Multiple-Site Labeling of Proteins with Unnatural Amino Acids

Authors


  • We thank Prof. P. G. Schultz for the pEVOL vector for in vivo expression of suppressor tRNA and CouRS-D8, Dr. Siew Pheng Lim for the expression system of the protein WNVpro, Dr. Takeshi Watanabe for the plasmid pChBD, Kekini Kuppan for the pCNF-RS protein, Dr. Isaac Ugwumba and Dr. Hiromasa Yagi for plasmid DNA of amber stop mutants of WNVpro and ERp29, Choy Theng Loh for one of the WNVpro constructs and an expression system of the D286R mutant of pCNF-RS, and Andrew Shafik and Dr. Chris Blake for help with S30 preparations and NMR spectroscopy, respectively. This work was supported by the Australian Research Council, including a Future Fellowship to T.H.

Abstract

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A cell-free protein synthesis system from which the release factor RF1 has been selectively removed enables the facile incorporation of unnatural amino acids into proteins at difficult and multiple sites by optimized use of orthogonal tRNA/aminoacyl-tRNA synthetase systems. 19F NMR spectroscopy of a protein labeled combinatorially with trifluoromethyl phenylalanine (red in picture) at multiple sites establishes resonance assignments with a minimal number of samples.

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