A of said pH7.0-9.0 can be selected from one or more in PBS, Tris-HCl, the borate buffer;
The buffer B of said pH2.0～4.0 can be selected from Na
2HPO
4In-citric acid, Gly-HCl, the phthalic acid-HCl damping fluid one or more, the consumption of buffer B are 3～10 times of protein A resin chromatography column volume;
(2) preparation of immunoaffinity chromatography medium
The working instructions of the CNBr activation Sepharose4B that publishes with reference to Pharmacia company carry out the preparation of immunoaffinity chromatography medium.Concrete steps are as follows:
With affinity chromatography medium pH is that the HCl solution of 2.0-3.0 carries out swelling, cleaning, the consumption of HCl solution is a 30-80 times of volume of affinity chromatography medium, coupling liquid with 10-20 times of volume of affinity chromatography medium cleans then, makes the pH of affinity chromatography medium reach 8.0-10.0.
Used coupling liquid is that the pH that contains 0.2-0.6M NaCl is the NaHCO of 8.0-10.0
3Solution.
The dissolving staphylococcal bacteria enzyme antibody lyophilized products that step (1) is collected is dissolved in the coupling liquid with weight ratio 1:200-400, mixed with the affinity chromatography medium that coupling liquid cleaned with the last step then, room temperature condition vibration 0.5-1.5 hour, or under 4 ℃ of conditions, placed 12-36 hour, make antibody and affinity chromatography medium coupling abundant; Clean the immunoaffinity chromatography medium make with coupling liquid then, coupling liquid consumption be the immunoaffinity chromatography medium volume that makes 5-20 doubly; Soak placement 2-4 hour with the Tris-HCl of immunoaffinity chromatography medium volume 1-5 0.05-0.1M doubly or the ethanolamine solutions of 1M again, be used for sealing the not avtive spot of binding antibody of immunoaffinity chromatography medium.
The weight ratio of dissolving staphylococcal bacteria enzyme antibody and affinity chromatography medium is 1:25-150;
Said affinity chromatography medium is selected from one or more in Sepharose, Mierocrystalline cellulose, soluble cotton, acrylamide polymer, multipolymer and derivative thereof, polymethacrylate derivative, polyethersulfone or the chitosan;
(3) staphylococcus lysozyme carries out purifying with the immunoaffinity chromatography medium
With the staphylococcus lysozyme sample with the flow velocity of per minute 1～4ml by in advance with the chromatography column of the good immunoaffinity chromatography medium that step (2) is housed of the buffer A balance of 0.05～0.2mol/LpH7.0-9.0; In the staphylococcus lysozyme sample, the content of staphylococcus lysozyme is 70-7000 μ g/ml, and based on the weight of staphylococcus lysozyme, applied sample amount is: the staphylococcus lysozyme of 1-1000 μ g/ml immunoaffinity chromatography medium;
Then with the immune affinity chromatographic column behind the last sample of buffer A cleaning of 0.05～0.2mol/L pH7.0-9.0;
Be 2.5～4.5 damping fluid C with the flow velocity of per minute 1～4ml with staphylococcus lysozyme wash-out and collect elution peak from the chromatography column with 0.1～0.5mol/L pH again; The consumption of damping fluid C is 3～10 times of chromatography column volume; Elutriant is collected in the damping fluid that 0.5～2mol/L pH7.0-9.0 is housed.
Said staphylococcus lysozyme sample is the intestinal bacteria culture supernatant that will contain the staphylococcus lysozyme gene according to Chinese patent CN1900290 preparation or according to the purification process of the Chinese patent CN1743453 crude product after through the ion-exchange step purifying, damping fluid dilution with the pH7.0 that contains 0.05～0.35mol/L NaCl～9.0, wherein, staphylococcus lysozyme content is 70-7000 μ g/ml;
The chromatography column of said damping fluid balance immunoaffinity chromatography medium comprises the steps:
With the buffer A of 0.05～0.2mol/L pH7.0-9.0 with the flow velocity of per minute 1～4ml by the chromatography column of immunoaffinity chromatography medium is housed, consumption is 3～10 times of chromatography column volume, by ultraviolet detection guarantee chromatography column balance to baseline;
The immune affinity chromatographic column that said buffer A cleaning is gone up behind the sample comprises the steps:
With the immune affinity chromatographic column behind the last sample of buffer A cleaning of 0.05～0.2mol/L pH7.0-9.0, flow velocity is per minute 1～4ml, and consumption is 3～10 times of chromatography column volume, guarantees that by ultraviolet detection chromatography column cleans to baseline.
