Mie Masayasu, Shimizu Shun, Takahashi Fumio, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-28 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Biochem Biophys Res Commun. 2008 Aug 15;373(1):48-52. doi: 10.1016/j.bbrc.2008.05.173. Epub 2008 Jun 9.
The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library.
信使核糖核酸（mRNA）的5′非翻译区（5′-UTR）作为翻译增强子发挥作用，可提高翻译效率。由于翻译起始减少，许多体外翻译系统在蛋白质翻译方面表现出效率降低。使用具有高翻译效率的5′-UTR序列可极大地提高这些系统中的蛋白质产量。在本研究中，我们利用核糖体展示技术开发了一种体外筛选系统，该系统有利于选择具有高翻译效率的5′-UTR。一个由带有His标签和海肾荧光素酶（R-luc）融合标签的5′-UTR组成的5′-UTR随机文库在兔网织红细胞中进行体外翻译。通过限制翻译时间，只有具有高翻译效率的mRNA才能被翻译。在翻译过程中，mRNA、核糖体和带有His标签的已翻译R-luc形成三元复合物。使用镍颗粒与已翻译的His标签一起收集它们。从三元复合物中提取的mRNA通过逆转录聚合酶链反应（RT-PCR）进行扩增并测序。最后，从随机的5′-UTR文库中获得了具有高翻译效率的5′-UTR。
