Journal
CHEMBIOCHEM
Volume 15, Issue 12, Pages 1800-1804Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201402033
Keywords
amino acids; click chemistry; genetic code expansion; optimization; orthogonal ribosomes; unnatural amino acids
Funding
- German Research Foundation (Emmy-Noether Programme)
- Freefloater-Programme of the University of Gottingen
- Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain
- Fonds der Chemischen Industrie
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Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.
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