The buffer A of said pH7.0-9.0 is selected from one or more in PBS, Tris-HCl, the borate buffer;
Said pH is that 2.5～4.5 damping fluid C is selected from acetic acid-sodium-acetate, Na
2HPO
4In-citric acid, the citric acid-sodium citrate damping fluid one or more.
Compound method reference " Chinese herbal medicine effective ingredients extracts and the separates " appendix ten of said various damping fluids: the preparation of damping fluid commonly used; Shanghai science tech publishing house, 1983.
The activated protein rate of recovery of above-mentioned steps is 85～95%, and specific activity is greater than 1000U/mg, and the protein purification multiple is more than the 3-5, and purity can reach 95-98%, and processing speed is fast, the rate of recovery is high and good reproducibility, post are imitated long; The adsorption column of the immunoaffinity chromatography medium that makes according to the present invention not only post is imitated long (under 4 ℃ of degree 20% ethanol preserve post imitate greater than 1 year) and joint efficiency constant substantially (1000 μ g/mlmedium).
Description of drawings
Fig. 1 is an antibody purification chromatography spectrogram.
Fig. 2 is mono-clonal, polyclonal antibody SDS-PAGE.
Fig. 3 is a monoclonal antibody affinity media purifying lysostaphin chromatography spectrogram.
Fig. 4 is a polyclonal antibody affinity media purifying lysostaphin chromatography spectrogram.
Fig. 5 is staphylococcus lysozyme SDS-PAGE.
Embodiment
The staphylococcus lysozyme Purification of Monoclonal Antibodies:
Chromatography media: 5ml protein A hightrap prepacked column, available from pharmacia Bioisystech Co., Ltd.
The monoclonal antibody sample: as antigen, the method for preparing monoclonal antibody immunity BalB/c mouse according to routine makes the mouse ascites 15ml that contains the staphylococcus lysozyme monoclonal antibody with staphylococcus lysozyme.Wherein, the content of staphylococcus lysozyme monoclonal antibody is
Tomography devices: AKTA explorer100 protein purification developing system, available from Pharmacia Bioisystech Co., Ltd.
The PBS damping fluid of balance, cleaning buffer solution: pH8.0.
Elution buffer: the Gly-HCl damping fluid that contains the pH2.8 of 0.1mol/L glycine.
Equilibrium process: with pH is 8.0 PBS damping fluid balance 5ml proteinA prepacked column, with the flow velocity of per minute 3ml, feeds prepacked column, and consumption is 6 times of prepacked column volume, by ultraviolet detection (A
280) guarantee chromatography column balance to baseline.
Last sample: the flow velocity with per minute 2ml makes 15ml monoclonal antibody sample by the good chromatography column of balance of last step.
Clean: it is intact to go up sample, is 8.0 PBS damping fluid with pH, cleans chromatography column with the flow velocity of per minute 2ml, and consumption is 8 times of prepacked column volume, cleans to baseline by ultraviolet detection (A280) assurance chromatography column.
Wash-out: clean to baseline, with the Gly-HCl damping fluid of pH2.8 that contains the 0.1mol/L glycine, with the flow velocity of per minute 2ml the staphylococcus lysozyme monoclonal antibody is eluted from chromatography column, the damping fluid consumption is 4 times of chromatography column volume.In collection tube, be added with the Tris-HCl damping fluid of the pH8.0 that contains 1mol/L Tris in advance, in order in and elutriant, collect 11 milliliters of elution peaks, make staphylococcus lysozyme monoclonal antibody lyophilized products 42.575mg (wherein including the salt component in staphylococcus lysozyme monoclonal antibody and the damping fluid) after the freeze-drying.
The result: the chromatography process is referring to Fig. 1.Among the figure, on behalf of antibody purification, peak 1 clean the peak, and the antibody purification elution peak is represented at peak 2.
