Abstract
In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3′-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.
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Kim, HC., Kwon, YC., Lee, KH. et al. Multi-hour translation of mRNA in a cell-free system. Biotechnol Bioproc E 16, 1152–1156 (2011). https://doi.org/10.1007/s12257-010-0417-0
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DOI: https://doi.org/10.1007/s12257-010-0417-0