CN103122029B - KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen - Google Patents
KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen Download PDFInfo
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- CN103122029B CN103122029B CN201110369655.2A CN201110369655A CN103122029B CN 103122029 B CN103122029 B CN 103122029B CN 201110369655 A CN201110369655 A CN 201110369655A CN 103122029 B CN103122029 B CN 103122029B
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Abstract
The invention discloses a kind of people to recombinate the affinity chromatographic purification process of SPINK6 albumen, comprising: KLK14 albumen of people being recombinated mixes with affinity chromatography medium, linked reaction, obtained people recombinates KLK14 albumen affinity chromatography medium; People is recombinated SPINK6 albumen by advance with the chromatography column that above-mentioned trypsinase affinity chromatography medium is housed that buffer A balances; Then to recombinate KLK14 albumen affinity column with the people after buffer A cleaning loading; Again by buffer B by SPINK6 albumen from wash-out chromatography column, and collect elution peak.Adopt method of the present invention, the recombinate activity recovery of SPINK6 albumen of people is 45-60%, and purity can reach 90%-95%, protein purification multiple be 10-20 doubly more than.The inventive method processing speed is fast, reproducible, post effect is long and joint efficiency is substantially constant.
Description
Technical field
The present invention relates to a kind of purification process of recombinant protein, be specifically related to people and recombinate the affinity chromatographic purification process of SPINK6 albumen.
Background technology
Tumour is that current human death leads one of the highest disease, and the result for the treatment of of existing drug on tumor is poor, particularly in the recurrence and transfer of tumour, lacks effective means.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 is primarily of subfamily compositions such as SPINK1, SPINK2, SPINK4, SPINK6.The SPINK6 gene order people source gene order that is NCBI disclosed in 2006, its sequence is at document: (MartinC.Wapenaar, Alienke J.Monsuur et al; Immunogenetics, 2007,59:349-357) in have detailed report.Studies have found that the anti-tumor activity (number of patent application: PCT/CN2009/074410) of SPINK6 albumen.This research experiment result shows: when SPINK6 gene is in JEG-3 during overexpression, JEG-3 is lost on soft agar, to form clone, in nude mouse, become knurl ability, the restructuring SPINK6 albumen reached with engineered method table also can the effective one-tenth knurl of inhibition tumor cell in nude mouse; Wherein (number of patent application: PCT/CN2009/074410) also constructs a kind of genetic engineering bacterium of the E.coli and yeast containing SPINK6 gene, and successful expression restructuring SPINK6 albumen.But illustrate purification process clearly not in detail in above-mentioned research method, exist in actual purge process purification efficiency low, reclaim rate variance, purity can only reach about 80%, still can not the purity requirement of fulfilling medicinal.Therefore the purifying process finding the restructuring SPINK6 albumen that a species specificity is good, product purity is high, sample adsorption amount is large is a difficult problem very in the urgent need to address in the art at present.
Summary of the invention
The object of the invention is to overcome the above-mentioned defect that prior art exists, the technical problem mainly solved is that openly a kind of KLK14 albumen affinitive layer purification people recombinates the method for spink6 albumen.
Technical scheme of the present invention is such:
Up-to-date result of study shows, SPINK6 albumen plays a significant role in skin keratin layer tissue as the specific inhibitor of kallikrein (KLK) family, and it is particularly evident to the restraining effect of KLK14.The present invention proves by experiment, when SPINK6 and KLK14 albumen 1: 1 is hatched, can suppress the activity of KLK14 substantially completely.On this basis, the present invention has the affine filler Purification of Human restructuring SPINK6 albumen of KLK14 albumen by coupling, is intended to obtain highly purified people and recombinates SPINK6 albumen thus meet follow-up experiment in vivo.
