EXPANSION OF THE GENETIC-CODE - SITE-DIRECTED P-FLUORO-PHENYLALANINE INCORPORATION IN ESCHERICHIA-COLI

Site-directed incorporation of the amino acid analogue p-fluoro-phenyl alanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(amber)(Phe)/phenylalanyl-tRNA synthetase pair was expressed in a n analogue-resistant E. coli strain to direct analogue incorporation a t a programmed amber stop codon in the DHFR marker protein. The progra mmed 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 positio n than the background incorporation at phenylalanine codons, showing h igh specificity of analogue incorporation. Protein expression yields o f 8-12 mg/L of culture, corresponding to about two thirds of the expre ssion level of the wild-type DHFR protein, are sufficient to provide f luorinated proteins suitable for F-19-NMR spectroscopy and other sampl e-intensive methods. The use of a nonessential ''21st'' tRNA/synthetas e pair will permit incorporation of a wide range of analogues, once th e synthetase specificity has been modified accordingly.