Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins
Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins
复制标题
DOI：
10.1093/nar/gng078
复制
发表时间：
2003-08-01
影响因子：
14.9
通讯作者：
Yanagawa, H
中科院分区：
文献类型：
--
作者：
Miyamoto-Sato, E;Takashima, H;Yanagawa, H
对于使用基因型（mRNA）-表型（蛋白质）融合形成和C-末端蛋白质标记作为选择后分析的高通量体外蛋白质选择，重要的是提高用于这两种技术的mRNA模板的稳定性和效率。在这里，我们描述了一个有效的单链连接（90%的输入mRNA）使用荧光素共轭聚乙二醇嘌呤霉素（荧光PEG Puro）间隔。这种连接提供了具有柔性Fluor-PEG Puro间隔区的稳定c-jun mRNA，用于在无细胞小麦胚芽翻译系统中有效融合形成（70%的输入mRNA具有PEG间隔区）。当使用包括SP 6启动子和Omega 29增强子（烟草花叶病毒Omega的一部分）的5'非翻译区时，3'端的A（8）序列（八个连续的腺苷酸残基）适合于融合形成，而XA（8）序列（XhoI和A（8）序列）适合于C-末端蛋白质标记。此外，我们报告了Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro（Boc）]间隔子增强了c-jun mRNA模板用于C-末端蛋白标记的稳定性和效率。这些mRNA模板应该是有用的嘌呤霉素为基础的技术（融合形成和C-末端蛋白质标记），以促进高通量的体外蛋白质选择，不仅进化蛋白质工程，但也蛋白质组探索。
For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.