CN1280134A - Technology for preparing soildifying metal ion-affine chromatograply glue - Google Patents
Technology for preparing soildifying metal ion-affine chromatograply glue Download PDFInfo
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- CN1280134A CN1280134A CN 00112143 CN00112143A CN1280134A CN 1280134 A CN1280134 A CN 1280134A CN 00112143 CN00112143 CN 00112143 CN 00112143 A CN00112143 A CN 00112143A CN 1280134 A CN1280134 A CN 1280134A
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Abstract
By using Sepharose 6B and imido-diacetic acid (IDA) as main raw materials and through activation, grafting, fixation and other reaction, Ni2+-affine chromatographic glue is synthesized, and the has high effect of purifying protein with His-tag label. Test in physicochemical index and protein separating function shows that the said metal ion-affine chromatographic glue is identical with available similar product, but it has much lower (about 1/10) cost.
Description
The present invention relates to genetically engineered downstream purified technology of protein.
The immobilized metal ion affinity chromatography technology is that a kind of separation and purification has the His-tag method of protein, thereby this glue can make protein be adsorbed on the glue securely by metal ion with the protein chelating that has purifying mark His-tag specifically, other impurity albumen since do not have above-mentioned purifying mark then can not with metal ion-chelant, also just can not be adsorbed onto on the glue; Use Washing Buffer (Washing Buffer (8x): 480mM imidazoles, 4M NaCl, 160mM Tris then; With dense HCl adjust pH is 7.9) will rinse out less than the impurity albumen of absorption; Use Eluting Buffer (Elute Buffer (4x): 4M imidazoles, 2M NaCl, 80mM Tris again; With dense HCl adjust pH is 7.9) needed target protein is eluted on chromatograph.Like this, just can obtain only to contain the elutriant of target protein matter, with this elutriant dialysis desalting, concentrated, lyophilize just can obtain the very high target protein matter of purity, and proteinic biological activity is unaffected again.Along with progressively finishing of " Human Genome Project ", " proteomic program " is about to be in full swing, and must use genetic engineering means to prepare various and human health proteins associated matter in " proteomic program " in a large number.And in genetically engineered, except clone, expression, the problem that is right after is exactly the target protein matter how purifying is expressed, and this is very important integral part in the downstream techn of gene engineering.Immobilized metal ion affinity chromatography glue just can satisfy these requirements, and it can be used for specific adsorption and have the protein of His-tag purifying mark and the target protein matter by expressing in the affinitive layer purification genetically engineered.Therefore, this affinity chromatography glue application prospect in life science field in the future and bio-pharmaceuticals is very wide.
At present, existing abroad a few the biological major company (as QIAGEN company, Amersham Pharmacia company etc.) of the technology of preparing of immobilized metal ion affinity chromatography glue works out and is producing relevant product.The related main raw material of these company's products also has iminodiacetic acid sodium (IDA-Na except carrier glue
2) or methyne nitrilotriacetic (NTA) etc., these raw materials are rare and price is very expensive.The technology of preparing route and the used other fees of adding external these major companies are also higher, so production cost is also just very high, price very expensive (general every 50ml price is 3000-4000 yuan).
Purpose of the present invention is exactly the technology of preparing that a kind of immobilized metal ion affinity chromatography glue will be provided, both easy to operate, process is simple, reliable results, cost is low again, cost is little, can prepare the related products that all is better than or is equal to foreign biomolecule major company at the aspects such as function of physical and chemical index, protein purification.This technology of preparing is initiated for us, with offshore company a great difference is arranged, as in the main preparation raw material of this glue, do not resemble and use carrier [agarose (Agrose) or Sepharose4B or Sepharose6B] and Iminodiacetic acid sodium salt (IDA-Na the external major company
2) or methyne nitrilotriacetic (NTA) synthesize Affi-Gel; But directly use iminodiethanoic acid (IDA) as starting raw material and the synthetic Affi-Gel of carrier (Sepharose4B or 6B) reaction.In addition, (as temperature, pH value, reaction times etc.) on some important reaction conditions also with above-mentioned biological major company and pertinent data on described differing widely.The cost of the glue of special the present invention's preparation is than low many of the cost of the glue of introducing preparation on foreign biomolecule major company and the pertinent data; By our forecasting of cost, only be that every 50ml is about 300-500 unit with the prediction price of the affinity chromatography glue of this technology of preparing production.
