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KMID : 1094720110160061152
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Biotechnology and Bioprocess Engineering
2011 Volume.16 No. 6 p.1152 ~ p.1156
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Multi-hour translation of mRNA in a cell-free system
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Kim Ho-Cheol
Kwon Yong-Chan
Lee Kyung-Ho
Kim Dong-Myung
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Abstract
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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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KEYWORD
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cell-free translation, mRNA stability, RNase, ribosomal protection
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