CN103122029A - Method for purifying human recombinant SPINK6 protein with KLK14 protein through affinity chromatography - Google Patents
Method for purifying human recombinant SPINK6 protein with KLK14 protein through affinity chromatography Download PDFInfo
- Publication number
- CN103122029A CN103122029A CN2011103696552A CN201110369655A CN103122029A CN 103122029 A CN103122029 A CN 103122029A CN 2011103696552 A CN2011103696552 A CN 2011103696552A CN 201110369655 A CN201110369655 A CN 201110369655A CN 103122029 A CN103122029 A CN 103122029A
- Authority
- CN
- China
- Prior art keywords
- albumen
- people
- klk14
- spink6
- buffer
- 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.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying a human recombinant SPINK6 protein through affinity chromatography. The method comprises the following steps of: mixing a human recombinant KLK14 protein with affinity chromatography media to carry out coupling reaction to prepare a human recombinant KLK14 protein affinity chromatography medium; and enabling the human recombinant SPINK6 protein to pass through a chromatographic column which has been balanced by buffer solution A in advance and is filled with a trypsin affinity chromatography medium, then washing a human recombinant KLK14 protein affinity chromatographic column after sample loading with the buffer solution A, then eluting the SPINK6 protein from the chromatographic column with buffer solution B and collecting the elution peak. The method has the advantages that the activity recovery of the human recombinant SPINK6 protein is 45-60%, the purity can reach 90-95% and the protein purification fold is over 10-20; and the method has the advantages of high treatment speed, good repeatability, long column efficiency and basically invariant joint efficiency.
Description
Technical field
The present invention relates to a kind of purification process of recombinant protein, be specifically related to the recombinate affinity chromatographic purification process of SPINK6 albumen of people.
Background technology
Tumour is one of the highest disease of present mankind's mortality ratio, and existing medicine is relatively poor to the result for the treatment of of tumour, is particularly lacking effective means aspect the recurrence of tumour and transfer.Market is in the urgent need to the medicine of novel treatment tumour.
Serine protease inhibitor Kazal type (SPINK) family and chronic pancreatitis, the disease relationships such as the esophageal carcinoma are close.This family mainly is comprised of subfamilies such as SPINK1, SPINK2, SPINK4, SPINK6.The SPINK6 gene order be NCBI in 2006 a disclosed people source gene order, its sequence is at document: (Martin C.Wapenaar, Alienke J.Monsuur et al; Immunogenetics, 2007, detailed report is arranged in 59:349-357).There is research to find the anti-tumor activity (number of patent application: PCT/CN2009/074410) of SPINK6 albumen.This research experiment result shows: when the SPINK6 gene in JEG-3 during overexpression, make JEG-3 lose and forming the clone on soft agar, become the knurl ability in nude mouse, the restructuring SPINK6 albumen that obtains with engineered method expression is the effective one-tenth knurl of inhibition tumor cell in nude mouse also; Wherein (number of patent application: PCT/CN2009/074410) also built a kind of genetic engineering bacterium of E.coli and yeast of the SPINK6 of containing gene, and successful expression restructuring SPINK6 albumen.But there is no the clear and definite detailed purification process of illustrating in above-mentioned research method, exist in actual purge process purification efficiency low, reclaim rate variance, purity can only reach 80% left and right, purity requirement that still can not fulfilling medicinal.Therefore the purifying process of seeking the restructuring SPINK6 albumen that a species specificity is good, product purity is high, the sample adsorptive capacity is large is a present difficult problem very in the urgent need to address in the art.
Summary of the invention
The objective of the invention is to overcome the defects that prior art exists, the technical problem that mainly solves is to disclose a kind of the recombinate method of spink6 albumen of KLK14 albumen affinitive layer purification people.
Technical scheme of the present invention is such:
Up-to-date result of study shows, SPINK6 albumen plays a significant role in the skin keratin layer tissue as the specific inhibitor of kallikrein (KLK) family, and its restraining effect to KLK14 is particularly evident.The present invention proves by experiment, when SPINK6 and KLK14 albumen are hatched at 1: 1, can suppress the activity of KLK14 substantially fully.On this basis, the present invention has the affine filler Purification of Human restructuring SPINK6 albumen of KLK14 albumen by coupling, satisfies experiment in follow-up body thereby be intended to obtain the highly purified people SPINK6 albumen of recombinating.
