利用FPLC技术建立重组碱性蛋白酶的快速纯化方案

Construction of Fast Purification Method of Recombinant Alkaline Protease with FPLC

在构建了以地衣芽孢杆菌为宿主的产碱性蛋白酶的工程菌BA0 71以后 ,为了给探索重组酶的性质及其稳定性奠定基础 ,利用快速蛋白液相层析 (FPLC)技术 ,建立了快速高效纯化碱性蛋白酶的方案。发酵液通过硫酸氨沉淀、DEAE A 5 0脱色及聚乙二醇浓缩得粗酶 ,再经过CM Sephadex C 5 0、Sephadex G 75柱层析后较好地得到了单一组份的重组碱性蛋白酶 ,酶纯度提高了 76.2倍。SDS PAGE显示重组碱性蛋白酶分子质量为 2 8ku。

In the preceding stage the alkaline protease gene engineering strain BA071 with Bacillus licheniformis as host cell was constructed,in order to further research the characterization of recombinant protease, constructed the fast purification method of recombinant protease with FPLC(fast protein liquid chromatography).The crude enzyme,treated with ammonium sulfate fractionation and decolored with DEAE A 50 and polyethylene glycol concentration,was purified with CM Sephadex C 50 and Sephadex G 75.The purified enzyme appears homologous on SDS PAGE.The purity of enzyme was increased 76.2 times.SDS PAGE analysis shows that the molecular weights of expressed recombinant products are about 28?ku.