重组大肠杆菌利用乳糖自诱导系统融合表达天蚕素AD和蛙BuforinⅡ

Fusion Expression of Cecropin AD and BuforinⅡ Using Lactose Self-induced System in Recombinant Escherichia coli

以包含质粒pET-Trx-CAD-BuforinⅡ的大肠杆菌BL21(DE3)为研究对象,开发了一种利用乳糖自诱导方式表达天蚕素AD和蛙BuforinⅡ的表达系统。首先,在LB培养基中研究了乳糖浓度、诱导时机和诱导时间对目的蛋白表达的影响。然后,利用"分解代谢物阻遏"原理构建乳糖自诱导表达系统,以"葡萄糖+主碳源"两种碳源的依次代谢将蛋白表达过程分为两个阶段:第一阶段为菌体生长阶段,菌体利用葡萄糖生长,乳糖无法进入细胞诱导蛋白表达;第二阶段为蛋白表达阶段,葡萄糖耗尽,菌体开始利用主碳源生长;同时乳糖进入细胞诱导蛋白表达。最后,利用优选的"葡萄糖+木糖"组合自诱导表达天蚕素AD和BuforinⅡ的融合蛋白,得到的目的蛋白占全菌总蛋白含量的45.2%。

A strategy was developed to express Cecropin AD and Buforin Ⅱ using the lactose self-induced system in recombinant Escherichia coli BL21 （DE3）, harboring plasmid pET-Trx-CAD-Buforin II. First, studies were done in LB culture to detect the effects of lactose concentration, induction-timing and induction-time on target protein ex- pression. Then, lactose self-induced system was constructed according to the ＂carbon catabolite repression＂ princi- ple. The protein expression process was divided into two phases： the first phase of cell growth, E. coli grew with glucose as carbon source. At this time, lactose could not enter into the cell to induce protein expression. The sec- ond phase of protein expression, E. coli grew using ＂main carbon source＂ after glucose depleted. Meanwhile, lac- tose enter into the cell to induce protein expression. The target protein content reached to 45.2% of total protein using the optimized ＂glucose ＋ xylose＂ self-induced system.