Particularly, the recombinate method of SPINK6 albumen of KLK14 albumen affinitive layer purification people of the present invention is characterized in that, it comprises step:
(1) preparation affinity chromatography medium
In the present invention, the working instructions of the Sepharose4B of the CNBr activation of publishing with reference to GE company carry out the preparation of affinity chromatography medium.Concrete steps are as follows: affinity media is carried out swelling, cleaning with the HCl solution of pH2.0-3.0, the consumption of HCl solution is 50-100 times of volume of affinity media, then the coupling liquid with the 10-20 of affinity media volume doubly cleans, and makes the pH of affinity chromatography medium reach 7.5-10.0;
Coupling liquid used is the NaHCO that contains 0.15-0.6M NaCl
3Solution (pH8.0-10.0);
People's KLK14 albumen of recombinating is dissolved in coupling liquid with weight ratio 1: 200-400, then mixed with the affinity media that coupling liquid cleaned with the upper step, room temperature condition vibration 0.5-2 hour, or placed 12-36 hour under 4 ℃ of conditions, make the people recombinate KLK14 albumen and the affinity media coupling abundant; Then clean with coupling liquid the affinity chromatography medium make, coupling liquid consumption be the affinity chromatography medium volume that makes 5-20 doubly; Use again Tris-HCl damping fluid (pH 8.0) or the 1-2M ethanolamine solutions of affinity media volume 1-5 0.5M-1M doubly to soak placement 2-4 hour, be used for the not combined avtive spot of sealing affinity chromatography medium;
Said people recombinates KLK14 albumen available from R﹠amp; The recombinate weight ratio of KLK14 albumen and affinity chromatography medium of D company, people is 1: 20-150;
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 chitosan.
(2) people's SPINK6 albumen of recombinating carries out purifying with affinity chromatography medium
Contain with 0.01-0.1mol/L pH7.0-9.0 the chromatography column that 0.05-0.15mol/L NaCl buffer A balance is equipped with affinity chromatography medium;
With the people recombinate the SPINK6 protein sample with the 10-75cm/h loading to the good chromatography column of pre-equilibration;
The said cm/h of unit is flow velocity unit, i.e. the damping fluid height by dielectric material per hour.Then the buffer A that contains 0.05-0.15mol/L NaCl with 0.01-0.1mol/L pH7.0-9.0 is cleaned the affinity column after loading;
The buffer B that contains 0.05-0.15mol/L NaCl with 0.1-0.5mol/L pH 2.5-4.5 again with the flow velocity of 10-55cm/h with people's SPINK6 albumen wash-out and collect elution peak from the chromatography column of recombinating; The consumption of buffer B is 3-10 times of chromatography column volume; Elutriant is collected in A in the damping fluid that 0.5-2mol/L pH7.0-9.0 is housed;
Said people recombinates the SPINK6 protein sample for (number of patent application: PCT/CN2009/074410) the recombinate elution peak (SPINK6 protein content be 0.01-0.08mg/ml, purity be 75%-80%) of pichia yeast fermented supernatant fluid after the ion-exchange step purifying and the buffer A of SPINK6 protein gene of people that contain of preparation mixes with volume ratio at 1: 1 according to patent;
Said buffer A balance affinity column comprises the steps:
The buffer A that 0.01-0.1mol/L pH7.0-9.0 is contained 0.05-0.15mol/L NaCl with velocity of medium by the chromatography column of affinity chromatography medium is housed, consumption be the chromatography column volume 3-10 doubly, guarantee that by ultraviolet detection chromatography column has cleaned to baseline;
The affinity column that said buffer A is cleaned after loading comprises the steps:
The buffer A that contains 0.05-0.15mol/L NaCl with 0.01-0.1mol/L pH7.0-9.0 is cleaned affinity column after loading with velocity of medium, consumption be the chromatography column volume 3-10 doubly, guarantee that by ultraviolet detection chromatography column has cleaned to baseline;
The buffer A of said pH7.0-9.0 is selected from one or more in PBS, Tris-HCl damping fluid;
The buffer B of said pH 2.5-4.5 is selected from acetic acid-sodium-acetate, NaHPO
4One or more of-citric acid, citric acid-sodium citrate damping fluid;
The compound method of said various damping fluids is with reference to " molecular cloning " second edition, appendix B: the reagent that uses in molecular cloning and the preparation of damping fluid; Shanghai Science Press, 1993.
