Abstract
We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Science 292, 498–500 (2001).
Chin, J.W. et al. Science 301, 964–967 (2003).
Wang, L. & Schultz, P.G. Angew. Chem. Int. Edn Engl. 44, 34–66 (2005).
Wang, L., Zhang, Z., Brock, A. & Schultz, P.G. Proc. Natl. Acad. Sci. USA 100, 56–61 (2003).
Chin, J.W., Martin, A.B., King, D.K., Wang, L. & Schultz, P.G. Proc. Natl. Acad. Sci. USA 99, 11020–11024 (2002).
Wu, N., Deiter, A., Cropp, T.A., King, D. & Schultz, P.G. J. Am. Chem. Soc. 126, 14306–14307 (2004).
Zhang, Z. et al. Science 303, 371–373 (2004).
Alfonta, L., Zhang, Z., Uryu, S., Loo, J.A. & Schultz, P.G. J. Am. Chem. Soc. 125, 14662–14663 (2003).
Wang, L. & Schultz, P.G. Chem. Biol. 8, 883–890 (2001).
Nakamura, Y., Gojobori, T. & Ikemura, T. Nucleic Acids Res. 28, 292 (2000).
Muskhelishvili, G., Buckle, M., Heumann, H., Kahmann, R. & Travers, A.A. EMBO J 16, 3655–3665 (1997).
Kobayashi, T. et al. Nat. Struct. Biol. 10, 425–432 (2003).
Plumbridge, J. & Söll, D. Biochimie 69, 539–541 (1987).
Chin, J.W. et al. J. Am. Chem. Soc. 124, 9026–9027 (2002).
Xie, J. et al. Nat. Biotechnol. 22, 1297–1301 (2004).
Acknowledgements
This work was supported by Department of Energy (ER46051). This is manuscript # 17854-CH of the Scripps Research Institute.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Rights and permissions
About this article
Cite this article
Ryu, Y., Schultz, P. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat Methods 3, 263–265 (2006). https://doi.org/10.1038/nmeth864
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth864
This article is cited by
-
Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad
Nature Communications (2024)
-
Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria
Scientific Reports (2023)
-
Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine
Nature Communications (2022)
-
Deactylation by SIRT1 enables liquid–liquid phase separation of IRF3/IRF7 in innate antiviral immunity
Nature Immunology (2022)
-
Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags
Amino Acids (2022)
