Isolation And Purification Of Phycobiliproteins And Comparison Of Their Anti-oxidative Activity With Recombinant Allophycocyanin

Phycobiliproteins are important light-harvesting pigment proteins found in some alga, including Phycocyanin, allophycocyanin,Phycoerythrin and Pycoerycyanin. Because they have potentially useful optical and optoelectronic properties that derive from their strong fluorescence, Phycobiliproteins are more and more regarded as fluorescence probes. Recently, Phycobiliproteins are discovered to have many other bioactivities, such as anti-oxidativity, anti-inflammatory, anti-tumor and cell-growth promoting. These properties might lead to applications in many other fields. But the complex isolation and purification and high price of Phycobiliproteins are long-standing problems. So isolation and purification of them is an emphasis of the study in phycobiliproteins. Recombinant Allophycocyanin(rAPC) can be gotten by subcloning and expressing Allophycocyanin gene(APC) in E.coli. It can inhibit H22 hepatomae and S180 tumor, enhance immunity of T cells, and produce more cytokine IL-2, γ-INF. In our work, we have studied the method of isolateion and purification of phycobiliproteins, and compared the anti-oxidative activity among phycocyanin, allophycocyanin and rAPC. We selected Spirulina platensis as the source of phycobiliproteins and freeze-thawed Spirulina platensis repeatedly between -20℃and 4℃for three times. Thus we can break most cells and perfectly keep the bioactiveities of phycobiliproteins. Then the blue supernatant was fractioned stepwise with ammonium sulphate, getting phycocyanin and allophycocyanin separately. Then the extracts were dialyzed. We select appropriate chromatography methods and eluting buffer, which not only improved the velocity of purifying phycobiliproteins, but also assured the purification ofphycobiliproteins. The crude product of phycocyanin was chromatographed on a hydroxylapatite (HA) column and eluted with 0.02M PBS(0.1 NaCl). We obtained C-phycocyanin with absorption ratio A620/A280=4.246. The crude product of allophycocyanin was chromatogramphed on a hydroxylapatite (HA) column twice and eluted with 0.1 M PBS(0.1 NaCl). We got allophycocyanin with absorption ratio A651/A280=3.6766. Then allophycocyanin was chromatogramphed on a DEAE-52 and eluted with 0.2NaCl(0.1MPBS). We got allophycocyanin with absorption ratio A651/A280=4.6. In addition, Fermenting a recombinant E.coli strain P1 was used to produce the recombinant allophycocyanin (rAPC). After affinity and hydrophobic chromatography, the purity of rAPC reached 98.03%. In conclusion of anti-oxidative experiment, phycocyanin was a better anti-oxidative agent, compared to allophycocyanin. The recombinant allophycocyanin ( rAPC ) has no...