Specifically, KLK14 albumen affinitive layer purification people of the present invention recombinates the method for SPINK6 albumen, and it is characterized in that, it comprises step:
(1) affinity chromatography medium is prepared
In the present invention, with reference to the working instructions of the Sepharose4B of the CNBr activation of GE company publication, carry out the preparation of affinity chromatography medium.Concrete steps are as follows: the HCl solution of affinity media pH2.0-3.0 is carried out swelling, cleaning, the consumption of HCl solution is 50-100 times of volume of affinity media, then clean with the coupling liquid of the 10-20 volume doubly of affinity media, make the pH of affinity chromatography medium reach 7.5-10.0;
Coupling liquid used is the NaHCO containing 0.15-0.6M NaCl
3solution (pH8.0-10.0);
KLK14 albumen of people being recombinated is dissolved in coupling liquid with weight ratio 1: 200-400, then cleaned with upper step coupling liquid affinity media mixes, at room temperature condition vibration 0.5-2 hour, or place 12-36 hour under 4 DEG C of conditions, make people recombinate KLK14 albumen and affinity media coupling abundant; Then with the affinity chromatography medium that the cleaning of coupling liquid is obtained, coupling liquid consumption be the 5-20 of obtained affinity chromatography medium volume doubly; Use the Tris-HCl damping fluid (pH 8.0) of affinity media volume 1-5 0.5M-1M doubly or 1-2M ethanolamine solutions to soak again and place 2-4 hour, for avtive spot not combined in closed affinity chromatography medium;
Said people recombinates KLK14 albumen purchased from R & D company, and the recombinate weight ratio of KLK14 albumen and affinity chromatography medium of people is 1: 20-150;
Said affinity chromatography medium be selected from Sepharose, Mierocrystalline cellulose, soluble cotton, acrylamide polymer, multipolymer and derivative thereof, polymethacrylate derivative, polyethersulfone or chitosan one or more.
(2) people's SPINK6 albumen affinity chromatography medium of recombinating carries out purifying
The chromatography column of affinity chromatography medium is housed containing 0.05-0.15mol/L NaCl buffer A balance with 0.01-0.1mol/L pH7.0-9.0;
People is recombinated SPINK6 protein sample with 10-75cm/h loading to the good chromatography column of pre-equilibration;
Said unit cm/h is flow rate, i.e. the damping fluid height by dielectric material per hour.Then the affinity column after loading is cleaned with 0.01-0.1mol/L pH7.0-9.0 containing the buffer A of 0.05-0.15mol/L NaCl;
Elution peak is collected with the flow velocity of 10-55cm/h SPINK6 albumen of people being recombinated from wash-out chromatography column containing the buffer B of 0.05-0.15mol/L NaCl again with 0.1-0.5mol/L pH 2.5-4.5; 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 SPINK6 protein sample for mixing with volume ratio 1: 1 containing the recombinate elution peak of pichia yeast fermented supernatant fluid after ion-exchange step purifying (SPINK6 protein content is 0.01-0.08mg/ml, and purity is 75%-80%) and the buffer A of SPINK6 protein gene of people of preparing according to patent (number of patent application: PCT/CN2009/074410);
Said buffer A balance affinity column comprises the steps:
By 0.01-0.1mol/L pH7.0-9.0 containing the buffer A of 0.05-0.15mol/L NaCl with velocity of medium by being equipped with the chromatography column of affinity chromatography medium, consumption be the 3-10 of chromatography column volume doubly, ensure that chromatography column has cleaned to baseline by ultraviolet detection;
Affinity column after said buffer A cleaning loading comprises the steps:
Clean affinity column loading after containing the buffer A of 0.05-0.15mol/L NaCl with velocity of medium with 0.01-0.1mol/L pH7.0-9.0, consumption is 3-10 times of chromatography column volume, ensures that chromatography column has cleaned to baseline by ultraviolet detection;
The buffer A of said pH7.0-9.0 be selected from PBS, Tris-HCl damping fluid one or more;
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;
Compound method reference " molecular cloning " second edition of said various damping fluid, Appendix B: the reagent used 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%, and purity can reach 90%-95%, protein purification multiple be 10-20 doubly more than, processing speed is fast, reproducible, post effect is long; The adsorption column of the affinity chromatography medium prepared according to the inventive method not only post effect long (at 4 DEG C, 20% ethanol is preserved post effect and is greater than a year) and joint efficiency substantially constant (0.35mg/ml affinity media).
