CN101921320A - A kind of separation purification method of recombinant protein A - Google Patents
A kind of separation purification method of recombinant protein A Download PDFInfo
- Publication number
- CN101921320A CN101921320A CN 201010227638 CN201010227638A CN101921320A CN 101921320 A CN101921320 A CN 101921320A CN 201010227638 CN201010227638 CN 201010227638 CN 201010227638 A CN201010227638 A CN 201010227638A CN 101921320 A CN101921320 A CN 101921320A
- Authority
- CN
- China
- Prior art keywords
- recombinant protein
- fragment
- igg
- purification method
- separation purification
- 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.)
- Pending
Links
Images
Abstract
The present invention relates to a kind of separation purification method of recombinant protein A, comprise the segmental preparation of IgG Fc in batches, segmental directed immobilization of Fc and utilization are the method for the affinity chromatography technical point of aglucon from purification of recombinant proteins A with the Fc fragment, belong to technical field of bioengineering.The final albumin A that obtains is more than 95% through SDS-PAGE and HPLC detection albumin A purity.This method has low cost, high-level efficiency and advantages of simple operation, both can be used for laboratory scale purifying protein A, also can be used for the production of industrialization albumin A.
Description
Technical field
The invention belongs to technical field of bioengineering, the method for the affinity chromatography technology purification of recombinant proteins A that to relate to a kind of Fc fragment with IgG be affinity ligand.
Background technology
Staphylococcus aureus protein A (Staphylococcus aureus protein A, SPA) be aureus cell wall associated protein, be the single chain polypeptide structure, have with human and other mammalian in the Fc section specificity bonded ability of immunoglobulin molecules, mainly adsorb IgG and contain the immunocomplex of IgG.Exactly because albumin A has specific binding capacity with human and other mammiferous panimmunity sphaeroprotein, therefore be widely used as immunology reagent (with the antibody test that is used for histological chemistry, Western, ELISA etc. after the multiple reporter molecules coupling); Link to each other with solid phase carrier, be used for the separation and purification of antibody; Be used for the treatment of diseases related blood-purifying adsorbing agent of antibody or the like clinically.
The method that obtains albumin A the earliest is that direct purification obtains from streptococcus aureus, yet there are some problems in this method, for example: native protein A only accounts for 1.7% of bacterial protein, be difficult to satisfy the needs of scale operation, and streptococcus aureus is pathogenic bacterium, and scale operation danger is bigger.For avoiding these problems, another technology of producing recombinant protein A based on gene engineering method has obtained exploitation recently.
The purposes of albumin A is wide, and demand is big, mainly relies on import but present case is an albumin A, and the Repligen Corporation as the U.S. costs an arm and a leg.And domesticly starting late aspect the albumin A research and development, yield poorly, the cost height, unstable product quality, can not satisfy the demand in market far away, wherein the problem of most critical is exactly that purifying process is still waiting to improve, and therefore is badly in need of setting up a kind of improved separation purifying technique that is suitable for scale operation, to reduce cost, improve the quality of products.
At present, the purification process to albumin A of report mainly contains two kinds, and a kind of is the method that adopts the IgG affinity chromatography, and second kind what then adopt is the method for multistep purifying.
As far back as 1972, people such as Hjelm just proposed to adopt the method purifying protein A of IgG affinity chromatography.Concrete grammar is: being carrier with the cross-linked agarose gel, adopting cyanogen bromide-activated technology, is that functional group is made affinity column with IgG, the affinitive layer purification albumin A.This method is simple to operate, and step chromatography operation can make the albumin A purity of purifying reach 95%.Yet, because IgG belongs to biomacromolecule, molecular weight big (Mr=150000), therefore sterically hindered for reducing, can only a spot of IgG of coupling on the unit volume carrier, cause the dynamic carrying capacity of affinity column low, be difficult to scale operation, be used for the laboratory scale purifying more.And what utilize during coupling IgG is a large amount of amino in IgG surface, belongs to the multiple spot coupling process, must cause immobilized albumen spatial orientation heterogeneity, has increased sterically hindered.
US 5075423 discloses the method for a kind of purifying protein A.This method is on the basis of IgG affinity chromatography, by increasing by a step ion exchange chromatography to remove the pollutent of trace.Yet, there is similar problem to aforesaid method, the IgG affinity column is sterically hindered big, and dynamically carrying capacity is low, and the cost height is difficult to large-scale application.