Elution peak is 1.905mg/ml through monoclonal antibody assay (lowry method), amount to 20.955mg, monoclonal antibody purity is analyzed through SDS*PAGE〉90%, the monoclonal antibody electrophorogram is seen Fig. 2, among the figure, swimming lane 1 is the lower molecular weight standard protein, and swimming lane 2 is the monoclonal antibody (handling through dithiothreitol (DTT)) of protein A purifying, and swimming lane 3 is the polyclonal antibody (handling through dithiothreitol (DTT)) of proteinA purifying.
The purifying of staphylococcus lysozyme polyclonal antibody:
Chromatography media: 5ml protein A hightrap prepacked column, available from pharmacia Bioisystech Co., Ltd.
The polyclonal antibody sample: as antigen, the preparation method of polyclonal antibody immunity new zealand rabbit according to routine makes the rabbit anti-serum 8ml that contains the staphylococcus lysozyme polyclonal antibody with staphylococcus lysozyme.Wherein, the content of staphylococcus lysozyme polyclonal antibody is
Tomography devices: AKTA explorer100 protein purification developing system, available from Pharmacia Bioisystech Co., Ltd.
Balance, cleaning buffer solution: the Tris-HCl damping fluid that contains the pH8.0 of 0.1mol/LTris.
Elution buffer: the Gly-HCl damping fluid that contains the pH2.8 of 0.1mol/L glycine.
Equilibrium process: with the pH that contains 0.1mol/L Tris is 8.0 Tris-HCl damping fluid balance 5mlprotein A prepacked column, and flow velocity is per minute 4ml, and consumption is 6 times of column volume, by ultraviolet detection (A
280) guarantee chromatography column balance to baseline;
Last sample: make sample by the good chromatography column of balance of last step with the flow velocity of per minute 3ml in the polyclonal antibody sample of 8ml.
Clean: it is intact to go up sample, is 8.0 Tris-HCl damping fluid with the pH that contains 0.1mol/L Tris, cleans chromatography column with the flow velocity of per minute 4ml, by ultraviolet detection (A
280) guarantee that chromatography column cleans to baseline.
Wash-out: clean to baseline, with the Gly-HCl damping fluid of pH2.8 that contains the 0.1mol/L glycine, with the flow velocity of per minute 4ml the staphylococcus lysozyme polyclonal antibody is eluted from chromatography column, the damping fluid consumption is 5 times of chromatography column volume.In collection tube, be added with the Tris-HCl damping fluid of the pH8.0 that contains 1mol/LTris in advance, in order in and elutriant, collect 10 milliliters of elution peaks, make staphylococcus lysozyme polyclonal antibody lyophilized products 342.395mg (wherein including the salt component in staphylococcus lysozyme monoclonal antibody and the damping fluid) after the freeze-drying.
The result: the chromatography process is referring to Fig. 1.Elution peak is 22mg/ml through polyclonal antibody assay (lowry method), amounts to 220mg, and polyclonal antibody purity is analyzed through SDS*PAGE〉90%.
Embodiment 3
The affinitive layer purification staphylococcus lysozyme
Affinity media: the chromatography column that Sepharose4B gel (available from Pharmacia Bioisystech Co., Ltd, through the CNBr activation) medium that 3 milliliters of couplings have the monoclonal antibody that embodiment 1 makes is housed.
Sample: according to the intestinal bacteria culture supernatant 10ml that contains the staphylococcus lysozyme gene of Chinese patent CN1900290 preparation, contain 964 unit staphylococcus lysozymes, the adding equal-volume contains the Tris-HCl damping fluid of the 0.1mol/L pH8.0 of 0.15mol/L NaCl, makes sample solution 20ml.
Tomography devices: AKTA explorer100 protein purification developing system, available from Pharmacia Bioisystech Co., Ltd.
Balance, cleaning buffer solution: the pH that contains 0.1mol/L NaCl is 8.0 Tris-HCl damping fluid;
Elution buffer: the acetate buffer solution that contains the pH4.3 of 0.1mol/L sodium-acetate.
Neutralization buffer: the Tris-HCl damping fluid that contains the pH8.0 of 1mol/LTris.
