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

Protein Science - Tập 7 Số 2 - Trang 419-426 - 1998
Rolf Furter1
1Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst, Massachusetts, 01003

Tóm tắt

AbstractSite‐directed incorporation of the amino acid analogue p‐fluoro‐phenylalanine (p‐F‐Phe) was achieved in Escherichia coli. A yeast suppressor tRNAPheamber/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 “21 st” tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.

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Protein Science

Tập 7 Số 2

419-426

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