The recombinate rate of recovery of SPINK6 albumen of the people that the present invention obtains is 45-60%, purity 90%-95%, protein purification multiple be 10-20 doubly more than, processing speed is fast, good reproducibility, post effect are long; According to the adsorption column of the affinity chromatography medium of the inventive method preparation not only post effect long (under 4 ℃, 20% ethanol was preserved the post effect greater than a year) and joint efficiency substantially constant (0.35mg/ml affinity media).
For the ease of understanding, below will describe in detail the recombinate method of SPINK6 albumen of KLK14 albumen affinitive layer purification people of the present invention by concrete drawings and Examples.It needs to be noted, specific examples and accompanying drawing are only in order to illustrate, obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in scope of the present invention.
Description of drawings
Fig. 1 is restructuring SPINK6 albumen affinity chromatography collection of illustrative plates.
Fig. 2 analyzes for restructuring SPINK6 protein SDS-PAGE.
Embodiment
The embodiment 1 preparation people KLK14 albumen affinity chromatography medium of recombinating
The Sepharose4B dry powder (1 gram dry powder swellable is to 3.5ml glue) of 0.5 gram CNBr activation is placed in 10mM HCl and carries out suction filtration with 150ml 10mM HCl in sintered glass funnel rapidly and cleaned 15 minutes and be evacuated to half-dried.13mg trypsinase is dissolved in the 5ml coupling buffer and slowly adds in pretreated glue (CNBr activates Sepharose-4B), and under room temperature, shaking table slowly vibrated 2 hours.After coupling, glue is cleaned in sintered glass funnel with the 20ml coupling buffer and remove unconjugated albumen and subsequently glue was placed in 1M Tris-HCl pH8.0 sealing avtive spot 1 hour, the consumption of confining liquid is 10ml.Containing 0.5MNaCl and 0.1M acetate buffer solution pH 4.0 with 0.1M Tris-HCl pH 8.0 contains 0.5M NaCl and alternately washs 3 circulations.Under the Tris-HCl system, mucilage binding is entered in 1cm * 10cm post at last, column volume is 2ml.Cleaning buffer solution 27ml after the collection coupling, measuring protein concentration by the lowry method is 0.1mg/ml, total amount is 2.7mg, then calculates the coupling rate.
The embodiment 2 people KLK14 affinitive layer purification people SPINK6 albumen of recombinating of recombinating
Affinity media: coupling has the recombinate Sepharose4B gel 1.5ml of KLK14 albumen of people, the 10ml volume chromatography column of packing in advance.
Sample: the people recombinates the SPINK6 protein sample for (number of patent application: PCT/CN2009/074410) preparation contains the people pichia yeast fermented supernatant fluid of spink6 protein gene elution peak (the SPINK6 protein content is 0.05mg/ml, and purity is 80%) 25ml and the sample buffer 50ml that mixes at 1: 1 with volume ratio of level pad after the ion-exchange step purifying that recombinate according to patent.
Tomography devices: AKTA explorer 100 protein purification systems, available from GE biotech company.
Balance, cleaning buffer solution: 0.05mol/L Tris-HCl (pH8.0) contains 0.1mol/L NaCl damping fluid.
Elution buffer: 0.1mol/L Gly-HCl (pH2.8) contains 0.15mol/L NaCl damping fluid.
Neutralization buffer: 1mol/L Tris-HCl (pH8.0) damping fluid.
Balance: with the level pad balance, the 1.5ml coupling being housed there is the recombinate Sepharose4B gel chromatography column of KLK14 albumen of people, and the post height is 2cm, with 5 column volumes of 50cm/h flow velocity balance, by ultraviolet detection (A280) guarantee chromatography column balance to baseline.
Loading: the 50ml sample buffer is passed through through the good tryptic Sepharose4B gel chromatography column of level pad balance with 40cm/h.During loading, sample keeps ice bath, begins to collect stream and wear the peak when ultraviolet detection changes.
Clean: loading is complete, and with the flow velocity cleaning chromatography column of cleaning buffer solution with 50cm/h, consumption is 5 times of chromatography column volume, treats ultraviolet detection (A
280) stop collecting stream during to baseline and wear the peak, receive altogether to such an extent that 55ml stream is worn liquid.
