Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems
Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems
复制标题
DOI：
10.1021/acssynbio.6b00010
复制
发表时间：
2016-12-01
影响因子：
4.7
通讯作者：
Huck, Wilhelm T. S.
中科院分区：
文献类型：
--
作者：
Hansen, Maike M. K.;Rosquelles, Marta Ventosa;Huck, Wilhelm T. S.
转录和翻译的去同步化导致了基因二级结构的形成，从而影响基因在体内的表达。然而，在无细胞蛋白表达中，二级结构对mRNA的失活常常被忽视。在无细胞表达系统中，转录和翻译速率通常不是高度同步的，导致过程之间的时间不匹配和蛋白质生产效率的下降。通过设计一个转录和翻译延伸独立进行的无细胞基因表达平台，我们确定序列依赖的mRNA二级结构是体外基因表达中mRNA失活的主要原因。
Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.