CAJ | 학술논문

以Phenyl-sepharose作为柱填料,用streamline柱层析技术从红藻(Palmaria palmata (Lannaeus) Kuntze)中规模分离捕光色素蛋白--R-藻红蛋白.由于不是传统的从层析柱上方进样,而是用泵将样品从streamline层析柱的下方加样(从下至上),因而解决了用一般层析柱分离R-藻红蛋白时海藻抽提液中大量的粘性多糖堵塞层析柱的难题.用P.palmata粗提液上样后,分别用0.2 mol/L、 0.1 mol/L和0.05 mol/L的(NH4)2SO4溶液从相反的方向(即从上到下)洗脱层析柱,发现这些洗脱液中的藻红蛋白纯度已经较高.然后将洗脱液透析去盐,用阴离子交换柱层析(Q-sepharose)进一步纯化.经过这两次柱层析后,R-藻红蛋白的纯度(OD565/OD280)超过3.5,高于一般认可的R-藻红蛋白的纯度标准3.2;产率为每克冷冻P.palmata可纯化0.122 mg高纯度的R-藻红蛋白,比使用一般分离方法的产率要高10倍.这些结果表明,使用本文报道的方法纯化藻红蛋白,将会使作为生化检测试剂的藻红蛋白市场价格大幅度下降.
이Phenyl-sepharose작위주전료,용streamline주층석기술종홍조(Palmaria palmata (Lannaeus) Kuntze)중규모분리포광색소단백--R-조홍단백.유우불시전통적종층석주상방진양,이시용빙장양품종streamline층석주적하방가양(종하지상),인이해결료용일반층석주분리R-조홍단백시해조추제액중대량적점성다당도새층석주적난제.용P.palmata조제액상양후,분별용0.2 mol/L、 0.1 mol/L화0.05 mol/L적(NH4)2SO4용액종상반적방향(즉종상도하)세탈층석주,발현저사세탈액중적조홍단백순도이경교고.연후장세탈액투석거염,용음리자교환주층석(Q-sepharose)진일보순화.경과저량차주층석후,R-조홍단백적순도(OD565/OD280)초과3.5,고우일반인가적R-조홍단백적순도표준3.2;산솔위매극냉동P.palmata가순화0.122 mg고순도적R-조홍단백,비사용일반분리방법적산솔요고10배.저사결과표명,사용본문보도적방법순화조홍단백,장회사작위생화검측시제적조홍단백시장개격대폭도하강.
R-phycoerythrin,a light-harvesting protein in some marine algae,and can be widely used in medicine,was isolated and purified from a red alga,Palmaria palmata (Lannaeus) Kuntze,using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography.Because the crude extract was applied to the column upwardly,the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga,this kind of blockage could hardly be overcome in ordinary chromatographic column.After applying the crude extract containing 0.5 mol/L (NH4)2SO4,(NH4)2SO4 solution of different concentrations (0.2 mol/L,0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted.The desalted eluates were then applied onto an ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin.Through these two steps,the purity ( OD565/OD280) of the R-phycoerythrin from P.palmata was up to 3.5,more than 3.2,the commonly accepted criterion for purity,and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P.palmata,much higher than that of phycobiliproteins purified with the previous methods.The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.``