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/tRNA(CUA) 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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alcohol Oxidoreductases / analysis
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Alcohol Oxidoreductases / chemistry
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Alcohol Oxidoreductases / genetics
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Bacterial Proteins / analysis
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / genetics
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Fluorine / analysis*
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Halogenation*
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Isotopes
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Nitroreductases / analysis
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Nitroreductases / chemistry
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Nitroreductases / genetics
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Nuclear Magnetic Resonance, Biomolecular / methods*
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PII Nitrogen Regulatory Proteins / analysis
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PII Nitrogen Regulatory Proteins / chemistry
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PII Nitrogen Regulatory Proteins / genetics
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Plasmids / chemistry
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Plasmids / genetics
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Protein Engineering / methods*
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Salmonella typhimurium / genetics
Substances
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Bacterial Proteins
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Isotopes
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PII Nitrogen Regulatory Proteins
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Fluorine
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Alcohol Oxidoreductases
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histidinol dehydrogenase
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Nitroreductases