In sum, present technique is compared with present external (domestic still do not have) existing technology, mainly contains following advantage:
1, production cost reduces greatly.As starting raw material, IDA compares IDA-Na with IDA for we
2Market price much lower (approximately being 1: 20) and be easy to get very much (domestic just have production); And IDA-Na
2Must be from external import, and price is very high.The present invention does initial raw material with Sepharose in addition, and is cheaply more many than the used agarose of offshore company (Agrose); Two aspects integrate accounting, and the cost of estimating this research is less than 1/10th of the production cost of offshore company.
2, preparation process is easy, and general technician one learns promptly can.
3, the proteic function of the physical and chemical index of finished product glue and separation and purification is fine.We adopt atomic absorption method that the affinity chromatography glue of the present invention's preparation has been measured chelating Ni
2+Amount, and compare with the product and the pertinent data results reported of external major company.The result shows that the physical and chemical index of the glue of the present invention's preparation is all more much higher than their index.In addition, we have specially designed a kind of protein of the His-tag of having mark (human B lymphocyte stimulating factor hBLyS), have been used for detecting the effect of separating purification of this synthetical glue; The result also shows, the metal ion Ni of the present invention's preparation
2+Affinity chromatography glue has in separation and purification aspect the protein of His-tag mark, and effect is very gratifying.
The objective of the invention is to realize by the following technical solutions:
The overall design and the reaction process of this technology of preparing are as follows:
Preparation process of the present invention is from iminodiethanoic acid and corresponding carrier, and this is the new technology that at home and abroad there is no at present.
Concrete preparation process:
1, carrier (Sepharose6B or Sepharose4B) being drained with the G-3 funnel, taken by weighing 50g and put into an Erlenmeyer flask, add CNBr or epoxy chloropropane (1-2 times of carrier colloid is long-pending), is 6.5-12 with the NaOH adjust pH, adds 0.0375gNaBH again
4At 22 ℃-60C stirring reaction after 1-2 hour, add 10ml 0.2M NaOH and 5mlCNBr or epoxy chloropropane, stirred 5-12 hour in the gap, glue is fully activated and the molecule in the glue particle on-the OH group connects epoxypropyl;
2, the carrier glue that activation is good is drained on the G-3 funnel, puts back in the Erlenmeyer flask again, adds IDA, at pH is (to use 0.2M Na under the condition of 6.5-12
2CO
3Solution is transferred pH) and arm IDA (iminodiethanoic acid) stirring reaction, temperature of reaction is 30 ℃-50 ℃; Reacted 2-6 hour;
3, the glue that has reacted is drained with the G-3 funnel;
4, the glue of draining returns back in the triangular flask, with the NiSO of the 1M of 2-5 times of volume
4Solution reacts with it; Reaction conditions is 30 ℃-50 ℃, stirring reaction 3-5 hour;
5, combine Ni
2+Glue on the G-3 funnel, fully drain, with rare HAc drip washing 2-3 time, use distilled water drip washing 2-3 time again, drain;
6, be stored in 4 ℃ of refrigerators with 20% ethanol; So just prepared immobilization Ni
2+Affinity chromatography glue; Take out portion of product and do the analysis experiment.
The evaluation that the present invention is prepared product:
1, atomic absorption detects:
Take out Affi-Gel (draining) 2g of foregoing invention preparation, with containing the solution 20ml of EDTA with all Ni on the 2g glue
2+Chelating gets off to enter solution, measures on atomic absorption instrument with calibration curve method then.
Result: get the Ni in the above-mentioned 2g of the containing glue
2+EDTA solution 0.5ml ddH
2After O is diluted to 250ml, record wherein [Ni with atomic absorption method
2+]=0.0168mmol/L.
Calculate: every gram is drained the Ni of chelating in the glue
2+Be 4.93mg or 84 μ mol; Every milliliter of Ni that precipitates chelating in the glue
2+Be 4.11mg or 70 μ mol.
From the said determination result, than the numeral 15.7 μ mol of Ni of pertinent data report
2+/ ml of gel
[1]And 8-12 μ mol Ni
2+/ ml glue
[2](QIAGEN company) is all high.Wherein, the calculation result of laboratory sample is than 8-12 μ mol Ni
2+/ ml of gel exceeds 5.83-8.75 doubly; Than 15.7 μ mol of Ni
2+/ mlof gel exceeds nearly 4.46 times.
2, the proteinic effect detection of separation and purification:
With reference to relevant document
[3], get the Ni of this research of 2ml preparation
2+Affinity chromatography glue is made affinity column, with Binding Buffer balance good after, other takes, and sample adds pillar on the total protein supernatant liquor that contains target protein matter hBLyS that e. coli strain bl21 expresses, rinse out non-adsorbable impurity albumen with Washing Buffer again, use elutriant wash-out target protein at last.The wash-out image of computer record is single sharp peak.