People such as Sjoquist have proposed the method purifying protein A of multistep operative combination in 1972, mainly comprise Acid precipitation, ammonium sulfate precipitation, ultrafiltration, DEAE weak anionic displacement chromatography and gel permeation chromatography.This method operation steps is many, the purge process complexity, and the rate of recovery is low, and the cycle grows up to this height, and gel permeation chromatography throughput is limited, and these have all limited the possibility of its large-scale application.
US 5314993 discloses a kind of method of energy large scale purification recombinant protein A, and this method comprises heat treated, three steps of ion exchange chromatography and ethanol sedimentation.This method technology is simple, but product recovery rate is not high.
People such as Hammond have also delivered a kind of method of energy large scale purification recombinant protein A, and this method comprises heat treated, removes the salt plug desalination, DEAE weak anionic displacement chromatography and phenyl hydrophobic interaction chromatography, and this method complicated operation, total yield is low.
WO 2007/035341A1 discloses the method for a kind of purifying protein A, and this method comprises filtration, diafiltration, and the successive chromatography operation of two steps, the first step is a kind of in anion-exchange chromatography, hydrophobic interaction chromatography or the ceramic hydroxyapatite chromatography, second step was cation-exchange chromatography.The same complicated operation of this method, efficient is low.
CN 1432578A discloses a kind of preparation method of protein A gene and expression product thereof of synthetic.Used purifying mode is a step molecular sieve purification, and treatment capacity is little, and product purity is not high, is not suitable for scale operation.
The disclosed recombinant protein A purification process of CN 101050464A and CN 101298476A is similar.It all is to have added the label of being made up of 6 Histidines when gene constructed, therefore adopts nickel ion chelating chromatography purification recombinant protein A.But this method need add specific enzyme in purge process excises histidine-taggedly, and the product purity that this method obtains is low.
In sum, though utilize IgG to have some defectives, obtain still to be higher than additive method on the product purity at single for the affinity chromatography method of aglucon.Therefore, the present invention is directed to IgG and be sterically hindered big, the deficiency such as carrying capacity is low of adsorption column that the affinity column of aglucon exists in the recombinant protein A purifying, the novel method of the affinity chromatography technology purification of recombinant proteins A that to have proposed a kind of Fc fragment with directed immobilization IgG be affinity ligand.
Summary of the invention
The invention provides a kind of Fc fragment with directed immobilization IgG is the preparation method of the affinity chromatography medium of affinity ligand, in the highly selective separation and purification that realizes recombinant protein A, significantly improves the carrying capacity and the work-ing life of chromatography column, reduces production costs.
Technical scheme of the present invention may further comprise the steps:
(1) adopting papain enzymolysis IgG is Fc fragment and Fab fragment, and passes through the fixedly affinity chromatography column purification Fc fragment of recombinant protein A;
(2) adopt sodium periodate oxidation Fc fragment sugar side chain, utilize the aldehyde radical that obtains after the reaction to realize the segmental directed immobilization of Fc;
(3) adopting sepharose is matrix, activate with carbonyl dimidazoles (CDI), fix by the Fc fragment of double-functional group spacerarm molecule after oxidation, synthesized can specificity in conjunction with the affinity chromatographic material of recombinant protein A, this material is 10-20mg/ml gel to the loading capacity of recombinant protein A.
Beneficial effect of the present invention is as follows:
1, adopting the Fc fragment is that affinity ligand is prepared into affinity column, because the Fc clip size only accounts for 1/3rd of complete IgG molecule, therefore can the more aglucon of coupling on the unit volume carrier, compare the dynamic carrying capacity of IgG affinity chromatography medium and obviously improve, thereby reduced production cost;
2, adopt sodium periodate oxidation Fc fragment sugar side chain, utilize the aldehyde radical that obtains after the reaction to realize the segmental directed immobilization of Fc.Owing to adopted directed coupling process, reduced sterically hinderedly, therefore can increase dynamic carrying capacity using under the condition of high flow rate more;
3, with respect to the traditional IgG affinity chromatography and the method for other multistep purifying, the characteristics of this technology are low-cost, high-level efficiency and easy and simple to handle, and this technology both can be used for laboratory scale purifying protein A, also can be used for the production of industrialization albumin A.As stated above, the final albumin A that obtains utilizes SDS-PAGE and HPLC detection product purity to reach more than 95%.
Description of drawings
Accompanying drawing 1 is for passing through the segmental SDS-PAGE gel electrophoresis of recombinant protein A affinity chromatography column purification Fc result, and wherein swimming lane 1 is IgG, and swimming lane 2 is a papain enzymolysis liquid, and swimming lane 3 is for penetrating, and swimming lane 4 is the distilled water lavage specimens, and swimming lane 5 is the Fc fragment behind the purifying.
