Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization.
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.
Hammill
Jared T
JT
Chemistry Department, Franklin and Marshall College, PO Box 3003, Lancaster, Pennsylvania 17604, USA.
Miyake-Stoner
Shigeki
S
Hazen
Jennifer L
JL
Jackson
Jennifer C
JC
Mehl
Ryan A
RA
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005461:Fluorine / Q000032:analysis*; D054879:Halogenation; D007554:Isotopes; D009601:Nitroreductases / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D051922:PII Nitrogen Regulatory Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics
D001426:Bacterial Proteins; D007554:Isotopes; D051922:PII Nitrogen Regulatory Proteins; D005461:Fluorine; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D009601:Nitroreductases