As seen, the purity of wash-out target protein matter hBLyS and glue all are good (washing fluid do not contain target protein hBLyS through electrophoresis detection) to the adsorption effect of target protein hBLyS.In addition, elutriant when directly carrying out electrophoresis detection, is also only seen a strap without dialysis, concentrated on the gel, and this illustrates that also the proteinic effect of this glue separation and purification is extraordinary.
Reference:
1、?Michele?C.?Smith,Thomas?C.?Furman,Charles?Pidgeon.Immobilized?IminodiaceticAcid?Metal?Peptide?Complexes.Identification?of?Chelating?Peptide?Purification?Handlesfor?Recombinant?Proteins.Inorg.Chem.1987,26,1965～1969
2, The QIAexpressionist (QIAGEN), 2nd Edition, 1992 (because are a brochure, so can only enclose relevant one page).
3, pET System Manual (Novagen), 7th Edition, 1997 (annotating :) because be a brochure, so do not enclose copy.
Claims (1)
1. the technology of preparing of an immobilized metal ion affinity chromatography glue, it is characterized in that carrier (Sepharose6B or Sepharose4B) is drained with the G-3 funnel, put into an Erlenmeyer flask, add CNBr or epoxy chloropropane, with the NaOH adjust pH is 6.5-12, adds NaBH again
4, at 22 ℃-60 ℃ after stirring reaction 1-2 hour, adding NaOH and CNBr or epoxy chloropropane, stirred 5-12 hour in the gap, and glue is fully activated; The carrier glue that activation is good is drained on the G-3 funnel, puts back in the Erlenmeyer flask again, adds IDA, at pH is (to use 0.2M Na under the condition of 6.5-12
2CO
3Solution is transferred pH) and arm IDA (iminodiethanoic acid) stirring reaction, temperature of reaction is 30 ℃-50 ℃, reacts 2-6 hour, and the glue that has reacted is drained with the G-3 funnel, and the glue of draining returns back in the triangular flask, with the NiSO of 2-5 times of volume
4Solution reacts with it; Reaction conditions is 30 ℃-50 ℃, stirring reaction 3-5 hour; Glue is fully drained on the G-3 funnel,, used distilled water drip washing 2-3 time again, drain with rare HAc drip washing 2-3 time; Ethanol with 20% is stored in 4 ℃ of refrigerators.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB00112143XA CN1160367C (en) | 2000-03-16 | 2000-03-16 | Technology for preparing soildifying metal ion-affine chromatograply glue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB00112143XA CN1160367C (en) | 2000-03-16 | 2000-03-16 | Technology for preparing soildifying metal ion-affine chromatograply glue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1280134A true CN1280134A (en) | 2001-01-17 |
| CN1160367C CN1160367C (en) | 2004-08-04 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB00112143XA Expired - Fee Related CN1160367C (en) | 2000-03-16 | 2000-03-16 | Technology for preparing soildifying metal ion-affine chromatograply glue |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389325C (en) * | 2003-02-25 | 2008-05-21 | 上海实业科华生物技术有限公司 | Preparation method of metal chelation chromatography stuffing |
| CN106749499A (en) * | 2016-11-29 | 2017-05-31 | 生工生物工程（上海）股份有限公司 | A kind of preparation method of carrying capacity Ni IDA Ago-Gels high |
| CN109675535A (en) * | 2018-12-18 | 2019-04-26 | 杨小丽 | Immobilization zinc ion affinity chromatographic material and its preparation method and application |
| CN112156499A (en) * | 2020-09-22 | 2021-01-01 | 上海碧云天生物技术有限公司 | Metal chelating chromatography filler and preparation method thereof |
-
2000
- 2000-03-16 CN CNB00112143XA patent/CN1160367C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389325C (en) * | 2003-02-25 | 2008-05-21 | 上海实业科华生物技术有限公司 | Preparation method of metal chelation chromatography stuffing |
| CN106749499A (en) * | 2016-11-29 | 2017-05-31 | 生工生物工程（上海）股份有限公司 | A kind of preparation method of carrying capacity Ni IDA Ago-Gels high |
| CN109675535A (en) * | 2018-12-18 | 2019-04-26 | 杨小丽 | Immobilization zinc ion affinity chromatographic material and its preparation method and application |
| CN109675535B (en) * | 2018-12-18 | 2021-10-22 | 杨小丽 | Immobilized zinc ion affinity chromatography material and preparation method and application thereof |
| CN112156499A (en) * | 2020-09-22 | 2021-01-01 | 上海碧云天生物技术有限公司 | Metal chelating chromatography filler and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN1160367C (en) | 2004-08-04 |
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