CN101240011A - Fast large preparation method for high-purity isophycocyanin - Google Patents
Fast large preparation method for high-purity isophycocyanin Download PDFInfo
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- CN101240011A CN101240011A CNA2008100144055A CN200810014405A CN101240011A CN 101240011 A CN101240011 A CN 101240011A CN A2008100144055 A CNA2008100144055 A CN A2008100144055A CN 200810014405 A CN200810014405 A CN 200810014405A CN 101240011 A CN101240011 A CN 101240011A
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Abstract
A rapid preparation of great amount of allophycocyanin with high purity, which pertains to separation and purification of phycocyanin technical field. The invention extracts C-phycocyanin and allophycocyanin by freeze dissolving and intensified swelling with low ions, fractionally precipitate allophycocyanin by ammonia sulfate, and absorb and enrich allophycocyanin with hydroxyapatite, prepares large amount relatively purified allophycocyanin by hydroxyapatite chromatography, and finally obtains highly purified allophycocyanin by ionexchange chromatography technique. The process eliminates conventional complex separating and purifying process combining molecular sieve chromatography with hydroxyapatite chromatography with three steps in total, solves the problem of extensive and rapid preparation. The process also dramatically lower preparation cost of CPC and APC, thus lays a foundation for CPC and APC application in ultrasensitive detection in biomedical.
Description
Technical field
The present invention relates to a kind of method for preparing the high purity Allophyxoxyanin fast in a large number, belong to the protein separation technical field.
Background technology
For cellular localization, interaction and the dynamic change thereof of studying protein and other, the researchist is badly in need of new technology and novel material and is realized " sign ", " reading " and " inquiry " to protein and other.Fluorescent mark now commonly used because the restriction of luminescent dye molecule fluorescent characteristic (fluorescence spectrum broad, quantum yield low), can not be applicable to the single-minded sign of high-throughout biomacromolecule far away.
Phycobiliprotein is that a class photosynthesis is caught photopigment-protein complex, mainly is present in blue-green algae, red algae, latent algae and the minority dinoflagellate.In photosynthesis, play a part to catch and transmit luminous energy, have intensive fluorescence.In blue-green algae and red algae, phycobiliprotein and a small amount of colourless albumen that is connected are formed supramolecular complex---phycobilisome, absorb efficiently and transmit luminous energy.At the mid-80, the scholar of American Studies algae photosynthesis proposition as fluorescent marker, is used for diagnostic reagent with phycobiliprotein.Because its unique advantage, the diagnostic reagent of phycobiliprotein and phycobiliprotein mark enters the world market in the early 1990s.
Compare with fluorescent marker commonly used, phycobiliprotein has following advantage: production process safety, nontoxic, and luminous energy absorbs strong, the fluorescent yield height surpasses 90%, and bias light interference and false positive rate are low, stable in the scope of pH4-11, can make double-colored, three looks and four color markers.So the range of application of this class reagent constantly enlarges.But,, be not applied to popular reagent for clinical diagnosis as yet owing to cost an arm and a leg.The whole imports of China also only limit to the diagnosis of carrying out with cell streaming instrument.
Phycobiliprotein and diagnostic reagent thereof are mainly by produced in USA, and Germany also has product to promote to China, and develop in Britain and China Taiwan.That develop product the earliest is (the Molecular Probes of U.S. molecular probe company, Inc.), now Sigma company has 6 kinds of phycobiliprotein products, 12 kinds of phycobiliprotein-Biotin/Avidin marker, RPE-IgG or RPE-IgM12 kind, 3 kinds of RPE monoclonal antibody markers.Disclosed laboratory method is continued to use in phycobiliprotein production.The kind of phycobiliprotein comprises Allophyxoxyanin (APC), Phycocyanins, C-(PC), phycoerythrocyanin (pec) (PEC) and phycoerythrin (PE) four big classes.The APC that wherein is present in the blue-green algae is one of phycobiliprotein fluorescent probe commonly used at present, because APC brightness is big, stoke shift is big.Blue-green algaes such as China's spirulina have begun the industrialization cultivation, and resource is very abundant, is the good material of separation and purification APC.What relation was purchased, sold in decision on the world market mainly is price factor.The principal element that influences the development and application of popular diagnostic reagent also is that the phycobiliprotein price is too high.Therefore, fast a large amount of separating and purifying technologies of exploitation APC are realized low-cost a large amount of preparations of high purity APC, have great importance in the application of popular diagnostic reagent for APC.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method for preparing the high purity Allophyxoxyanin fast in a large number is provided.