Wash-out: clean to baseline, people's SPINK6 albumen of recombinating is eluted from chromatography column with the flow velocity of 20cm/h with elution buffer, begin to collect elution peak when ultraviolet detection changes, the damping fluid consumption is long-pending 4 times of chromatographic column cylinder.Add in advance neutralization buffer in the elutriant collection tube, collect elution peak 2.5ml, experimental result as shown in Figure 1, wherein peak 1 is restructuring SPINK6 albumen elution peak, the peak is worn for restructuring SPINK6 protein stream in peak 2.By SDS-PAGE, restructuring SPINK6 albumen is carried out Purity, result as shown in Figure 2.In figure, swimming lane M is lower molecular weight standard protein (Mark), and swimming lane 1,2,3 is the recombinate SPINK6 albumen of KLK14 affinity column purifying of people.This result is analyzed with electrophoresis analysis software bandscan5.0, the result demonstration, after the people recombinated the KLK14 affinity purification, the purity of SPINK6 albumen arrived to 95.3%.Measure the restructuring SPINK6 protein content that respectively goes on foot in purge process in damping fluid by the BCA method, as calculated, wherein containing protein content in sample-loading buffer is 1.25mg, and stream is worn 0.55mg, 0.7mg in elution buffer, and protein recovery reaches 56%.The check of table 1SDS-PAGE electrophoresis purity.
The check of table 1 SDS-PAGE electrophoresis purity
Claims (6)
- The method of SPINK6 albumen, is characterized in that 1.KLK14 albumen affinitive layer purification people recombinates, and it comprises step:(1) preparation affinity chromatography medium:People's KLK14 albumen of recombinating is mixed with affinity chromatography medium, and linked reaction makes people's KLK14 albumen affinity chromatography medium of recombinating;(2) people's SPINK6 albumen of recombinating carries out purifying with affinity chromatography mediumThe people is recombinated SPINK6 albumen by the recombinate chromatography column of KLK14 albumen affinity chromatography medium of the people that step (1) is housed with the buffer A balance of pH7.0-9.0;Then clean people after the loading KLK14 albumen affinity column of recombinating with the buffer A of pH7.0-9.0;The buffer B that contains 0.05-0.15mol/L NaCl with pH 2.5-4.5 again is SPINK6 albumen wash-out from the chromatography column, and collects elution peak;The buffer A of described pH7.0-9.0 is selected from one or more in PBS, Tris-HCl damping fluid;The buffer B that described pH 2.5-4.5 contains 0.05-0.15mol/L NaCl is selected from acetic acid-sodium-acetate, NaHPO 4One or more of-citric acid, citric acid-sodium citrate damping fluid.
- 2. method according to claim 1, is characterized in that, described affinity chromatography medium is selected from one or more in Sepharose, Mierocrystalline cellulose, soluble cotton, acrylamide polymer, polyethersulfone or chitosan.
- 3. method according to claim 1, it is characterized in that, the recombinate chromatography column of KLK14 albumen affinity chromatography medium of the buffer A balance people of described pH7.0-9.0 comprises step: with the buffer A of pH7.0-9.0 by the recombinate chromatography column of KLK14 albumen affinity chromatography medium of people is housed, consumption be the chromatography column volume 3-10 doubly.
- 4. method according to claim 1, it is characterized in that, the buffer A of described pH7.0-9.0 is cleaned people after the loading KLK14 albumen affinity column of recombinating and comprised step: clean people after the loading KLK14 albumen affinity column of recombinating with the buffer A of pH7.0-9.0, consumption is 3-10 times of chromatography column volume.
- 5. method according to claim 1, is characterized in that, in described step (2), people's SPINK6 albumen of recombinating is 10-75cm/h by the recombinate flow velocity of KLK14 albumen affinity column of people.
