Abstract
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 µg/mL of translated protein (comprising 25 µg in 50 µL 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acids / chemistry
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Amino Acids / genetics
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Amino Acids / metabolism*
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Binding Sites / genetics
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Blotting, Western
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Cell-Free System / metabolism*
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Codon, Terminator / genetics
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Molecular Sequence Data
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Molecular Structure
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Mutation
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Protein Biosynthesis*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism*
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Tandem Mass Spectrometry
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Tyrosine / genetics
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Tyrosine / metabolism
Substances
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Amino Acids
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Codon, Terminator
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Recombinant Proteins
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Green Fluorescent Proteins
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Tyrosine
Grants and funding
The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement no. 260647 and from the Edmond J. Safra Center for the Design and Engineering of Functional Biopolymers. LA holds the Elaine S. and Alvin W. Wene Career Development Chair in Biotechnology Engineering. SS acknowledges the Harbour Foundation for a Fay and Bert Harbour Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript