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An enhanced system for unnatural amino acid mutagenesis in E. coli.
J Mol Biol. 2010 Jan 15; 395(2):361-74.JM

Abstract

We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.

Authors+Show Affiliations

Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

19852970

Citation

Young, Travis S., et al. "An Enhanced System for Unnatural Amino Acid Mutagenesis in E. Coli." Journal of Molecular Biology, vol. 395, no. 2, 2010, pp. 361-74.
Young TS, Ahmad I, Yin JA, et al. An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol. 2010;395(2):361-74.
Young, T. S., Ahmad, I., Yin, J. A., & Schultz, P. G. (2010). An enhanced system for unnatural amino acid mutagenesis in E. coli. Journal of Molecular Biology, 395(2), 361-74. https://doi.org/10.1016/j.jmb.2009.10.030
Young TS, et al. An Enhanced System for Unnatural Amino Acid Mutagenesis in E. Coli. J Mol Biol. 2010 Jan 15;395(2):361-74. PubMed PMID: 19852970.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An enhanced system for unnatural amino acid mutagenesis in E. coli. AU - Young,Travis S, AU - Ahmad,Insha, AU - Yin,Jun A, AU - Schultz,Peter G, Y1 - 2009/10/21/ PY - 2009/07/08/received PY - 2009/10/06/revised PY - 2009/10/14/accepted PY - 2009/10/27/entrez PY - 2009/10/27/pubmed PY - 2010/3/4/medline SP - 361 EP - 74 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 395 IS - 2 N2 - We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/19852970/An_enhanced_system_for_unnatural_amino_acid_mutagenesis_in_E__coli_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(09)01270-4 DB - PRIME DP - Unbound Medicine ER -