Mishra Sanjiv K, Shrivastav Anupama, Mishra Sandhya
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), G.B. Marg, Bhavnagar, Gujarat 364021, India.
Protein Expr Purif. 2011 Dec;80(2):234-8. doi: 10.1016/j.pep.2011.06.016. Epub 2011 Jul 1.
C-phycoerythrin was isolated and purified from marine Pseudanabaena sp. using two step chromatographic methods. Phycobiliproteins in the marine Pseudanabaena was extracted in 100 mM phosphate buffer (pH 7.2) and precipitated by salting out. The precipitated C-phycoerythrin was purified by gel filtration with Sephadex G-150, and then it was purified by ion exchange chromatography on DEAE cellulose, which was developed by linear ionic strength gradients. Purified phycoerythrin showed absorption maxima at 568 and 541 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A₅₆₈/A₂₈₀, a criterion for purity (purity ratio) achieved was 6.86. It showed a single band on examination by polyacrylamide gel electrophoresis (PAGE). The polypeptide analysis of the purified C-phycoerythrin by SDS-PAGE demonstrated that it contained two chromophore-carrying subunits. The yield of purified C-phycoerythrin obtained was 13.6 mg/g of the cell dry weight with 47% of yield. Obtaining highly pure C-phycoerythrin allows one to evaluate its fluorescence properties for future applications in biochemical and biomedical research.
采用两步色谱法从海洋伪鱼腥藻中分离纯化出C-藻红蛋白。将海洋伪鱼腥藻中的藻胆蛋白在100 mM磷酸盐缓冲液(pH 7.2)中提取,并用盐析法沉淀。沉淀的C-藻红蛋白先用Sephadex G-150进行凝胶过滤纯化,然后在DEAE纤维素上进行离子交换色谱纯化,采用线性离子强度梯度洗脱。纯化后的藻红蛋白在568和541 nm处有最大吸收峰,在578 nm处有最大荧光发射峰。获得的纯度标准吸光度比值A₅₆₈/A₂₈₀为6.86。经聚丙烯酰胺凝胶电泳(PAGE)检测显示为单一条带。通过SDS-PAGE对纯化后的C-藻红蛋白进行多肽分析表明,它含有两个携带发色团的亚基。获得的纯化C-藻红蛋白产量为13.6 mg/g细胞干重,产率为47%。获得高纯度的C-藻红蛋白有助于评估其荧光特性,以便未来在生物化学和生物医学研究中应用。