Preparation of site-specifically labeled fluorinated proteins for 19f-nmr structural characterization

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

Research output: Contribution to journalArticlepeer-review

110 Scopus citations

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.

Original languageEnglish
Pages (from-to)2601-2607
Number of pages7
JournalNature Protocols
Volume2
Issue number10
DOIs
StatePublished - Oct 2007

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS 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).

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (all)

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