For the ease of understanding, by by concrete drawings and Examples, the recombinate method of SPINK6 albumen of KLK14 albumen affinitive layer purification people of the present invention is described in detail below.It needs to be noted, specific examples and accompanying drawing are only to illustrate, obvious those of ordinary skill in the art according to illustrating, can make various correction and change to the present invention herein within the scope of the invention, and these are revised and change and also include in scope of the present invention.
Accompanying drawing explanation
Fig. 1 is restructuring SPINK6 albumen affinity chromatography collection of illustrative plates.
Fig. 2 is the analysis of restructuring SPINK6 protein SDS-PAGE.
Embodiment
Embodiment 1 is prepared people and to be recombinated KLK14 albumen affinity chromatography medium
The Sepharose4B dry powder (1 gram of dry powder swellable is to 3.5ml glue) of 0.5 gram of CNBr activation is placed in 10mM HCl and carries out suction filtration with 150ml 10mM HCl in sintered glass funnel rapidly and cleans 15 minutes and be evacuated to half-dried.13mg trypsinase be dissolved in 5ml coupling buffer and slowly add in pretreated glue (CNBr activates Sepharose-4B), under room temperature, shaking table slowly vibrates 2 hours.After coupling, in sintered glass funnel, carry out the unconjugated albumen of cleaning removing with 20ml coupling buffer to glue and subsequently glue is placed in the closed avtive spot of 1MTris-HCl pH8.0 1 hour, the consumption of confining liquid is 10ml.Washing 3 circulation is replaced containing 0.5MNaCl and 0.1M acetate buffer solution pH 4.0 containing 0.5M NaCl with 0.1M Tris-HClpH 8.0.Finally entered by mucilage binding in 1cm × 10cm post under Tris-HCl system, column volume is 2ml.Cleaning buffer solution 27ml after collection coupling, measuring protein concentration by lowry method is 0.1mg/ml, and total amount is 2.7mg, then calculates Conjugate ratio.
The embodiment 2 people KLK14 affinitive layer purification people that recombinates recombinates SPINK6 albumen
Affinity media: coupling has people to recombinate the Sepharose4B gel 1.5ml of KLK14 albumen, loads 10ml volume chromatography column in advance.
Sample: people recombinates SPINK6 protein sample for recombinating the sample buffer 50ml that the elution peak of pichia yeast fermented supernatant fluid after ion-exchange step purifying (SPINK6 protein content is 0.05mg/ml, and purity the is 80%) 25ml of spink6 protein gene and level pad mix with volume ratio 1: 1 containing people of preparing according to patent (number of patent application: PCT/CN2009/074410).
Tomography devices: AKTA explorer 100 protein purification system, purchased from GE biotech company.
Balance, cleaning buffer solution: 0.05mol/L Tris-HCl (pH8.0) is containing 0.1mol/L NaCl damping fluid.
Elution buffer: 0.1mol/L Gly-HCl (pH2.8) is containing 0.15mol/L NaCl damping fluid.
Neutralization buffer: 1mol/L Tris-HCl (pH8.0) damping fluid.
Balance: with equilibration buffer, 1.5ml coupling being housed has people to recombinate the Sepharose4B gel chromatography column of KLK14 albumen, post height is 2cm, balance 5 column volumes with 50cm/h flow velocity, ensure that chromatography column has balanced to baseline by ultraviolet detection (A280).
Loading: 50ml sample buffer is passed through through the good tryptic Sepharose4B gel chromatography column of equilibration buffer with 40cm/h.During loading, sample keeps ice bath, starts to collect stream and wear peak when ultraviolet detection changes.
Cleaning: loading is complete, with cleaning buffer solution with the flow velocity of 50cm/h cleaning chromatography column, consumption is 5 times of chromatography column volume, treats ultraviolet detection (A
280) wear peak to stopping collecting stream during baseline, receive 55ml stream wears liquid altogether.
