Study on protein network using novel ribosome-display method

对于核糖体展示方法的发展，核糖体、信使核糖核酸和多肽三元复合体的稳定性至关重要。为了提高稳定性，考察了在溶菌酶特异性单抗HyHEL10scFv的C末端插入间隔区序列对稳定性的影响。结果表明，在核糖体展示程序的选择过程中，引入SEC M的翻译抑制序列F X W I X G I R A G P，可以有效地恢复mRNA的表达。这可能是由于SECM引入的核糖体复合体的稳定性所致。此外，PURESYSTEM是非常有用的，因为该系统中不存在进行核糖体回收过程的tmRNA，这与通过粗细胞提取的系统完全不同。在过量的dhfr m RNA中，系统中的特定因子(EF、核糖体和m RNA)对HyHEL110 m RNA的遗漏实验表明，m RNA的恢复实际上是通过核糖体三元复合体介导的。此外，我们在单轮选择中成功地将目标分子的浓度提高了12000倍以上。这表明了PURESYSTEM用于核糖体展示的有效性，因为到目前为止，使用细胞提取物的核糖体展示方法的浓缩效率只有不到1000倍的报道。

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.