Department of Bioengineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan.
Anal Biochem. 2011 Feb 1;409(1):105-11. doi: 10.1016/j.ab.2010.10.002. Epub 2010 Oct 13.
We designed a new approach for selection of translation enhancer sequences that enables efficient protein synthesis in cell-free systems. The selection is based on a gel shift assay of a messenger RNA (mRNA)-protein fusion product that is synthesized in a cell-free translation system using an mRNA display method. A library of randomized 20-nt-long sequences, with all possible combinations of the four nucleotides, upstream of a coding region was screened by successive rounds of screening in which the translation time of the succeeding round was reduced compared with the previous round. An efficient translation enhancer sequence capable of more rapid initiation of cell-free protein synthesis, with a minimal translation time of 5 min, than a natural longer enhancer sequence (Xenopus β-globin 5'UTR) was selected using rabbit reticulocyte extract as a model cell-free translation system. Furthermore, a successful screening of cap-independent translation enhancer sequence and a significant sequence similarity of the selected candidates validated the efficiency of the combined mRNA display and gel shift assay method for the rapid development of advanced cell-free translation systems.
我们设计了一种新的方法来选择翻译增强序列，使无细胞系统中的蛋白质合成更有效率。这种选择是基于一种信使 RNA（mRNA）-蛋白质融合产物的凝胶迁移分析，该产物是在使用 mRNA 显示方法的无细胞翻译系统中合成的。通过连续几轮筛选，对编码区上游的 20 个核苷酸长的随机序列文库进行筛选，其中下一轮的翻译时间比上一轮缩短。与天然较长的增强子序列（爪蟾β-珠蛋白 5'UTR）相比，使用兔网织红细胞提取物作为模型无细胞翻译系统，选择了一种能够更快速起始无细胞蛋白质合成的有效翻译增强子序列，其最小翻译时间为 5 分钟。此外，对无帽依赖翻译增强序列的成功筛选以及所选候选物的显著序列相似性验证了组合的 mRNA 显示和凝胶迁移分析方法在快速开发先进的无细胞翻译系统方面的效率。