Immunoaffinity chromatography medium coupling process:
With 1g Sepharose4B dry powder (available from Pharmacia Bioisystech Co., Ltd, through CNBr activation) carry out swelling, cleaning with the HCl solution of 200ml pH2.0, clean with 50ml coupling liquid then, make the pH of Sepharose4B gel reach 8.3 (the solvable 3.5ml of the being expanded into gels of 1g Sepharose4B dry powder.Used coupling liquid is that the pH that contains 0.2M NaCl is 8.3 NaHCO
3Solution.
The dissolving staphylococcal bacteria enzyme antibody 10mg that embodiment 1 is made is dissolved in the 4ml coupling liquid, mixes with the Sepharose4B gel that coupling liquid cleaned with the last step then, room temperature condition vibration 1 hour, makes antibody and affinity chromatography medium coupling abundant; Clean the immunoaffinity chromatography medium that makes with 35ml coupling liquid then; Placed 2 hours with the Tris-HCl solution soaking of 12ml0.1mol/L again, be used for sealing the not avtive spot of binding antibody of immunoaffinity chromatography medium.
Balance: with the level pad balance chromatography column that 3 milliliters of couplings have the Sepharose4B gel media of the monoclonal antibody that embodiment 1 makes is housed, with 5 column volumes of flow velocity balance of per minute 2ml, by ultraviolet detection (A
280) guarantee chromatography column balance to baseline.
Last sample: make the staphylococcus lysozyme sample by using the good chromatography column of damping fluid balance in advance with the flow velocity of per minute 1ml sample solution 20ml.
Clean: it is intact to go up sample, and with the flow velocity cleaning chromatography column of cleaning buffer solution with per minute 2ml, consumption is 8 times of chromatography column volume; By ultraviolet detection (A
280) guarantee that chromatography column cleans to baseline.
Wash-out: clean to baseline, with the flow velocity of elution buffer with per minute 1ml staphylococcus lysozyme is eluted from chromatography column, the damping fluid consumption is 4 times of chromatography column volume.In collection tube, be added with the Tris-HCl damping fluid of the pH8.0 that contains 1mol/LTris in advance, in order in and elutriant, collect 6 milliliters of elution peaks.Test-results such as Fig. 3 and table 1.Among Fig. 3, peak 1 cleans the peak for staphylococcus lysozyme, and peak 2 is the staphylococcus lysozyme elution peak.
Table 1 staphylococcus lysozyme determination of recovery rates
The staphylococcus lysozyme that makes through above-mentioned steps shows that through non-reduced SDS-PAGE electrophoresis result purity can reach more than 95%, and the rate of recovery is 92%, and specific activity can reach the 1000U/mg (see figure 5).
Embodiment 4
The affinitive layer purification staphylococcus lysozyme:
Affinity media: the chromatography column that Sepharose4B gel (available from Pharmacia Bioisystech Co., Ltd, through the CNBr activation) medium that 6 milliliters of couplings have the polyclonal antibody that embodiment 2 makes is housed.
Sample: contain the purification process of the intestinal bacteria culture supernatant of staphylococcus lysozyme gene according to Chinese patent CN1743453, through the staphylococcus lysozyme crude product solution 1ml behind the ion-exchange step purifying, contain the borate buffer dissolving of the pH8.0 of 0.30mol/L NaCl with 1ml, activity is 1500U after testing.
Tomography devices: AKTA explorer100 protein purification developing system, available from Pharmacia Bioisystech Co., Ltd.
Balance, cleaning buffer solution: the borate buffer that contains the pH8.5 of 0.15mol/L Sodium Tetraborate.
Elution buffer: the Gly-HCl damping fluid that contains the pH3.2 of 0.1mol/L glycine.
Neutralization buffer: the borate buffer that contains the pH8.5 of 2mol/L Sodium Tetraborate.
Immunoaffinity chromatography medium coupling process:
2g Sepharose4B dry powder (available from Pharmacia Bioisystech Co., Ltd, through CNBr activation) is carried out swelling, cleaning with the HCl solution of 450ml pH3.0, clean with 90ml coupling liquid then, make the pH of Sepharose4B gel reach 8.5.
