Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract. Issue 22 (15th November 2019)

• Record Type: Journal Article
• Authors: Ogawa, Atsushi; Takamatsu, Masashi
• Is Part Of: Bioorganic & medicinal chemistry letters. Volume 29:Issue 22(2019)
• Publication Date: 2019-11-15
• Subjects: Cell-free translation -- Wheat germ extract -- Internal ribosome entry site -- Translational enhancer -- Start codon; Bioorganic chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1016/j.bmcl.2019.126729
• Languages: English
• ISSNs: 0960-894X

Abstract

Graphical abstract: Abstract: Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5′ terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3–4-fold higher than that of its wild type, and also 3–4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.