- 6. method according to claim 1, is characterized in that, in described step (2), the buffer B that pH 2.5-4.5 contains 0.05-0.15mol/L NaCl with the flow velocity of 10-55cm/h with people's SPINK6 albumen wash-out from the chromatography column of recombinating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110369655.2A CN103122029B (en) | 2011-11-18 | 2011-11-18 | KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110369655.2A CN103122029B (en) | 2011-11-18 | 2011-11-18 | KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103122029A true CN103122029A (en) | 2013-05-29 |
| CN103122029B CN103122029B (en) | 2015-10-28 |
Family
ID=48453201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110369655.2A Active CN103122029B (en) | 2011-11-18 | 2011-11-18 | KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103122029B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779618A (en) * | 2016-04-19 | 2016-07-20 | 中南大学湘雅二医院 | Novel target gene for diagnosing and treating tongue squamous carcinoma and application thereof |
| CN109789385A (en) * | 2016-10-03 | 2019-05-21 | 通用电气医疗集团生物工艺研发股份公司 | New chromatographic medium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302504A (en) * | 2007-05-11 | 2008-11-12 | 上海高科联合生物技术研发有限公司 | Method for purifying lysostaphin by antibody affinity chromatography |
-
2011
- 2011-11-18 CN CN201110369655.2A patent/CN103122029B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302504A (en) * | 2007-05-11 | 2008-11-12 | 上海高科联合生物技术研发有限公司 | Method for purifying lysostaphin by antibody affinity chromatography |
Non-Patent Citations (3)
| Title |
|---|
| ULF MEYER-HOFFERT ET AL: "Isolation of SPINK6 in Human Skin SELECTIVE INHIBITOR OF KALLIKREIN-RELATED PEPTIDASES", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 285, no. 42, 28 July 2010 (2010-07-28) * |
| 李巧云等: "人激肽释放酶蛋白14在宫颈癌中的表达", 《第三军医大学学报》, vol. 29, no. 14, 30 July 2007 (2007-07-30) * |
| 顾双等: "卵巢上皮性癌组织中激肽释放酶7及14的表达及临床意义", 《山东大学学报(医学版)》, vol. 46, no. 2, 16 February 2008 (2008-02-16) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779618A (en) * | 2016-04-19 | 2016-07-20 | 中南大学湘雅二医院 | Novel target gene for diagnosing and treating tongue squamous carcinoma and application thereof |
| CN109789385A (en) * | 2016-10-03 | 2019-05-21 | 通用电气医疗集团生物工艺研发股份公司 | New chromatographic medium |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103122029B (en) | 2015-10-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6593721B2 (en) | A chromatographic method to isolate and purify high-purity recombinant human serum albumin | |
| JP6420756B2 (en) | Affinity chromatography matrix | |
| JP3676817B2 (en) | Novel factor IX purification method | |
| JP2019165747A (en) | Mutated immunoglobulin-binding polypeptides | |
| JP5772816B2 (en) | Affinity chromatography packing and method for isolating immunoglobulins | |
| JP2019034963A (en) | Novel antibody purification method using cation exchanger | |
| US20090318674A1 (en) | Process for purification of antibodies | |
| KR20070001969A (en) | Antibody purification | |
| CN102911250B (en) | Method for purifying acidic recombinant protein medicament | |
| CN101302504B (en) | Method for purifying lysostaphin by antibody affinity chromatography | |
| CN101602805B (en) | Agarose compatible medium for purifying immune globulin of hand-foot-and-mouth disease and preparation method thereof | |
| JPS6098988A (en) | Purification of lpf-ha | |
| WO2021109177A1 (en) | Immunoglobulin binding protein, preparation method and application thereof | |
| WO2014034457A1 (en) | Mix-mode antibody affinity separation matrix and purification method using same, and target molecule | |
| CN103122029A (en) | Method for purifying human recombinant SPINK6 protein with KLK14 protein through affinity chromatography | |
| Platis et al. | Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine | |
| JP2012001462A (en) | Column for antibody purification | |
| CN104841375A (en) | Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon | |
| CN103122028B (en) | Trypsinase affinitive layer purification people recombinates the method for SPINK6 albumen | |
| CN103122346A (en) | Method for purifying recombinant KLK14 protein | |
| Olson | Affinity chromatography | |
| CN102443055B (en) | A kind of purifying process of recombinant soluble human TRAIL | |
| WO2023024214A1 (en) | Method for purifying fusion protein of recombinant human serum albumin | |
| WO2011015919A1 (en) | A highly efficient process of purification and production of recombinant infliximab | |
| WO2011015920A2 (en) | A highly efficient process of purification and production of recombinant trastuzumab |
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 |