Used coupling liquid is that the pH that contains 0.5M NaCl is 8.5 NaHCO
3Solution.The dissolving staphylococcal bacteria enzyme antibody 80mg that embodiment 2 is made is dissolved in the 16ml coupling liquid, mixes with the Sepharose4B gel that coupling liquid cleaned with the last step then, places 24 hours under 4 ℃ of conditions, makes antibody and affinity chromatography medium coupling abundant; Clean the immunoaffinity chromatography medium that makes with 50ml coupling liquid then; Ethanolamine solutions with 15ml1mol/L soaks placement 3 hours again, is used for sealing the not avtive spot of binding antibody of immunoaffinity chromatography medium.
Balance: the borate buffer balance with the pH8.5 that contains the 0.15mol/L Sodium Tetraborate is equipped with the chromatography column that 6 milliliters of couplings have the Sepharose4B gel media of staphylococcus lysozyme polyclonal antibody, with 6 column volumes of per minute 3ml balance, by ultraviolet detection (A
280) guarantee chromatography column balance to baseline.
Last sample: make sample pass through to use in advance the good chromatography column of damping fluid balance with the flow velocity of per minute 1ml in the sample of 2ml.
Clean: upward sample is intact, and with the borate buffer of the pH8.5 that contains the 0.15mol/L Sodium Tetraborate, with the flow velocity cleaning chromatography column of per minute 2ml, consumption is for passing through ultraviolet detection (A
280) guarantee that chromatography column cleans to baseline.
Wash-out: clean to baseline,, staphylococcus lysozyme is eluted from chromatography column, collect 7 milliliters of elution peaks with the flow velocity of per minute 1ml with the Gly-HCl damping fluid of 0.1mol/L pH3.2.The damping fluid consumption is 6 times of chromatography column volume.
Test-results such as Fig. 4 and table 2.Among Fig. 4, peak 1 cleans the peak for staphylococcus lysozyme, and peak 2 is the staphylococcus lysozyme wash-out.
The recovery test of table 2 immunoaffinity chromatography medium purification staphylococcus lysozyme
The staphylococcus lysozyme that makes through above-mentioned steps shows that through non-reduced SDS-PAGE electrophoresis result purity can reach more than 95%, and the rate of recovery is 89%, can further bring up to 455U/mg from 311U/mg than vigor, and purity liquid reaches 95% above (see figure 5).
Among Fig. 5, swimming lane 1 is the staphylococcus lysozyme through the monoclonal antibody affinitive layer purification, swimming lane 2 is the staphylococcus lysozyme through how anti-affinitive layer purification, swimming lane 3 is the staphylococcus lysozyme through preparation method's purifying of Chinese patent CN1743453, swimming lane 4 is lower molecular weight standard protein (Mark), swimming lane 5 is the staphylococcus lysozyme of the ion exchange chromatography step purifying in Chinese patent CN1743453 method, the intestinal bacteria culture supernatant that contain staphylococcus lysozyme gene of swimming lane 6 for making according to Chinese patent CN1900290 preparation method.
Claims (8)
1. the method for a purifying lysostaphin by antibody affinity chromatography is characterized in that, comprises the steps:
(1) processing of dissolving staphylococcal bacteria enzyme antibody:
The sample that will contain the dissolving staphylococcal bacteria enzyme antibody with the good chromatography column that the proteinA resin is housed of buffer A balance, cleans the proteinA chromatography column of going up behind the sample with buffer A by in advance then; The buffer B of using pH2.0～4.0 again is dissolving staphylococcal bacteria enzyme antibody wash-out from the chromatography column, and collects elution peak, freeze-drying;
(2) preparation of immunoaffinity chromatography medium
The lyophilized products that step (1) is collected is mixed with affinity chromatography medium, and linked reaction makes the immunoaffinity chromatography medium;
(3) staphylococcus lysozyme carries out purifying with the immunoaffinity chromatography medium
With the staphylococcus lysozyme sample by in advance with the chromatography column of the good immunoaffinity chromatography medium that step (2) is housed of buffer A balance;
Then with the immune affinity chromatographic column behind the last sample of buffer A cleaning;
The damping fluid C that uses pH2.5～4.5 again is staphylococcus lysozyme wash-out from the chromatography column, and collects elution peak;
Described buffer A is selected from more than one in PBS, Tris-HCl or the borate buffer, and the pH of buffer A is 7.0-9.0;
Described buffer B is selected from Na
2HPO
4In-citric acid, Gly-HCl or phthalic acid-HCl damping fluid more than one;
Described damping fluid C is selected from acetic acid-sodium-acetate, Na
2HPO
4In-citric acid or the citric acid-sodium citrate damping fluid more than one.
