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

doi:10.1038/nprot.2007.379

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

Pharmaceutical Sciences

Research output: Contribution to journal › Article › peer-review

126 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 language	English
Pages (from-to)	2601-2607
Number of pages	7
Journal	Nature Protocols
Volume	2
Issue number	10
DOIs	https://doi.org/10.1038/nprot.2007.379
State	Published - 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).

Funding

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).

Funders	Funder number
ACS-PRF	42214-GB4
F&M Hackman and Eyler funds	NSF-MCB-0448297
Research Corporation for Science Advancement	CC6364

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1038/nprot.2007.379

Cite this

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title = "Preparation of site-specifically labeled fluorinated proteins for 19f-nmr structural characterization",

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.",

author = "Hammill, {Jared T.} and Shigeki Miyake-Stoner and Hazen, {Jennifer L.} and Jackson, {Jennifer C.} and Mehl, {Ryan A.}",

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AU - Hazen, Jennifer L.

AU - Jackson, Jennifer C.

AU - Mehl, Ryan A.

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

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

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