Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.
Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
Furter
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Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst 01003, USA.
D001426:Bacterial Proteins / Q000096:biosynthesis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics*; D010135:p-Fluorophenylalanine / Q000737:chemistry*
D001426:Bacterial Proteins; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D010135:p-Fluorophenylalanine