2. method according to claim 1 is characterized in that, said buffer A balance is equipped with the method for the chromatography column of protein A resin, comprises the steps: to pass through chromatography column with the buffer A of pH7.0-9.0, and consumption is 3～10 times of chromatography column volume.
3. method according to claim 1, it is characterized in that, the method of the proteinA resin chromatography column behind the sample is gone up in said buffer A cleaning, comprise the steps: that consumption is 3～10 times of protein A resin chromatography column volume with the protein A resin chromatography column behind the last sample of buffer A cleaning of pH7.0-9.0.
4. method according to claim 1 is characterized in that said affinity chromatography medium is selected from more than one in Sepharose, Mierocrystalline cellulose, soluble cotton, acrylamide polymer, polyethersulfone or the chitosan.
5. method according to claim 1, it is characterized in that, the chromatography column of said damping fluid balance immunoaffinity chromatography medium comprises the steps: buffer A with pH7.0-9.0 by the chromatography column of immunoaffinity chromatography medium is housed, and consumption is 3～10 times of chromatography column volume.
6. method according to claim 1, it is characterized in that, said buffer A is cleaned the immune affinity chromatographic column of going up behind the sample and is comprised the steps: to clean the immune affinity chromatographic column of going up behind the sample with the buffer A of pH7.0-9.0, and consumption is 3～10 times of chromatography column volume.
7. method according to claim 1 is characterized in that, in the step (1), the consumption of buffer B is 3～10 times of protein A resin chromatography column volume, and in the step (3), the consumption of damping fluid C is 3～10 times of chromatography column volume.
8. method according to claim 1 is characterized in that, in the step (3), damping fluid C with the flow velocity of per minute 1～4ml with staphylococcus lysozyme wash-out from the chromatography column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100405614A CN101302504B (en) | 2007-05-11 | 2007-05-11 | Method for purifying lysostaphin by antibody affinity chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100405614A CN101302504B (en) | 2007-05-11 | 2007-05-11 | Method for purifying lysostaphin by antibody affinity chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101302504A CN101302504A (en) | 2008-11-12 |
| CN101302504B true CN101302504B (en) | 2011-12-21 |
Family
ID=40112608
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100405614A Active CN101302504B (en) | 2007-05-11 | 2007-05-11 | Method for purifying lysostaphin by antibody affinity chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101302504B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122346B (en) * | 2011-11-18 | 2016-01-13 | 复旦大学 | The purification process of restructuring KLK14 albumen |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ616921A (en) * | 2009-07-15 | 2015-06-26 | Genentech Inc | Gram-positive bacteria specific binding compounds |
| CN103122029B (en) * | 2011-11-18 | 2015-10-28 | 复旦大学 | KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen |
| DK3041857T3 (en) | 2013-09-04 | 2019-07-22 | Emd Millipore Corp | PROTEIN A-CHROMATOGRAPHY |
| KR101891522B1 (en) | 2013-12-12 | 2018-08-24 | 이엠디 밀리포어 코포레이션 | Protein separations using an acrylamide containing filter |
| CN110573526B (en) * | 2017-04-13 | 2022-03-01 | 威尔科学公司 | Antibody of royal jelly major protein and application thereof |
| CN111423509B (en) * | 2019-01-10 | 2021-07-09 | 瑞阳(苏州)生物科技有限公司 | Affinity chromatography purification method of anti-HSA single domain antibody and fusion protein thereof |
| CN113046343A (en) * | 2019-12-28 | 2021-06-29 | 常州天地人和生物科技有限公司 | Preparation and application method of lysozyme affinity filler |
| CN114380916A (en) * | 2020-09-27 | 2022-04-22 | 信达生物制药(苏州)有限公司 | Method for processing purification intermediate of antibody molecule |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1743453A (en) * | 2004-09-02 | 2006-03-08 | 上海高科联合生物技术研发有限公司 | Lysostaphin industrial purifying process |
