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

Protein Sci. 1998 Feb;7(2):419-26. doi: 10.1002/pro.5560070223.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / biosynthesis
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Mutagenesis, Site-Directed
  • RNA, Fungal / chemistry
  • RNA, Fungal / genetics*
  • RNA, Transfer, Phe / chemistry
  • RNA, Transfer, Phe / genetics*
  • p-Fluorophenylalanine / chemistry*

Substances

  • Bacterial Proteins
  • RNA, Fungal
  • RNA, Transfer, Phe
  • p-Fluorophenylalanine