Accompanying drawing 2 is SDS-PAGE gel electrophoresis result behind the purifying, and wherein swimming lane 1 is low molecular weight protein (LMWP) marker, and swimming lane 2 is the recombinant protein A behind the purifying.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme and accompanying drawing.
Embodiment 1
The segmental preparation of Fc
(1) papain enzymolysis IgG
An amount of papoid certain hour of PBS damping fluid dissolving with 10mM pH 7.4 filters.Add a certain amount of activator halfcystine and sequestrant EDTA, make its final concentration reach 0.02mol/L and 0.01mol/L respectively, add IgG and make its concentration remain on 0.5-10mg/ml, regulate the pH value between the 6-7 behind the adding reactant, 40 ℃ of water-bath 3-6h.Adding the terminator iodo-acid amide at last makes its concentration reach 0.03mol/L.
(2) utilize recombinant protein A affinity chromatography column purification Fc fragment
Getting with the recombinant protein A is the affinity chromatography filler dress post of aglucon, with 5 times of column volume level pads (10mM PBS, pH 7.4) balance pillar, detects the 280nm absorbancy, till the baseline balance.With the slow post of crossing of the enzymolysis solution of above-mentioned gained, then with 5 times of column volume level pad flushing pillars, use the distilled water flushing again instead, with flush away because of hydrophobic interaction but not the foreign protein of special absorption, use elution buffer (100mM citric acid, the pH 2.3) wash-out of 2-4 times of column volume at last, collect elution peak, and use neutralization buffer (500mM Tris-HCl, pH 8.0) to be neutralized to neutrality immediately.
The segmental directed immobilization of Fc
(1) the segmental oxidizing reaction of Fc
To the above-mentioned elutriant desalination of dialysing, change over to after concentrating in the Erlenmeyer flask of aluminium-foil paper parcel, adding sodium periodate to final concentration is 2-10mg/ml, slight wobble, dialyse fast behind the room temperature lucifuge reaction 20-40min and remove sodium periodate, and the solution after the dialysis is concentrated with termination reaction.
(2) CDI method activated agarose gel
Get the Sepharose CL-4B gel behind the 5ml bulk deposition, with the sanitas in the distilled water flush away glue etc.Taking by weighing 0.5g CDI is dissolved in about 10ml acetone.Washed gel is added in the CDI acetone soln 30 ℃, 150rpm, constant temperature shaking table reaction 1h.Reacted gel washes repeatedly with acetone, removes the imidazoles that produces in the reaction process.
(3) connection of arm
Add the quadrol, 1 of 2ml in the 10ml anhydrous propanone, 6-hexanediamine or diamino dipropyl imines (DADPA) add activatory CDI gel again, 30 ℃, 150rpm, constant temperature shaking table reaction 2h.With the reacted gel of a large amount of distilled water flushings, use 150mM pH 8.2 borate buffer suction filtrations again.
(4) Fc is segmental fixing
Get the gel after a certain amount of activation, add the borate buffer of isopyknic 150mM pH 8.2, add and contain the segmental solution of Fc after the oxidation, 30 ℃, 150rpm, constant temperature shaking table reaction 3h.Reacted gel with a large amount of distilled water suction filtrations after, put into the unreacted aldehyde radical of ethanolamine solutions sealing, 30 ℃, 150rpm, the constant temperature shaking table spends the night.Add sodium borohydride at last, 30 ℃, 150rpm, about shaking table reaction 30min, the gel after the reduction cleans with a large amount of distilled water suction filtrations, puts into then to contain 0.02% sodium azide solution, and 4 ℃ of preservations are stand-by.Synthetic is that the aglucon density of the affinity chromatography medium of aglucon is 5-15mg/ml gel with the Fc fragment.
The separation and purification of recombinant protein A
(1) with the centrifugal 15min of fermented liquid 4000rpm, remove the supernatant substratum, thalline is dissolved in lysate (the 50mM Tris of 5 times of volumes, 100mM NaCl, pH 8.0) in, it is 0.2mg/mL that the adding N,O-Diacetylmuramidase makes its final concentration, ultrasonication behind 37 ℃ of temperature bath 15min, the centrifugal 20min of bacterium liquid 10000rpm with after the fragmentation obtains supernatant liquor.
(2) getting with the Fc fragment of IgG is the affine filler dress post of aglucon, with 5 times of column volume level pads (pH 7.4 for 10mM PBS, 0.5M NaCl) balance pillar, detects the 210nm absorbancy, till the baseline balance.With the slow post of crossing of the albumen supernatant liquor after the above-mentioned processing, then with 5-10 times of column volume level pad flushing pillar, till 210nm place absorbancy is got back to baseline and is stablized, again with elution buffer (the 100mM citric acid of 2-4 times of column volume, pH 2.3) wash-out, collect elution peak, and use neutralization buffer (500mM Tris-HCl, pH 8.0) to be neutralized to neutrality immediately.
