Efficient protein selection based on ribosome display system with purified components
Efficient protein selection based on ribosome display system with purified components
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DOI：
10.1016/j.bbrc.2006.11.017
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发表时间：
2007-01-05
影响因子：
3.1
通讯作者：
Ueda, Takuya
中科院分区：
文献类型：
--
作者：
Ohashi, Hiroyuki;Shimizu, Yoshihiro;Ueda, Takuya
利用PURE（Protein synthesis Using Recombinant Elements）系统，我们开发了一种高效、高度可控的核糖体展示方法，用于功能蛋白的筛选。PURE系统由负责大肠杆菌中基因表达的纯化因子和酶组成。我们对核糖体展示系统进行了详细的分析和优化，并证明了稳定的mRNA/核糖体/多肽三元复合物的形成。由于复合物的形成是成功的核糖体展示的基础，这些改进导致mRNA回收率的显著增加。结果，在单轮选择中实现了类似于12，000倍的单链抗体（scFv）cDNA富集。仅用三轮亲和选择就实现了从竞争mRNA中1：10（10）稀释液中特异性选择scFv mRNA。这些发现，连同在所附的文件[T. Matsuura，H.柳田，J.牛田，I.浦部，T. Yomo，Nascent chain，RNA，and ribosome complex generated by pure translation system（参见随附论文）。证明PURE系统可以为可靠和可重复的核糖体展示提供基础。(c)2006年爱思唯尔公司All rights reserved.
Using the PURE (Protein synthesis Using Recombinant Elements) system, we developed an efficient and highly controllable ribosome display method for selection of functional protein. The PURE system is composed of purified factors and enzymes that are responsible for gene expression in Escherichia coli. We performed the detailed analyses and optimization of the ribosome display system and demonstrated the formation of stable mRNA/ribosome/polypeptide ternary complexes. As complex formation is fundamental to successful ribosome display, these improvements resulted in a dramatic increase in the mRNA recovery rate. As a result, a similar to 12,000-fold enrichment of single-chain antibody (scFv) cDNA was achieved in a single round of selection. Specific selection of scFv mRNA from a 1:10(10) dilution in competitor mRNA was achieved with only three rounds of affinity selection. These findings, together with the results in the accompanying paper [T. Matsuura, H. Yanagida, J. Ushioda, I. Urabe, T. Yomo, Nascent chain, RNA, and ribosome complexes generated by pure translation system (see the accompanying paper).], demonstrate that the PURE system can provide a basis for reliable and reproducible ribosome display. (c) 2006 Elsevier Inc. All rights reserved.