大肠杆菌细胞抽提物制备方案的简化与优化  

Simplification and optimization of the preparation of Escherichia coli extract for cell-free protein expression

作　　者：郭新娟[1,2] 权春善[3] 赵朋超[1,2] 王丽娜[1,2] 范圣第[3] 

机构地区：[1]中国科学院大连化学物理研究所,辽宁大连116023 [2]中国科学院大学,北京100049 [3]大连民族学院生物技术与资源利用国家民委-教育部重点实验室,辽宁大连116600

出　　处：《生物工程学报》2013年第4期532-535,共4页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.21152002);中央高校专项基金(No.DC12010118)资助~~

摘　　要：无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。Cell-flee protein expression system is a new method to express target protein in vitro and has been widelyapplied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation ofEscherichia coli cell extract. A simple centrifugation step （12 000×g, 10 rain） followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity （209 μg/mL）. Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.

关 键 词：蛋白质表达 无细胞表达系统 细胞抽提物 大肠杆菌 体外表达 

分 类 号：Q78[生物学—分子生物学]