Expansion of the genetic code: Site-directed p-fluoro-phenylalanine incorporation in Escherichia coli
✍ Scribed by Rolf Furter
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2008
- Tongue
- English
- Weight
- 866 KB
- Volume
- 7
- Category
- Article
- ISSN
- 0961-8368
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✦ Synopsis
Abstract
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 ^19^F‐NMR spectroscopy and other sample‐intensive methods. The use of a nonessential “21 st” tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.