Journal
PLOS ONE
Volume 8, Issue 7, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0068363
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Funding
- European Research Council under the European Union's Seventh Framework Programme [260647]
- Edmond J. Safra Center for the Design and Engineering of Functional Biopolymers
- European Research Council (ERC) [260647] Funding Source: European Research Council (ERC)
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Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 mu g/mL of translated protein (comprising 25 mu g in 50 mL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.
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