The existing relatively method of the present invention only needs just can obtain highly purified Allophyxoxyanin solution in a large number through two step column chromatographies.
A kind of method for preparing the high purity Allophyxoxyanin fast in a large number, step is as follows:
(1) extraction of Allophyxoxyanin
Blue-green algae is dissolved with the method disintegration of freezing molten and low ionic strength swelling, and supernatant liquor is got in centrifugation, promptly gets the Allophyxoxyanin slurries.
(2) preliminary purification of Allophyxoxyanin
Use ammonium sulfate precipitation method, Allophyxoxyanin is precipitated out from the Allophyxoxyanin slurries, centrifugal collecting precipitation is used 20mM phosphoric acid buffer (pH6.8～7.0) dissolving and dialysis again, removes ammonium sulfate, gets the Allophyxoxyanin crude extract.
(3) hydroxyapatite chromatography extracts Allophyxoxyanin
Use hydroxyapatite adsorption and enrichment Allophyxoxyanin in the Allophyxoxyanin crude extract, carry out wash-out with phosphoric acid buffer then, get Allophyxoxyanin solution.This step can realize that a large amount of of low abundance Allophyxoxyanin concentrate and enrichment.
(4) ion-exchange prepares the high purity Allophyxoxyanin
Keep stable properties according to Allophyxoxyanin in the pH of broad scope, utilize ion-exchange chromatography, apparatus has the damping fluid of constant ionic strength, continuous pH gradient to carry out wash-out, promptly can obtain highly purified Allophyxoxyanin.
Blue-green algae in the described step (1) is a spirulina.
The concrete operations step of the extraction of described step (1) Allophyxoxyanin is: with the blue-green algae frond by weight volume ratio 1: 1 (g/ml or kg/l) add 0.02mol/L phosphoric acid buffer (pH6.8～7.0), freezing down at-20 ℃, under 20～25 ℃, melt 2～3h then.Behind multigelation 2-4 time, the blue-green algae suspension under 4 ℃, the centrifugal 10min～15min of 8000rpm～10000rpm, supernatant liquor is the Allophyxoxyanin slurries.
The concrete operations step of the preliminary purification of described step (2) Allophyxoxyanin is: in the Allophyxoxyanin slurries, add solid ammonium sulfate, to concentration be 30% (w/v), place 4～5h, then under 4 ℃, centrifugal 10min～the 15min of 10000rpm～11000rpm gets supernatant liquor, adds solid ammonium sulfate in supernatant liquor, to ultimate density be 60% (W/V), place more than the 4h, then under 4 ℃, the centrifugal 10min～15min of 10000rpm～11000rpm, abandon supernatant, get precipitation; Precipitation is dissolved in the phosphoric acid buffer (pH6.8～7.0) of 20mM, uses the phosphoric acid buffer (pH6.8～7.0) of 20mM to dialyse then, collect dialyzate, promptly get the Allophyxoxyanin crude extract.The ratio of above-mentioned precipitation and phosphoric acid buffer does not have strict the qualification, as long as precipitation can be dissolved fully.
The concrete operations of described step (3) hydroxyapatite chromatography extraction Allophyxoxyanin are as follows: the hydroxyapatite that will cross with 20mM phosphoric acid buffer (pH6.8～7.0) balance in advance, add in the Allophyxoxyanin crude extract, the hydroxyapatite of the full Allophyxoxyanin of absorption is taken out from solution, use 20mM phosphoric acid buffer (pH6.8～7.0) flushing hydroxyapatite, C-Phycocyanins, C-remaining on the hydroxyapatite is rinsed out, damping fluid with ionic homeostasis intensity carries out wash-out again, Allophyxoxyanin is eluted, promptly get Allophyxoxyanin solution.Because the difference of adsorptive power, Allophyxoxyanin has been adsorbed onto on the hydroxyapatite, and the C-Phycocyanins, C-does not adsorb, and still remains in the solution.