| CN1900290A (en) * | 2005-07-22 | 2007-01-24 | 上海高科联合生物技术研发有限公司 | Method for E, coli to express lysostaphin in high efficiency via external secretion |
-
2007
- 2007-05-11 CN CN2007100405614A patent/CN101302504B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1743453A (en) * | 2004-09-02 | 2006-03-08 | 上海高科联合生物技术研发有限公司 | Lysostaphin industrial purifying process |
| CN1900290A (en) * | 2005-07-22 | 2007-01-24 | 上海高科联合生物技术研发有限公司 | Method for E, coli to express lysostaphin in high efficiency via external secretion |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122346B (en) * | 2011-11-18 | 2016-01-13 | 复旦大学 | The purification process of restructuring KLK14 albumen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101302504A (en) | 2008-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101302504B (en) | Method for purifying lysostaphin by antibody affinity chromatography | |
| Kline | Handbook of affinity chromatography | |
| CN107108700B (en) | Modified kappa light chain-binding polypeptides | |
| TW200530265A (en) | Protein a chromatography | |
| NZ548127A (en) | Method for antibody purification | |
| US5075423A (en) | Purification of protein a by affinity chromatography followed by anion exchange | |
| Hanora et al. | Capture of bacterial endotoxins using a supermacroporous monolithic matrix with immobilized polyethyleneimine, lysozyme or polymyxin B | |
| JP7479151B2 (en) | Compositions and methods for reducing bioburden in chromatography | |
| JP2018513840A (en) | Sanitization method for affinity chromatography matrix | |
| AU759633B2 (en) | Method for detection of (legionella) bacteria employing purified antigen-specific antibodies | |
| CN101921320A (en) | A kind of separation purification method of recombinant protein A | |
| Sjöberg et al. | Purification and characterization of CS2, a sialic acid-specific haemagglutinin of enterotoxigenic Escherichia coli | |
| CN106146627B (en) | Fc Specific binding proteins, IgG affinity chromatography medium and the preparation method and application thereof | |
| Xiao et al. | Preparing a highly specific inert immunomolecular-magnetic beads for rapid detection and separation of S. aureus and group G Streptococcus | |
| Platis et al. | Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine | |
| Lee et al. | Large-scale purification of a murine antimelanoma monoclonal antibody | |
| CN108355618B (en) | Bovine-derived hyaluronidase affinity medium and adsorption method thereof | |
| Jack et al. | Immunoaffinity chromatography | |
| CN101782578A (en) | Preparation method and application of immunomagnetic microspheres coated with staphylococal protein | |
| JP2854872B2 (en) | Insoluble carrier with antimicrobial peptide derived from horseshoe crab blood cell membrane as ligand | |
| CN105063142B (en) | A method of immunoglobulin G Fc segment is prepared with Mixed-Modechromatography filler | |
| CN101935665A (en) | Preparation method and application of recombinant protein A gene and expression product thereof | |
| JP3476242B2 (en) | Purification method of influenza HA protein | |
| CN103122029A (en) | Method for purifying human recombinant SPINK6 protein with KLK14 protein through affinity chromatography | |
| CN103122028B (en) | Trypsinase affinitive layer purification people recombinates the method for SPINK6 albumen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: KUNSHAN BOQING BIO-TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHANGHAI GAOKE UNION BIOTECHNOLOGY DEVELOPMENT CO., LTD. Effective date: 20130123 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 201206 PUDONG NEW AREA, SHANGHAI TO: 215316 SUZHOU, JIANGSU PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20130123 Address after: 215316 No. 2199 Ring Road, Yushan Town, Jiangsu, Suzhou Patentee after: Kunshan Biogreen Technology Co., Ltd. Address before: 201206, No. 3, building B, block 501, Jingang Road, Shanghai, Pudong New Area Patentee before: Shanghai Gaoke Union Biotechnology Development Co., Ltd. |