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In Vivo Incorporation of Unnatural Amino Acids into Proteins

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Final rept. 1 Aug 1998-30 Jun 1999

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A new orthogonal suppressor tRNA was derived from tRNA2 to the G1n, which is not a substrate for any E. coil aminoacyl-tRNA synthetase, yet functions with the E. coi translational machinery. Importantly, S. cerevisiae E. coli glutaminyl-tRNA synthetase G1nRS aminioacylates the yeast orthogonal tRNA in vitro and in E. coi, but does not charge E. coli tRNA. This suppressor tRNA and yeast G1nRS thus represent a completely orthogonal pair in E. coi suitable for the delivery of unnatural amino acids into proteins in vivo. A general method was developed to select for mutant synthetases capable of charging any ribosomally-accepted molecule onto an orthogonal suppressor tRNA. Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids. Taken together, these steps clear the way for the final phase of our efforts. Selections for mutant yeast G1nRS enzymes that accept unnatural amino acids will be undertaken. These include 1 a two-step selection with a positive selection based on suppression of b-lactamase in the presence of unnatural amino acids, and a negative barnase selection in the absence of amino acid 2 a screen based on recognition of a suppressed OmpA epitope and 3 a screen based on suppression of GFP.

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  • Biochemistry
  • Anatomy and Physiology

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