Preferably, the damping fluid of described ionic homeostasis intensity is 100mM phosphoric acid buffer (pH6.8～7.0).
It is as follows that described step (4) ion-exchange prepares the concrete operations of high purity Allophyxoxyanin: get the Allophyxoxyanin solution that step (3) makes, at 20mmol/L acetate buffer solution (pH5.0, contain 0.05mol/L NaCl) middle dialysis, get the solution after the dialysis then, on sepharose FF (DEAE Sepharose Fast Flow) ion-exchange chromatography, with the 20mmol/L acetate buffer solution (pH5.0 that contains 0.05mol/L NaCl,) wash-out, use 20mmol/L acetate buffer solution (pH5.0 at last, contain 0.05mol/L NaCl) and 20mmol/L acetate buffer solution (pH3.6, containing 0.05mol/L NaCl) each 100mL carries out gradient elution, elution speed is 60～70mL/h, detect wavelength 280nm, collect the cyan liquid of wash-out, obtain the Allophyxoxyanin of purifying.
Preferably, described DEAE Sepharose Fast Flow ion-exchange chromatography is that specification is 0.6 * 10cm through 20mmol/L acetate buffer solution (pH5.0 contains 0.05mol/L NaCl) pre-balance.
Above operation steps is this area routine operation if no special instructions.
Detect the purifying Allophyxoxyanin A that obtains through absorption spectrum
650/ A
280Reach 5.0 (Fig. 1).It is generally acknowledged A
650/ A
280Reach more than 4.5, just think that Allophyxoxyanin is purified, therefore, the Allophyxoxyanin that this ion exchange chromatography obtains is high purifying.The Native-PAGE electrophoresis detection has only a band, shows that the APC of separation and purification does not have other proteic pollution (Fig. 3).
The method of prior art for preparing Allophyxoxyanin be with the cytoclasis crude extract through a hydroxyapatite column, use Sephadex G-100 gel permeation chromatography again, and then experience the hydroxyapatite column chromatography one time.The Allophyxoxyanin purity that obtains in the correlation technique can reach 5.0.Compare with the three steps operation of correlation technique, the present invention be with crude extract through a hydroxyapatite chromatography, experience the primary ions displacement chromatography again, only need the operation of two steps, just can obtain a large amount of high-purity Allophyxoxyanin solution goods, and effectively reduce cost.
During the prior art for preparing Allophyxoxyanin, this step of employed hydroxyapatite chromatography is in advance hydroxyapatite to be contained in the chromatography column, then crude extract is gone up column chromatography.Because hydroxyapatite column has the slow-footed characteristics of chromatography, the process of last sample expends time in very much, and this step is the very number time taking step in the Allophyxoxyanin leaching process.The present invention is directed to this deficiencies in the prior art, the method of invention is: the hydroxyapatite that will cross with 20mM phosphoric acid buffer (pH6.8～7.0) balance in advance, adding damping fluid is similarly in the Allophyxoxyanin crude extract of 20mM phosphoric acid buffer (pH6.8～7.0), at this moment, because under this specific ionic strength of 20mM phosphoric acid buffer (pH6.8～7.0) and pH value condition, hydroxyapatite is different to the adsorptive power of C-Phycocyanins, C-and Allophyxoxyanin, therefore Allophyxoxyanin can be adsorbed onto on the hydroxyapatite, and the C-Phycocyanins, C-does not adsorb, and still remains in the solution.The hydroxyapatite of the full Allophyxoxyanin of absorption is taken out from solution, use 20mM phosphoric acid buffer (pH6.8～7.0) flushing hydroxyapatite, C-Phycocyanins, C-remaining on the hydroxyapatite is rinsed out.Again these hydroxyapatites are installed in the chromatography column, carry out wash-out, Allophyxoxyanin is eluted with the damping fluid of ionic homeostasis intensity, just can purer in a large number Allophyxoxyanin.Compared with prior art, the present invention when carrying out hydroxyapatite chromatography, changed of the prior art on this step of sample, but under specific ionic strength and specific pH condition, use hydroxyapatite in crude extract, Allophyxoxyanin to be carried out selective adsorption, thereby reach the purpose of absorption and enrichment Allophyxoxyanin.The present invention can not only significantly save time this step of hydroxyapatite chromatography, but also can improve the service efficiency of hydroxyapatite.
