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Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli

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    Research Article

    Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli
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    † Department of Molecular and Cell Biology, Science for Life Laboratory, Uppsala University, Se-751 24 Uppsala, Sweden
    ‡ Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, 69117, Germany
    ∥ Biological Chemistry, Deutsche Krebsforschungszentrum, Heidelberg, 69120, Germany
    *E-mail: [email protected]
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2017, 6, 2, 233–255
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    https://doi.org/10.1021/acssynbio.6b00138
    Published October 24, 2016
    Copyright © 2016 American Chemical Society
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    Abstract

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    Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

    Copyright © 2016 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00138.

    • Table S1 with the sequences of M.mazei tRNAPyl and PylRSAF; Table S2 with primer sequences used in the study, supplementary methods, Figures S1–S36 (incl. NcAA and dye structures, NcAA incorporation sites in LacI and OmpC, controls for the labeling) (PDF)

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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2017, 6, 2, 233–255
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.6b00138
    Published October 24, 2016
    Copyright © 2016 American Chemical Society
    Request reuse permissions

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