Protein Science, Vol 7, Issue 2 419-426, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Expansion of the genetic code: Site-directed p-fluoro-phenylalanine incorporation in Escherichia coli

R. FURTER
Present address: Bank J. Vontobel & Co Ltd, Bahnhofstr. 3, CH-8022 Zurich, Switzerland.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(amber)(Phe)/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 (19)F-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.
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