Excellent results of the present invention also is, with the blue-green algae is material, through freezing molten and low ionic strength swelling rapid extraction Allophyxoxyanin (APC), after ammonium sulfate precipitation, use the hydroxyapatite chromatography method to carry out preliminary purification, we can keep the characteristics of stable properties in the pH of broad scope according to Allophyxoxyanin (APC) at last, utilize DEAESepharose Fast Flow ion-exchange chromatography, damping fluid with constant ionic strength, pH gradient carries out wash-out, can obtain highly purified Allophyxoxyanin (APC).
Compare in conjunction with the complex separations purifying procedure of hydroxyapatite chromatography with traditional application sieve chromatography; present method has overcome a difficult problem that is difficult to a large amount of fast preparation Allophyxoxyanins of mass-producing; easy and simple to handle; time saving and energy saving; simple to equipment requirements, productive rate height, the easy preparation cost for preparing and greatly reduce APC in a large number; thereby lay a good foundation for APC being applied to overdelicate biomedical the detection, have good application prospects and economic benefit.
Description of drawings
The absorption spectrum of the Allophyxoxyanin of Fig. 1 separation and purification.Maximum absorption band is 650nm, at the 620nm place one acromion is arranged.A
650/ A
280Reached 5.0, shown highly purified.
The fluorescence emission spectrum of the Allophyxoxyanin of Fig. 2 separation and purification.Excite with 650nm, fluorescence emission peak is positioned at 661nm, and is identical with the standard fluorescence emission peak.
The Native-PAGE electrophoretogram of the spirulina Allophyxoxyanin of Fig. 3 separation and purification.In Native-PAGE, have only a band, the Allophyxoxyanin that further specifies separation and purification does not have other proteic pollution.
Embodiment
Embodiment 1:
A kind of method for preparing the high purity Allophyxoxyanin fast in a large number, step is as follows:
(1) raw material is a spirulina.The cultivation after-filtration is collected ,-20 ℃ of freezing preservations.
(2) extraction of Allophyxoxyanin: get the spirulina frond 10g of freezing preservation, the spirulina frond is added 0.02mol/L phosphoric acid buffer (pH6.8～7.0) 10ml, freezing under-20 ℃, under 20 ℃, melt 2.5h then.Behind the multigelation 3 times, the spirulina suspension under 4 ℃, the centrifugal 15min of 10000rpm, supernatant liquor is the Allophyxoxyanin slurries.
(3) preliminary purification of Allophyxoxyanin: in the Allophyxoxyanin slurries that above-mentioned steps (2) obtains, add solid ammonium sulfate, to concentration be 30% (w/v), place 4h, under 4 ℃, the centrifugal 15min of 10000rpm discards precipitation then, collects the solution part.Continue in solution, to add solid ammonium sulfate again, to final concentration be 60% (W/V), place more than the 4h, then under 4 ℃, the centrifugal 15min of 10000rpm, collecting precipitation part.Precipitation is dissolved in the phosphoric acid buffer (pH6.98) of 20mM, then the phosphoric acid buffer (pH6.98) of 20mM is dialysed, collect dialyzate, i.e. Allophyxoxyanin crude extract.
(4) hydroxyapatite chromatography extracts Allophyxoxyanin: the hydroxyapatite that will cross with 20mM phosphoric acid buffer (pH6.98) balance in advance, adding damping fluid is in the Allophyxoxyanin crude extract of 20mM phosphoric acid buffer (pH6.98), adsorbs.The hydroxyapatite of the full Allophyxoxyanin of absorption is taken out from solution, use 20mM phosphoric acid buffer (pH6.98) flushing hydroxyapatite, C-Phycocyanins, C-remaining on the hydroxyapatite is rinsed out.Use 100mM phosphoric acid buffer (pH6.98) to carry out wash-out again, Allophyxoxyanin is eluted, promptly get Allophyxoxyanin solution.
