用膨化柱填料的方法从红藻中规模分离纯化R-藻红蛋白(英)

以Phenyl_sepharose作为柱填料 ,用streamline柱层析技术从红藻 (Palmariapalmata (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 .2mol/L、0 .1mol/L和 0 .0 5mol/L的 (NH4) 2 SO4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/OD2 80 )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 .12 2mg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。

Large_scale Isolation and Purification of R_phycoerythrin from Red Alga Palmaria palmata Using the Expanded Bed Adsorption Method

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 (NH 4) 2SO 4, (NH 4) 2SO 4 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 ( OD 565 /OD 280 ) 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.