The final albumin A that obtains is more than 95% through SDS-PAGE and HPLC detection albumin A purity.
Claims (5)
1. the separation purification method of a recombinant protein A, its feature comprises the steps:
(1) adopting papain enzymolysis IgG is Fc fragment and Fab fragment, and passes through the fixedly affinity chromatography column purification Fc fragment of recombinant protein A;
(2) adopt sodium periodate oxidation Fc fragment sugar side chain, utilize the aldehyde radical that obtains after the reaction to realize the segmental directed immobilization of Fc;
(3) adopting sepharose is matrix, with the carbonyl dimidazoles activation, fixes by the Fc fragment of double-functional group spacerarm molecule after with oxidation, has synthesized the affinity chromatographic material of specificity in conjunction with recombinant protein A.
2. the separation purification method of a kind of recombinant protein A according to claim 1, its feature also is: the condition of described papain enzymolysis IgG is: the final concentration of activator halfcystine and sequestrant EDTA is respectively 0.02mol/L and 0.01mol/L, the concentration that adds IgG remains on 5-10mg/ml, enzymolysis pH value 6-7, hydrolysis temperature 37-45 ℃, enzymolysis time 6h; The concentration that adds the terminator iodo-acid amide at last is 0.03mol/L.
3. the separation purification method of a kind of recombinant protein A according to claim 1, its feature also is: described sodium periodate oxidation Fc fragment sugar side chain condition is: solution I gG concentration is 0.5-10mg/ml, and the final concentration that adds sodium periodate is 2-10mg/ml.
4. the separation purification method of a kind of recombinant protein A according to claim 1, its feature also is: double-functional group spacerarm molecule is a quadrol, 1,6-hexanediamine or diamino dipropyl imines.
5. the separation purification method of a kind of recombinant protein A according to claim 1, its feature also is: synthetic is that the aglucon density of the affinity chromatography medium of aglucon is 5-15mg/ml with the Fc fragment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010227638 CN101921320A (en) | 2010-07-15 | 2010-07-15 | A kind of separation purification method of recombinant protein A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010227638 CN101921320A (en) | 2010-07-15 | 2010-07-15 | A kind of separation purification method of recombinant protein A |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101921320A true CN101921320A (en) | 2010-12-22 |
Family
ID=43336554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010227638 Pending CN101921320A (en) | 2010-07-15 | 2010-07-15 | A kind of separation purification method of recombinant protein A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101921320A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676562A (en) * | 2012-04-21 | 2012-09-19 | 大连理工大学 | Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies |
| CN102731631A (en) * | 2011-04-07 | 2012-10-17 | 常州荣君生物医药科技有限公司 | Affinity separation material for protein A and application of same |
| CN104977401A (en) * | 2014-04-01 | 2015-10-14 | 苏州浩欧博生物医药有限公司 | Purifying preparation method of allergen positive serum |
| CN105063142A (en) * | 2015-08-07 | 2015-11-18 | 山西瑞亚力科技有限公司 | Method used for preparing immunoglobulin G Fc fragments with mixed-mode chromatography filling material |
| CN108025282A (en) * | 2015-08-28 | 2018-05-11 | 通用电气医疗集团生物工艺研发股份公司 | The isolation medium and method of separation antibody |
| CN112630420A (en) * | 2020-12-07 | 2021-04-09 | 北京科技大学 | Method for realizing directional coupling by using glycosyl of antibody and solid phase carrier material |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5075423A (en) * | 1988-07-22 | 1991-12-24 | Imre Corporation | Purification of protein a by affinity chromatography followed by anion exchange |
| CN1432578A (en) * | 2003-02-26 | 2003-07-30 | 本元正阳基因技术股份有限公司 | Recombinant protein A gene and prepn and application of its expression product |
| CN101050464A (en) * | 2006-03-17 | 2007-10-10 | 上海中信国健药业有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101268181A (en) * | 2005-09-20 | 2008-09-17 | 马林克罗特贝克公司 | Protein A production and purification without using animal derived components |
| CN101298476A (en) * | 2008-06-06 | 2008-11-05 | 广州康盛生物科技有限公司 | Artificial recombinant tetrad protein A, construction method and use thereof |
-
2010
- 2010-07-15 CN CN 201010227638 patent/CN101921320A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5075423A (en) * | 1988-07-22 | 1991-12-24 | Imre Corporation | Purification of protein a by affinity chromatography followed by anion exchange |
