胡萝卜抗冻蛋白在大肠杆菌中的高效表达

High Efficient Expression of Carrot Antifreeze Protein in E.coli

以pET - 11a为载体构建了胡萝卜抗冻蛋白非融合表达的重组质粒 ,并在大肠杆菌菌株BL2 1(DE3,pLysS)中成功地进行了高效表达。表达的蛋白质以包涵体的形式存在 ,相对分子质量为 36 80 0。经正交试验得到了优化的诱导表达条件。包涵体经各种缓冲液洗涤纯化、溶解、复性和超滤浓缩后 ,经SDS -PAGE电泳检测 ,纯度达单一带水平。生物学活性研究表明 ,重组表达的胡萝卜抗冻蛋白具有很强的热滞活性和提高细菌耐寒性的作用。

Using pET-11a as vector, a recombinant plasmid containing coding seque nce of carrot antifreeze protein(AFP) was constructed and high efficiently expre ssed in the E. coli BL21(DE3, pLysS) strain. A non-fusion expression protoc ol was adopted to express carrot AFP, the expressed carrot AFP existed in the fo rm of inclusion body in the total proteins, and the molecular weight of the reco mbinant protein is 36 800. An optimal inductive condition was obtained by a desi gned orthogonal experiments protocol. The AFP inclusion body was dissolved after washed with kinds of washing buffers, and the AFP in the supernatant was refold ed and concentrated by ultrafiltration. The purified AFP displayed a single band by SDS-PAGE analysis, and could increase low temperature resistance of bacteria with thermal hysteresis activity.