大肠杆菌无细胞蛋白质合成系统的初步研究  

Preliminary study on preparation of E.coli cell-free system for protein expression

作　　者：陈海琴[1] 徐志南[1] 汪诚[1] 廖玉华 岑沛霖[1] 

机构地区：[1]浙江大学生物工程研究所,浙江杭州310027 [2]浙江养生堂天然药物研究所,浙江杭州310007

出　　处：《浙江大学学报（工学版）》2007年第5期843-847,共5页Journal of Zhejiang University：Engineering Science

基　　金：国家自然科学基金资助项目(20276066)

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.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.

关 键 词：蛋白质 生物合成 无细胞系统 绿色荧光蛋白(GFP) 

分 类 号：Q7[生物学—分子生物学]