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Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization

Nature Protocols volume 2, pages 2601–2607 (2007)Cite this article

Abstract

A straightforward protocol for the site-specific incorporation of a 19F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNACUA that incorporates L-4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.

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Figure 1: tfmF and pDule plasmid.
Figure 2
Figure 3: Analysis of crude cell pellet expressions for NTR and NTR-tfmF from Table 2.
Figure 4: Analysis of native and tfmF-protein produced for HDH, NTR and NtrCr.

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Acknowledgements

We thank Cinthia Kinsland for her helpful discussions on molecular biology and media. This work was supported by F&M Hackman and Eyler funds and NSF-MCB-0448297, Research Corporation (CC6364) and ACS-PRF (42214-GB4).

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  1. Chemistry Department, Franklin and Marshall College, PO Box 3003, Lancaster, 17604, Pennsylvania, USA

    Jared T Hammill, Shigeki Miyake-Stoner, Jennifer L Hazen, Jennifer C Jackson & Ryan A Mehl

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Correspondence to Ryan A Mehl.

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Hammill, J., Miyake-Stoner, S., Hazen, J. et al. Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nat Protoc 2, 2601–2607 (2007). https://doi.org/10.1038/nprot.2007.379

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