【摘要】 在近几年来被广泛研究和应用的大肠杆菌无细胞蛋白质合成系统中,大肠杆菌抽提物的制备、底物浓度的配比以及能量再生体系的选择是最关键的几个因素.以绿色荧光蛋白(GFP)为报导基因,以缺失核酸酶I基因的大肠杆菌A19为实验菌株,通过对大肠杆菌无细胞抽提物制备过程中菌体收集时间、细胞破碎压力、后处理条件等参数的摸索和确定、无细胞蛋白质合成系统中底物浓度配比的正交实验优化以及系统中能量再生体系的比较和选择,制备得到了GFP表达量为154μg/mL的大肠杆菌无细胞蛋白质合成系统,与优化前相比产量提高了29倍.更多还原

【Abstract】 The preparation of cell extraction,the optimization of substrates concentration and the energy regeneration system are the key factors for construction of cell-free system for protein expression.The cell extraction was prepared from RNase I-defective strain E.coli A19.The cell growth phase,the pressure for cell disruption and the storage condition of cell extraction were optimized.The optimal substrate concentrations and the energy regeneration system were selected.Under the optimized conditions,the green fluorescent protein(GFP) reporter gene was expressed in the E.coli cell-free system with high expression level(Ca.154 μg/mL),which is 29 times higher than the expression level before optimization.更多还原