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Study on protein network using novel ribosome-display method

利用新型核糖体展示方法研究蛋白质网络

基本信息

批准号：
14208080
负责人：
UEDA Takuya
金额：
$ 32.45万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14208080/
关键词：
Protein Synthesis Evolutionary Engineering Protein Interaction Ribosome Release Factor 翻訳開始開始因子

项目摘要

For the development of ribosome display method, the stability of ternary complex consisting of ribosome, mRNA and polypeptide is very crucial. To increase the stability, influence of insertion of spacer sequence at the C-terminus of HyHEL10scFv (lysozyme specific monoclonal antibody) was examined. It turned out that the introduction of translation arrest sequence of Sec M, F X X X X W I X X X X G I R A G P, caused efficient recovery of mRNA during the selection of ribosome-display procedure. It is likely that this was due to the stability of the ribosome complex by the SecM introduction. Moreover, it indicates that PURESYSTEM is very useful because no tmRNA which conducts ribosome recycling process, exists in the system, which completely different from the system by crude cell extract.. Omission experiment of particular factors (EF, ribosome, and mRNA) from the system towards HyHEL110 mRNA among excess amount of DHFR mRNA showed that the recovery of mRNA is actually mediated via ribosomal ternary complex. Moreover we succeeded in the concentration of a target molecule more than12000-fold in a single round selection. It is thought that this shows the effectiveness of PURESYSTEM for ribosome-display because only less than 1000 times concentration efficiency in the ribosome-disply method using cell-extract has been reported so far.

在核糖体展示技术的发展过程中，核糖体、mRNA和多肽组成的三元复合物的稳定性至关重要。为了提高稳定性，检查了在HyHEL 10 scFv（溶菌酶特异性单克隆抗体）的C-末端插入间隔序列的影响。结果表明，Sec M的翻译阻滞序列F X X X X X W I X X X G I R A G P的引入在核糖体展示过程的选择过程中引起了mRNA的有效回收。这可能是由于SecM引入引起的核糖体复合物的稳定性。此外，这表明PURESYSTEM是非常有用的，因为在该系统中不存在进行核糖体再循环过程的tmRNA，这与粗细胞提取物的系统完全不同。在过量的DHFR mRNA中，从系统中对HyHEL 110 mRNA的特定因子（EF、核糖体和mRNA）的省略实验表明，mRNA的恢复实际上是通过核糖体三元复合物介导的。此外，我们成功地在一个单一的轮选择超过12000倍的目标分子的浓度。据认为，这表明PURESYSTEM用于核糖体展示的有效性，因为迄今为止在使用细胞提取物的核糖体展示方法中仅报道了不到1000倍的浓缩效率。