Wash-out: clean to baseline, elutes from chromatography column with the flow velocity of 20cm/h SPINK6 albumen of people being recombinated with elution buffer, starts to collect elution peak when ultraviolet detection changes, and damping fluid consumption is long-pending 4 times of chromatographic column cylinder.In elutriant collection tube, add neutralization buffer in advance, collect elution peak 2.5ml, as shown in Figure 1, wherein peak 1 is restructuring SPINK6 albumen elution peak to experimental result, and peak 2 is that restructuring SPINK6 protein stream wears peak.Carry out Purity by SDS-PAGE to restructuring SPINK6 albumen, result as shown in Figure 2.In figure, swimming lane M is lower molecular weight standard protein (Mark), and swimming lane 1,2,3 to be recombinated the SPINK6 albumen of KLK14 affinity column purifying for people.Analyzed by this result electrophoresis analysis software bandscan5.0, result shows, and after people recombinates KLK14 affinity purification, the purity of SPINK6 albumen can reach to 95.3%.Measure the restructuring SPINK6 protein content in purge process in each step damping fluid by BCA method, as calculated, wherein containing protein content in sample-loading buffer is 1.25mg, and stream wears 0.55mg, and 0.7mg in elution buffer, protein recovery reaches 56%.The inspection of table 1SDS-PAGE electrophoresis purity.
Table 1 SDS-PAGE electrophoresis purity is checked
Claims (1)
1.KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen, and it is characterized in that, it comprises step:
Affinity media: coupling has people to recombinate the Sepharose4B gel 1.5ml of KLK14 albumen, loads 10ml volume chromatography column in advance;
Sample: people's SPINK6 protein sample of recombinating is the elution peak of pichia yeast fermented supernatant fluid after ion-exchange step purifying of the gene containing encoding human restructuring spink6 albumen, and SPINK6 protein content is 0.05mg/ml, and purity is 80%, 25ml elution peak;
Tomography devices: AKTA explorer 100 protein purification system;
Balance, cleaning buffer solution: containing the 0.05mol/L Tris-HCl pH8.0 damping fluid of 0.1mol/L NaCl;
Elution buffer: containing the 0.1mol/L Gly-HCl pH2.8 damping fluid of 0.15mol/L NaCl;
Neutralization buffer: the damping fluid of 1mol/L Tris-HCl, pH8.0;
Balance: with equilibration buffer, 1.5ml coupling being housed has people to recombinate the Sepharose4B gel chromatography column of KLK14 albumen, and post height is 2cm, balances 5 column volumes with 50cm/h flow velocity, has been balanced to baseline by ultraviolet detection guarantee chromatography column;
Loading: by 50ml sample buffer with 40cm/h by the Sepharose4B gel chromatography column through the good KLK14 albumen of equilibration buffer, during loading, sample keeps ice bath, starts to collect stream and wear peak when ultraviolet detection changes;
Cleaning: loading is complete, with cleaning buffer solution with the flow velocity of 50cm/h cleaning chromatography column, consumption is 5 times of chromatography column volume, treats ultraviolet detection A
280wearing peak to stopping collecting stream during baseline, receiving 55ml stream wears liquid altogether;
Wash-out: clean to baseline, elutes from chromatography column with the flow velocity of 20cm/h SPINK6 albumen of people being recombinated with elution buffer, starts to collect elution peak when ultraviolet detection changes, and damping fluid consumption is long-pending 4 times of chromatographic column cylinder; In elutriant collection tube, add neutralization buffer in advance, collect elution peak 2.5ml, analyze known, peak 1 is restructuring SPINK6 albumen elution peak, and peak 2 is that restructuring SPINK6 protein stream wears peak.
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| CN201110369655.2A CN103122029B (en) | 2011-11-18 | 2011-11-18 | KLK14 albumen affinitive layer purification people recombinates the method for SPINK6 albumen |
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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 |
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| 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 |
|---|
| Isolation of SPINK6 in Human Skin SELECTIVE INHIBITOR OF KALLIKREIN-RELATED PEPTIDASES;Ulf Meyer-Hoffert et al;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20100728;第285卷(第42期);摘要 * |
| 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;顾双等;《山东大学学报(医学版)》;20080216;第46卷(第2期);全文 * |
| 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》.2007,第29卷(第14期),全文. * |
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