CN1271085C - Rapid separating purification of R-phycoerythrin - Google Patents
Rapid separating purification of R-phycoerythrin Download PDFInfo
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- CN1271085C CN1271085C CN 200510042028 CN200510042028A CN1271085C CN 1271085 C CN1271085 C CN 1271085C CN 200510042028 CN200510042028 CN 200510042028 CN 200510042028 A CN200510042028 A CN 200510042028A CN 1271085 C CN1271085 C CN 1271085C
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Abstract
The present invention relates to a rapid separation and purification method for R-phycoerythrin, which belongs to the technical field of the separation and the purification of ocean red alga. Ocean red algae are used as materials of the present invention, and R-phycoerythrin is rapidly extracted by using frozen dissolution and low ionic-strength expansion. Initial purification is then carried out by ammonium sulfate precipitation, and the R-phycoerythrin is finally eluted by a buffer solution with constant ionic strength and pH gradients according to the characteristic of stability maintenance in a wider pH range of the R-phycoerythrin by using DEAE Sepharose Fast Flow ion exchange chromatography technology The R-phycoerythrin with high purity can be obtained by a one-step method. The method eliminates complicated separation and purification procedures of traditional sieve chromatography combined with ion exchange chromatography of ionic strength elution, and overcomes the problem of difficult scale rapid preparation. The present invention has the advantages of easy operation, high yield, easy large-scale preparation and lower RPE preparation cost; consequently, the present invention establishes a basis for the application of RPE to supersensitive biomedicine detection.
Description
(1) technical field
The present invention relates to a kind of rapid separation and purification method of R-phycoerythrin, belong to marine red alga separating and purifying technology field.
(2) 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, form the supramolecular structure phycobilisome by 2-3 kind phycobiliprotein, form transmission ofenergy sequence efficiently.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.), at present Sigma (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, wherein, Phycocyanins, C-and phycoerythrin are according to different CPC, RPC, CPE, b-PE, BPE and the RPE (R-phycoerythrin) of being divided into again of its spectral response curve.The RPE that wherein is present in the large-scale red algae in ocean is present the most frequently used phycobiliprotein fluorescent probe.Because RPE pigment group is many, brightness is big, and stoke shift is big.The resource of the coastal large-scale red algae of China is very abundant, and wherein some is the good material of separation and purification R-phycoerythrin (RPE).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, the rapidly and efficiently separating and purifying technology of exploitation RPE realizes low-cost a large amount of preparations of high purity RPE, has great importance in the application of popular diagnostic reagent for RPE.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of rapid separation and purification method of R-phycoerythrin is provided.
The rapidly and efficiently separating and purifying method of R-phycoerythrin of the present invention, step is as follows:
(1) raw material is a marine red alga, is selected from multitube algae or Ceramium kondoi.
(2) extraction of R-phycoerythrin is adopted and is frozen molten and method low ionic strength swelling, frond is disintegrated dissolve, and phycoerythrin discharges.
The red algae frond 10～12g of freezing preservation added 0.02/L phosphoric acid buffer (pH6.8～7.0) in 1: 1 by volume, under 20～25 ℃, melt 2～3h, filtered through gauze, filtrate is under 4 ℃, centrifugal 10min～the 15min of 8000rpm～10000rpm, supernatant liquor is the coarse body fluid of R-phycoerythrin.
(3) preliminary purification of R-phycoerythrin: the using sulfated ammonium precipitator method, R-phycoerythrin in the coarse body fluid of R-phycoerythrin is precipitated out from solution, centrifugal collecting precipitation is used 20mM phosphoric acid buffer (pH6.8～7.0) dissolving and dialysis again, removes ammonium sulfate.
(4) single stage method prepares high purity R-phycoerythrin: keep stable properties according to phycobiliprotein in the pH of broad scope, utilize ion-exchange chromatography, apparatus has the damping fluid of constant ionic strength, pH gradient to carry out wash-out, and single stage method can obtain highly purified R-phycoerythrin.
Above-mentioned raw materials multitube algae and Ceramium kondoi are the large-scale red algae in ocean, are to gather from coastal tideland, gather the back and clean freezing with seawater.
The preliminary purification concrete operations of above-mentioned steps (3) R-phycoerythrin are: add solid ammonium sulfate in the coarse body fluid of the R-phycoerythrin that above-mentioned steps (2) obtains, to concentration be 55～60% (w/v), place 4～5h, then under 4 ℃, centrifugal 10min～the 15min of 10000rpm～11000rpm, the collecting precipitation part.Precipitation is dissolved in the phosphoric acid buffer (pH6.8～7.0) of 20mM, then the phosphoric acid buffer (pH6.8～7.0) of 20mM is dialysed.Collect dialyzate.
