Kim Jong-Myung, Shin Ho-Joon, Kim Kyongmin, Lee Myung-Shin
Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.
Mol Biotechnol. 2007 May;36(1):32-7. doi: 10.1007/s12033-007-0017-1.
The size and diversity of ribosome display libraries depends upon stability of the complex formed between the ribosome, mRNA and translated protein. To investigate if mRNA secondary structure improves stability of the complex, we tested a pseudoknot, originating from the genomic RNA of infectious bronchitis virus (IBV), a member of the positive-stranded coronavirus group. We used the previously-isolated anti-DNA scFv, 3D8, as a target protein. During in vitro translation in rabbit reticulocyte lysate, we observed that incorporation of the pseudoknot into the mRNA resulted in production of a translational intermediate that corresponded to the expected size for ribosomal arrest at the pseudoknot. Complexes containing the mRNA pseudoknot exhibited a higher efficiency of affinity selection than that those without, indicating that the pseudoknot improves stability of the mRNA-ribosome-antibody complex in a eukaryotic translation system. Thus, in order to improve the efficiency of selection, this relatively short pseudoknot sequence could be incorporated into ribosome display.
核糖体展示文库的大小和多样性取决于核糖体、信使核糖核酸（mRNA）和翻译后的蛋白质之间形成的复合物的稳定性。为了研究mRNA二级结构是否能提高复合物的稳定性，我们测试了一个来自传染性支气管炎病毒（IBV）基因组RNA的假结，IBV是正链冠状病毒组的成员。我们使用先前分离的抗DNA单链抗体片段（scFv）3D8作为靶蛋白。在兔网织红细胞裂解物的体外翻译过程中，我们观察到将假结掺入mRNA导致产生了一种翻译中间体，其大小与假结处核糖体停滞的预期大小相符。含有mRNA假结的复合物比不含假结的复合物表现出更高的亲和选择效率，这表明假结提高了真核翻译系统中mRNA-核糖体-抗体复合物的稳定性。因此，为了提高选择效率，可以将这个相对较短的假结序列掺入核糖体展示中。