(5) ion exchange method prepares the high purity Allophyxoxyanin: the Allophyxoxyanin solution of step (4) is dialysed in 20mmol/L acetate buffer solution (pH5.0), dialysis is got DEAE Sepharose Fast Flow ion-exchange chromatography on the dialyzate after finishing, this ion-exchange chromatography is through 20mmol/L acetate buffer solution (pH5.0, contain 0.05mol/L NaCl) pre-balance, specification is 0.6 * 10cm, then with 20mmol/L acetate buffer solution (pH5.0) wash-out that contains 0.05mol/L NaCl, use 20mmol/L acetate buffer solution (pH5.0 at last, contain 0.05mol/L NaCl) and 20mmol/L acetate buffer solution (pH3.6, containing 0.05mol/LNaCl) each 100mL carries out gradient elution, elution speed is 60～70mL/h, collect blue liquid, obtain highly purified Allophyxoxyanin.
Claims (8)
1. methods that prepare fast in a large number the high purity Allophyxoxyanin, step is as follows:
(1) extraction of Allophyxoxyanin
Blue-green algae is dissolved with the method disintegration of freezing molten and low ionic strength swelling, and supernatant liquor is got in centrifugation, promptly gets the Allophyxoxyanin slurries;
(2) preliminary purification of Allophyxoxyanin
Use ammonium sulfate precipitation method, Allophyxoxyanin is precipitated out from the Allophyxoxyanin slurries, centrifugal collecting precipitation is used the 20mM phosphoric acid buffer of pH6.8～7.0 again, and dissolving is dialysis also, removes ammonium sulfate, the Allophyxoxyanin crude extract;
(3) hydroxyapatite chromatography extracts Allophyxoxyanin
Use hydroxyapatite adsorption and enrichment Allophyxoxyanin in the Allophyxoxyanin crude extract, carry out wash-out with phosphoric acid buffer then, get Allophyxoxyanin solution;
(4) ion-exchange prepares the high purity Allophyxoxyanin.
2. the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 1 is characterized in that the blue-green algae in the described step (1) is a spirulina.
3. the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 1, it is characterized in that, the concrete operations step of the extraction of described step (1) Allophyxoxyanin is: the 0.02mol/L phosphoric acid buffer that the blue-green algae frond is added pH6.8～7.0, freezing down at-20 ℃, under 20～25 ℃, melt 2～3h then; Behind multigelation 2-4 time, the blue-green algae suspension under 4 ℃, the centrifugal 10min～15min of 8000rpm～10000rpm, supernatant liquor is the Allophyxoxyanin slurries.
4. the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 1, it is characterized in that, the concrete operations step of the preliminary purification of described step (2) Allophyxoxyanin is: add solid ammonium sulfate in the Allophyxoxyanin slurries, to concentration be 30%, be weightmeasurement ratio, place 4～5h, then under 4 ℃, centrifugal 10min～the 15min of 10000rpm～11000rpm, get supernatant liquor, in supernatant liquor, add solid ammonium sulfate, to ultimate density be 60%, be weightmeasurement ratio, place more than the 4h, then under 4 ℃, the centrifugal 10min～15min of 10000rpm～11000rpm, abandon supernatant, get precipitation; Precipitation is dissolved in the phosphoric acid buffer of 20mM of pH6.8～7.0, dialyses with the phosphoric acid buffer of the 20mM of pH6.8～7.0 then, collect dialyzate, promptly get the Allophyxoxyanin crude extract.
5, the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 1, it is characterized in that, the concrete operations of described step (3) hydroxyapatite chromatography extraction Allophyxoxyanin are as follows: the hydroxyapatite that will cross with the 20mM phosphoric acid buffer balance of pH6.8～7.0 in advance, add in the Allophyxoxyanin crude extract, the hydroxyapatite of the full Allophyxoxyanin of absorption is taken out from solution, use the 20mM phosphoric acid buffer flushing hydroxyapatite of pH6.8～7.0, C-Phycocyanins, C-remaining on the hydroxyapatite is rinsed out, damping fluid with ionic homeostasis intensity carries out wash-out again, Allophyxoxyanin is eluted, promptly get Allophyxoxyanin solution.