The ratio of above-mentioned precipitation and phosphoric acid buffer does not have strict the qualification, as long as precipitation can be dissolved fully.
It is as follows that above-mentioned steps (4) single stage method prepares the concrete operations of high purity R-phycoerythrin:
Get the dialyzate 1～2ml of step (3), last DEAE Sepharose Fast Flow ion-exchange chromatography, this ion-exchange chromatography is through 20mmol/L acetate buffer solution (pH5.6, contain 0.05mol/L NaCl) pre-balance, specification is 0.6 * 10cm, then with the 20mmol/L acetate buffer solution (pH5.6 that contains 0.05mol/L NaCl,) wash-out, use 20mmol/L acetate buffer solution (pH5.6 at last, contain 0.05mol/L NaCl) and 20mmol/L acetate buffer solution (pH4.0, containing 0.05mol/LNaCl) each 100mL carries out gradient elution, elution speed is 60～70mL/h, and elution curve is seen Fig. 1, detects wavelength 280, according to the red liquid of elution curve collection protein peak 5, obtain the R-phycoerythrin (Fig. 2) of purifying.
Detect the purifying R-phycoerythrin A that obtains through absorption spectrum
565/ A
280Reached 5.6.It is generally acknowledged A
565/ A
280Reach more than 4.5, just think that the R-phycoerythrin is purified, therefore, the R-phycoerythrin that this single stage method ion exchange chromatography obtains is high purifying.Simultaneously, A
620/ A
280And A
650/ A
280Ratio＜0.001, and the maximum absorption band of C-algae basket albumen (CPC) and different algae basket albumen (APC) lays respectively at 620nm and 650nm, the RPE that separation and purification is described does not have CPC and pollution APC.The Native-PAGE electrophoresis detection has only a band, shows that further the RPE of separation and purification does not have other proteic pollution.
Excellent results of the present invention is, with the marine red alga is material, application is through freezing molten and low ionic strength swelling rapid extraction R-phycoerythrin (RPE), carry out preliminary purification through ammonium sulfate precipitation then, in the pH of broad scope, keep stable properties according to R-phycoerythrin (RPE) at last, utilize DEAE Sepharose Fast Flow ion-exchange chromatography, carry out wash-out with the damping fluid of constant ionic strength, pH gradient, single stage method can obtain highly purified R-phycoerythrin (RPE).Present method has been saved the complex separations purifying procedure of the ion exchange chromatography of traditional application sieve chromatography coupled ion intensity wash-out; overcome and be difficult to the mass-producing difficult problem of preparation fast; easy and simple to handle; time saving and energy saving; simple to equipment requirements, yield height, the easy preparation cost for preparing and greatly reduce RPE in a large number; thereby lay a good foundation for RPE being applied to overdelicate biomedical the detection, have good application prospects and economic benefit.
(4) description of drawings
Fig. 1 is embodiment 1 multitube algae RPE ion exchange chromatography elution curve (detecting wavelength 280), and peak 5 is the R-phycoerythrin.
Fig. 2 is the absorption spectrum (solid line) and the fluorescence emission spectrum (dotted line) of the R-phycoerythrin of separation and purification in the peak 5 of Fig. 1.Absorption peak is respectively 498nm, 545nm, 565nm.Excite with 498nm, fluorescence emission peak is positioned at 578nm, and is identical with the standard fluorescence emission peak.
Fig. 3 is the Native-PAGE electrophoretogram of the multitube algae R-phycoerythrin of embodiment 1 separation and purification.
(5) embodiment
The rapidly and efficiently separating and purifying method of embodiment 1:R-phycoerythrin, step is as follows:
(1) raw material is the multitube algae.Gather from coastal tideland, gather the back and clean freezing with seawater.
(2) extraction of R-phycoerythrin, the multitube algae frond 10g of freezing preservation, 1: 1 by volume adding 0.02/L phosphoric acid buffer (pH6.98), under 24 ℃, melt 2h, filtered through gauze, filtrate under 4 ℃, the centrifugal 12min of 10000rpm, supernatant liquor is the coarse body fluid of R-phycoerythrin.
(3) preliminary purification of R-phycoerythrin: in the coarse body fluid of the R-phycoerythrin that above-mentioned steps (2) obtains, add solid ammonium sulfate, to concentration be 58% (w/v), place 4h, then under 4 ℃, the centrifugal 12min 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.
