Rational optimization of amber suppressor tRNAs toward efficient incorporation of a non-natural amino acid into protein in a eukaryotic wheat germ extract

Org Biomol Chem. 2016 Mar 7;14(9):2671-8. doi: 10.1039/c5ob02533h.


Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 μg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Molecular Structure
  • Phenylalanine / analogs & derivatives*
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Plant Extracts / chemistry*
  • Protein Biosynthesis*
  • Proteins / chemistry*
  • Proteins / metabolism
  • RNA, Transfer / genetics*
  • Suppression, Genetic*
  • Triticum / chemistry*


  • 3-(4-acetylphenyl)-2-aminopropanoic acid
  • Plant Extracts
  • Proteins
  • Phenylalanine
  • RNA, Transfer