6, the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 5 is characterized in that the damping fluid of described ionic homeostasis intensity is the 100mM phosphoric acid buffer of pH6.8～7.0.
7, the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 1, it is characterized in that, it is as follows that described step (4) ion-exchange prepares the concrete operations of high purity Allophyxoxyanin: get the Allophyxoxyanin solution that step (3) makes, at pH5.0, contain in the 20mmol/L acetate buffer solution of 0.05mol/L NaCl and dialyse, get the solution after the dialysis then, on DEAE Sepharose Fast Flow ion-exchange chromatography, use pH5.0, the 20mmol/L acetate buffer solution wash-out that contains 0.05mol/L NaCl, use pH5.0 again, the 20mmol/L acetate buffer solution and the pH3.6 that contain 0.05mol/L NaCl, each 100mL of 20mmol/L acetate buffer solution that contains 0.05mol/L NaCl carries out gradient elution, elution speed is 60～70mL/h, detect wavelength 280nm, collect the cyan liquid of wash-out, obtain the Allophyxoxyanin of purifying.
8, the method for preparing the high purity Allophyxoxyanin fast in a large number as claimed in claim 7, it is characterized in that, described DEAE Sepharose Fast Flow ion-exchange chromatography is through pH5.0, contains the 20mmol/L acetate buffer solution pre-balance of 0.05mol/L NaCl, and specification is 0.6 * 10cm.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2451567A (en) * | 2007-07-26 | 2009-02-04 | Hoya Corp | Separation of phycobilin-based pigments using an adsorbent, the surface of which comprises a calcium phosphate-based compound, and phosphate elution buffers |
| CN101891809A (en) * | 2010-06-18 | 2010-11-24 | 中国科学院海洋研究所 | Preparation and application of phycocyanin extract |
| CN106963785A (en) * | 2017-04-20 | 2017-07-21 | 广州市天河华南发展有限公司 | A kind of spirulina extract and its preparation method and application |
| CN107858334A (en) * | 2017-11-23 | 2018-03-30 | 浙江海洋大学 | One kind extraction and purification Catechol 2, method of 3 dioxygenases from microalgae |
| CN115666610A (en) * | 2020-05-19 | 2023-01-31 | 瓦克萨科技有限公司 | Photosynthetic controlled spirulina extracts for the treatment of cytokine storm syndrome |
| CN116349890A (en) * | 2015-09-25 | 2023-06-30 | 费尔曼塔格公司 | Acidic compositions comprising phycocyanin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1268516A (en) * | 1999-03-26 | 2000-10-04 | 中国科学院水生生物研究所 | Preparation method of phycocyanin |
-
2008
- 2008-02-28 CN CN2008100144055A patent/CN101240011B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2451567A (en) * | 2007-07-26 | 2009-02-04 | Hoya Corp | Separation of phycobilin-based pigments using an adsorbent, the surface of which comprises a calcium phosphate-based compound, and phosphate elution buffers |
| CN101891809A (en) * | 2010-06-18 | 2010-11-24 | 中国科学院海洋研究所 | Preparation and application of phycocyanin extract |
| CN116349890A (en) * | 2015-09-25 | 2023-06-30 | 费尔曼塔格公司 | Acidic compositions comprising phycocyanin |
| CN106963785A (en) * | 2017-04-20 | 2017-07-21 | 广州市天河华南发展有限公司 | A kind of spirulina extract and its preparation method and application |
| CN107858334A (en) * | 2017-11-23 | 2018-03-30 | 浙江海洋大学 | One kind extraction and purification Catechol 2, method of 3 dioxygenases from microalgae |
| CN115666610A (en) * | 2020-05-19 | 2023-01-31 | 瓦克萨科技有限公司 | Photosynthetic controlled spirulina extracts for the treatment of cytokine storm syndrome |
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