(4) single stage method prepares high purity R-phycoerythrin: the dialyzate 2ml that gets step (3), last DEAE SepharoseFast Flow ion-exchange chromatography, this ion-exchange chromatography is through 20mmol/L acetate buffer solution (pH5.6, contain 0.05mol/LNaCl) pre-balance, specification is 0.6 * 10cm, then with the 20mmol/L acetate buffer solution (pH5.6 that contains 0.05mol/LNaCl,) wash-out, use 20mmol/L acetate buffer solution (pH5.6 at last, contain 0.05mol/L NaCl) and 20mmol/L acetate buffer solution (pH4.0, containing 0.05mol/L NaCl) each 100mL carries out gradient elution, elution speed is 60～70mL/h, elution curve is seen Fig. 1, collects the red liquid of protein peak 5, obtains the R-phycoerythrin of purifying.
The R-phycoerythrin absorption spectrum of above-mentioned purifying is seen Fig. 2, and absorption peak is respectively 498nm, 545nm, 565nm, and A565/A280 reaches 5.6, shows highly purified.Excite with 498nm, fluorescence emission peak is positioned at 578nm, and is identical with the standard fluorescence emission peak.The R-phycoerythrin Native-PAGE electrophoretogram of above-mentioned purifying is seen Fig. 3, has only a band, and the R-phycoerythrin that further specifies separation and purification does not have other proteic pollution.
Embodiment 2: as described in embodiment 1, different is:
(1) raw material is a Ceramium kondoi, gathers from coastal tideland, cleans freezing with seawater.
(2) get the Ceramium kondoi 12g of refrigeration, added 0.02/L phosphoric acid buffer (pH6.8) in 1: 1 by volume, under 22 ℃, melt 3h, filtered through gauze, filtrate under 4 ℃, the centrifugal 15min of 8000rpm, supernatant liquor is the coarse body fluid of R-phycoerythrin.
(3) in the coarse body fluid of the R-phycoerythrin that above-mentioned steps (2) obtains, add solid ammonium sulfate, to concentration be 55% (w/v), place 5h, then under 4 ℃, the centrifugal 10min of 11000rpm, collecting precipitation part.Precipitation is dissolved in the phosphoric acid buffer (pH6.8) of 20mM, then the phosphoric acid buffer (pH6.8) of 20mM is dialysed.Collect dialyzate.
(4) with embodiment 1.
Claims (3)
1.R-the separation purification method of phycoerythrin, step is as follows:
(1) raw material is a marine red alga, is selected from multitube algae or Ceramium kondoi;
(2) extraction of R-phycoerythrin, the red algae frond 10～12g of freezing preservation, added 0.02/L phosphoric acid buffer pH6.8～7.0 in 1: 1 by volume, under 20～25 ℃, melt 2～3h, filtered through gauze, filtrate is under 4 ℃, centrifugal 10min～the 15min of 8000rpm～10000rpm, supernatant liquor is the coarse body fluid of R-phycoerythrin;
(3) preliminary purification of R-phycoerythrin: add solid ammonium sulfate in the coarse body fluid of the R-phycoerythrin that above-mentioned steps (2) obtains, centrifugal collecting precipitation with 20mM phosphoric acid buffer pH6.8～7.0 dissolvings and dialysis, is removed ammonium sulfate again;
(4) single stage method prepares high purity R-phycoerythrin: utilize ion-exchange chromatography, the damping fluid that apparatus has constant ionic strength, a pH gradient carries out wash-out to the dialyzate of step (3) gained, obtains highly purified R-phycoerythrin.
2. the separation purification method of R-phycoerythrin as claimed in claim 1 is characterized in that, adding solid ammonium sulfate to concentration in the step (3) is 55～60%w/v, place 4～5h, then under 4 ℃, the centrifugal 10min～15min of 10000rpm～11000rpm, collecting precipitation part.
3. the separation purification method of R-phycoerythrin as claimed in claim 1, it is characterized in that, step (4) ion-exchange chromatography is the dialyzate 1～2ml that gets step (3), last DEAE Sepharose Fast Flow ion-exchange chromatography, this ion-exchange chromatography is through 20mmol/L acetate buffer solution pre-balance, specification is 0.6 * 10cm, then with the 20mmol/L acetate buffer solution pH5.6 wash-out that contains 0.05mol/L NaCl, carry out gradient elution with 20mmol/L acetate buffer solution pH5.6 and each 100mL of 20mmol/L acetate buffer solution pH4.0 at last, elution speed is 60～70mL/h, according to the elution curve that detects wavelength 280, collect the red liquid of protein peak 5 as shown in Figure 1, obtain the R-phycoerythrin of purifying.
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| CN101240009B (en) * | 2008-02-28 | 2010-06-02 | 山东大学 | Method for fast separating and purifying R-phycoerythrin, R-phycocyanin |
| CN106117326A (en) * | 2015-12-09 | 2016-11-16 | 烟台大学 | A kind of centrifuging combines the method that anion-exchange chromatography medium prepares phycoerythrin |
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