无细胞蛋白质合成系统的研究进展研究.pdfVIP

I027 无细胞蛋白质合成系统的研究进展 徐志南 陈海琴 岑沛霖 浙江大学生物工程研究所，浙江 杭州 310027 ） 要 无细胞蛋白质合成系统是一种以外源mRNA 或DNA 为模板，通过在细胞抽提物的酶系中补 充底物和能源物质来合成蛋白质的体外系统。与传统的体内重组表达系统相比, 体外无细胞合成系统具 有多种优点, 如可表达对细胞有毒害作用的蛋白质，通过加入人工合成的氨基乙酰tRNA 合成含有非天 然氨基酸 （如D-氨基酸）的蛋白质，能够减少内源蛋白酶对目标蛋白质的攻击，能够直接以PCR 产物 作为模板同 平行合成多种蛋白质，开展高通量药物筛选和蛋白质组学的研究，等等。本文综述了无细 胞蛋白质合成系统的发展历史、系统中合成蛋白质所需的能量供应、遗传模板的稳定性和微型无细胞生 物反应器等多方面的研究，并探讨了无细胞蛋白质合成系统中存在的难点、研究方向和广泛的应用前景。 最后，还总结了浙江大学生物工程研究所对无细胞体系的研究和将其用于合成人防御素及 HIV 蛋白方 面所取得的最新成果. 关键词 蛋白质 多肽 生物合成 无细胞系统 中图分类号 Q7 ADVANCES IN CELL-FREE PROTEIN BIOSYNTHESIS XU Zhinan, CHEN Haiqin and CEN Peilin (Bioengineering Research Institute, Zhej iang University, Hangzhou 310027, zhej iang, China) Abstract The cell-free protein synthesis system enables the direct in vitro synthesis of protein from template DNA or mRNA by using the existing enzymes in the cell extract, which is supplemented with substrates and energy sources. Comparing with conventional recombinant cell technology, cell-free protein synthesis system has several advantages, such as the synthesis of some specific proteins with cytotoxicity or having several unnaturally modified amino acids (D-amino acids), simultaneous synthesis of many different protein molecules by using PCR-generated linear DNA template, which satisfy the high-throughput demand of proteomics and drug screening, etc. This review aims to retrieve the development history of cell-free synthesis system, and to outline the research advances in energy regeneration, the stability of mRNA and the mini-bioreactor for this system. Moreover, the present challenge, research directions and potential application prospects in this cell-free system will also be discussed. Furthermore, the work of establishing the E coli cell-free system and the expression of human beta-defensin-2