Lee Sea On, Xie Qi, Fried Stephen D
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United States.
ACS Cent Sci. 2021 Oct 27;7(10):1736-1750. doi: 10.1021/acscentsci.1c00574. Epub 2021 Sep 24.
The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling "loopable" translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.
在重组系统中，具有重复氨基酸序列的长蛋白质的表达常常面临挑战。为克服这一障碍，我们报告了一种基因构建体，它通过重新排列来自T4噬菌体的I类自剪接内含子的拓扑结构使mRNA环化，从而实现“可环化”翻译。我们使用基于荧光的检测方法来探究环化mRNA的翻译效率，确定了几种优化该系统蛋白质表达的条件。我们的数据表明，环化mRNA的翻译可能主要受核糖体起始速率的限制；因此，我们使用改良的易错PCR方法，构建了一个将突变集中在环化mRNA起始区域的文库，并发现了表达水平显著更高的突变体。将我们的合理改进与通过定向进化发现的改进相结合，我们报告了一种可环化翻译器，其蛋白质表达水平达到标准线性翻译水平的1.5倍以内。总之，我们的工作证明了可环化翻译是创建大肽链的一个有前景的平台，在新型蛋白质材料开发中具有潜在应用价值。