| CN1432578A (en) * | 2003-02-26 | 2003-07-30 | 本元正阳基因技术股份有限公司 | Recombinant protein A gene and prepn and application of its expression product |
| CN101268181A (en) * | 2005-09-20 | 2008-09-17 | 马林克罗特贝克公司 | Protein A production and purification without using animal derived components |
| CN101050464A (en) * | 2006-03-17 | 2007-10-10 | 上海中信国健药业有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101298476A (en) * | 2008-06-06 | 2008-11-05 | 广州康盛生物科技有限公司 | Artificial recombinant tetrad protein A, construction method and use thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102731631A (en) * | 2011-04-07 | 2012-10-17 | 常州荣君生物医药科技有限公司 | Affinity separation material for protein A and application of same |
| CN102731631B (en) * | 2011-04-07 | 2014-04-30 | 上海荣君生物医药科技有限公司 | Affinity separation material for protein A and application of same |
| CN102676562A (en) * | 2012-04-21 | 2012-09-19 | 大连理工大学 | Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies |
| CN102676562B (en) * | 2012-04-21 | 2013-11-06 | 大连理工大学 | Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies |
| CN104977401A (en) * | 2014-04-01 | 2015-10-14 | 苏州浩欧博生物医药有限公司 | Purifying preparation method of allergen positive serum |
| CN105063142A (en) * | 2015-08-07 | 2015-11-18 | 山西瑞亚力科技有限公司 | Method used for preparing immunoglobulin G Fc fragments with mixed-mode chromatography filling material |
| CN105063142B (en) * | 2015-08-07 | 2019-09-03 | 山西瑞亚力科技有限公司 | A method of immunoglobulin G Fc segment is prepared with Mixed-Modechromatography filler |
| CN108025282A (en) * | 2015-08-28 | 2018-05-11 | 通用电气医疗集团生物工艺研发股份公司 | The isolation medium and method of separation antibody |
| CN112630420A (en) * | 2020-12-07 | 2021-04-09 | 北京科技大学 | Method for realizing directional coupling by using glycosyl of antibody and solid phase carrier material |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1925898B (en) | Antibody purification technique | |
| CN101921320A (en) | A kind of separation purification method of recombinant protein A | |
| JP2019165747A (en) | Mutated immunoglobulin-binding polypeptides | |
| US10792654B2 (en) | Solid phase for mixed-mode chromatographic purification of proteins | |
| CN103228328A (en) | Affinity chromatography matrix | |
| CN102234332B (en) | Process for separating and purifying recombinant human serum albumin and fusion protein thereof | |
| JP6335785B2 (en) | Mixed-mode antibody affinity separation matrix and purification method and target molecule using the same | |
| CN105555795A (en) | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom | |
| CN101302504B (en) | Method for purifying lysostaphin by antibody affinity chromatography | |
| Kish et al. | Peptide-based affinity adsorbents with high binding capacity for the purification of monoclonal antibodies | |
| CN110090635B (en) | GSH affinity chromatography medium for purifying GST-tag fusion protein, and preparation method and application thereof | |
| Van Sommeren et al. | Comparison of three activated agaroses for use in affinity chromatography: effects on coupling performance and ligand leakage | |
| US6177548B1 (en) | Enhanced aggregate removal from bulk biologicals using ion exchange chromatography | |
| CN108355618B (en) | Bovine-derived hyaluronidase affinity medium and adsorption method thereof | |
| JPS62253396A (en) | Affinity chromatography material and purification of proteinantigen of bordetella bacteria using said material | |
| CN112206754B (en) | Affinity chromatography medium and preparation method and application thereof | |
| CN104841375A (en) | Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon | |
| CN107999035A (en) | Chromatography media using tryptamines as functional ligand | |
| CN101921818B (en) | Method for producing recombinant protein A | |
| CN101362792B (en) | Affinity separation polymer of lactoferrin and affinity purification method of lactoferrin | |
| US6555661B1 (en) | Simple, environmentally benign, method for purifying protein A | |
| WO2019075892A1 (en) | Method for purifying placenta-like chondroitin sulfate a or derivative thereof by affinity chromatography | |
| CN105301230B (en) | Antibody fluorescence labeling method based on hydrophobic charge induction magnetic microspheres | |
| CN111871396A (en) | Albumin electrostatic affinity dual-mode chromatography medium and preparation method and application thereof | |
| Patchornik et al. | Free nonimmobilized ligands as a tool for purification of proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20101222 |